FABS-IN-TANDEM IMMUNOGLOBULIN AND USES THEREOF
20230220080 · 2023-07-13
Inventors
Cpc classification
A61K39/395
HUMAN NECESSITIES
C07K16/2863
CHEMISTRY; METALLURGY
A61P29/00
HUMAN NECESSITIES
C07K2317/14
CHEMISTRY; METALLURGY
C07K16/2809
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
C07K2317/64
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/22
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K39/395
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
C07K16/22
CHEMISTRY; METALLURGY
C07K16/24
CHEMISTRY; METALLURGY
Abstract
The present invention provides multivalent and multispecific binding proteins that are capable of binding two or more antigens, or two or more epitopes. The present invention also provides methods of making and using such multivalent and multispecific binding proteins, including methods of using such binding proteins for prevention or treatment of various diseases, or for detecting specific antigens in vitro or in vivo.
Claims
1. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain from N- to C-terminal order comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding the same epitope of c-Met, and the same epitope of EGFR as that of bispecific binding protein FIT013a, wherein the bispecific binding protein FIT013a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 240; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 249; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 254.
2. The bispecific binding protein of claim 87, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 242, a VL.sub.A CDR2 of SEQ ID NO: 243, and a VL.sub.A CDR3 of SEQ ID NO: 244; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 246, a VH.sub.B CDR2 of SEQ ID NO: 247, and a VH.sub.B CDR3 of SEQ ID NO: 248; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 251, a VH.sub.A CDR2 of SEQ ID NO: 252, and a VH.sub.A CDR3 of SEQ ID NO: 253; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 256, a VL.sub.B CDR2 of SEQ ID NO: 257, and a VL.sub.B CDR3 of SEQ ID NO: 258; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 242, a VL.sub.A CDR2 of SEQ ID NO: 243, and a VL.sub.A CDR3 of SEQ ID NO: 244; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 246, a VH.sub.B CDR2 of SEQ ID NO: 247, and a VH.sub.B CDR3 of SEQ ID NO: 248; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 251, a VH.sub.A CDR2 of SEQ ID NO: 252, and a VH.sub.A CDR3 of SEQ ID NO: 253; wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 256, a VL.sub.B CDR2 of SEQ ID NO: 257, and a VL.sub.B CDR3 of SEQ ID NO: 258.
3. The bispecific binding protein of claim 87, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 241, and a VH.sub.B having the sequence of SEQ ID NO: 245, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 250, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 255.
4. The bispecific binding protein of claim 87, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 240; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 249; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 254.
5. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain from N- to C-terminal order comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding one or more epitopes on epidermal growth factor receptor (EGFR), and binding one or more epitopes on programmed death-ligand 1 (PD-L1).
6. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain from N- to C-terminal order comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding one or more epitopes on human epidermal growth factor receptor 3 (Her3) and binding one or more epitopes on insulin-like growth factor 1 receptor (IGF-1R).
7. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain from N- to C-terminal order comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding one or more epitopes on delta-like 4 (DLL4), and binding one or more epitopes on vascular endothelial growth factor (VEGF).
8. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain from N- to C-terminal order comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding one or more epitopes on human epidermal growth factor receptor 3 (Her3), and binding one or more epitopes on epidermal growth factor receptor (EGFR).
9. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain from N- to C-terminal order comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding one or more epitopes on programmed cell death 1 (PD-1), and binding one or more epitopes on programmed death-ligand 1 (PD-L1).
10. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain from N- to C-terminal order comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding one or more epitopes on human epidermal growth factor receptor 3 (Her3), and binding one or more epitopes on programmed cell death 1 (PD-1).
11. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain from N- to C-terminal order comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding one or more epitopes on mesenchymal-epithelial transition factor (c-Met), and binding one or more epitopes on programmed death-ligand 1 (PD-L1).
12. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain from N- to C-terminal order comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding one or more epitopes on B and T lymphocyte associated (BTLA) and binding one or more epitopes on programmed cell death 1 (PD-1).
13. The binding protein of any one of claim 5 to claim 12, wherein the binding protein comprises three polypeptide chains, wherein the second polypeptide chain comprises VH.sub.A and CH1, or VL.sub.B and CL, and wherein the third polypeptide chain comprises VL.sub.B and CL, or VH.sub.A and CH1.
14. The binding protein of any one of claim 5 to claim 12, wherein the binding protein comprises two polypeptide chains, and wherein the second polypeptide chain comprises VH.sub.A, CH1, VL.sub.B, and CL, or VL.sub.B, CL, VH.sub.A, and CH1.
15. The bispecific binding protein of claim 5, wherein the binding protein is capable of binding the same epitope of EGFR, and the same epitope of PD-L1 as that of bispecific binding protein FIT012b, wherein the bispecific binding protein FIT012b comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 202; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 211; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 216.
16. The bispecific binding protein of claim 15, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A comprises a VL.sub.A CDR1 of SEQ ID NO: 204, a VL.sub.A CDR2 of SEQ ID NO: 205, and a VL.sub.A CDR3 of SEQ ID NO: 206; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B comprises a VH.sub.B CDR1 of SEQ ID NO: 208, a VH.sub.B CDR2 of SEQ ID NO: 209, and a VH.sub.B CDR3 of SEQ ID NO: 210; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A comprises a VH.sub.A CDR1 of SEQ ID NO: 213, a VH.sub.A CDR2 of SEQ ID NO: 214, and a VH.sub.A CDR3 of SEQ ID NO: 215; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B comprises a VL.sub.B CDR1 of SEQ ID NO: 218, a VL.sub.B CDR2 of SEQ ID NO: 219, and a VL.sub.B CDR3 of SEQ ID NO: 220; (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A comprises a VL.sub.A CDR1 of SEQ ID NO: 204, a VL.sub.A CDR2 of SEQ ID NO: 205, and a VL.sub.A CDR3 of SEQ ID NO: 206, the VH.sub.B comprises a VH.sub.B CDR1 of SEQ ID NO: 208, a VH.sub.B CDR2 of SEQ ID NO: 209, and a VH.sub.B CDR3 of SEQ ID NO: 210, the VH.sub.A comprises a VH.sub.A CDR1 of SEQ ID NO: 213, a VH.sub.A CDR2 of SEQ ID NO: 214, and a VH.sub.A CDR3 of SEQ ID NO: 215, and the VL.sub.B comprises a VL.sub.B CDR1 of SEQ ID NO: 218, a VL.sub.B CDR2 of SEQ ID NO: 219, and a VL.sub.B CDR3 of SEQ ID NO: 220.
17. The bispecific binding protein of claim 15, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 203, and a VH.sub.B having the sequence of SEQ ID NO: 207, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 212, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 217.
18. The bispecific binding protein of claim 15, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 202; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 211; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 216.
19. The bispecific binding protein of claim 5, wherein the binding protein is capable of binding the same epitope of EGFR, and the same epitope of PD-L1 as that of bispecific binding protein FIT012d, wherein the bispecific binding protein FIT012d comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO 221; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 230; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 235.
20. The bispecific binding protein of claim 19, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 223, a VL.sub.A CDR2 of SEQ ID NO: 224, and a VL.sub.A CDR3 of SEQ ID NO: 225; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 227, a VH.sub.B CDR2 of SEQ ID NO: 228, and a VH.sub.B CDR3 of SEQ ID NO: 229; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 232, a VH.sub.A CDR2 of SEQ ID NO: 233, and a VH.sub.A CDR3 of SEQ ID NO: 234; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 237, a VL.sub.B CDR2 of SEQ ID NO: 239, and a VL.sub.B CDR3 of SEQ ID NO: 239; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 223, a VL.sub.A CDR2 of SEQ ID NO: 224, and a VL.sub.A CDR3 of SEQ ID NO: 225; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 227, a VH.sub.B CDR2 of SEQ ID NO: 228, and a VH.sub.B CDR3 of SEQ ID NO: 229; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 232, a VH.sub.A CDR2 of SEQ ID NO: 233, and a VH.sub.A CDR3 of SEQ ID NO: 234; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 237, a VL.sub.B CDR2 of SEQ ID NO: 238, and a VL.sub.B CDR3 of SEQ ID NO: 239;
21. The bispecific binding protein of claim 19, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 222, and a VH.sub.B having the sequence of SEQ ID NO: 226, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 231, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 236.
22. The bispecific binding protein of claim 19, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO 221; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 230; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 235.
23. The bispecific binding protein of claim 6, wherein the binding protein is capable of binding the same epitopes of Her3, and the same epitope of IGF-1R as that of bispecific binding protein FIT016a, wherein the bispecific binding protein FIT016a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 278; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 287; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 292.
24. The bispecific binding protein of claim 23, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 280, a VL.sub.A CDR2 of SEQ ID NO: 281, and a VL.sub.A CDR3 of SEQ ID NO: 282; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 284, a VH.sub.B CDR2 of SEQ ID NO: 285, and a VH.sub.B CDR3 of SEQ ID NO: 286; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 289, a VH.sub.A CDR2 of SEQ ID NO: 290, and a VH.sub.A CDR3 of SEQ ID NO: 291; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 294, a VL.sub.B CDR2 of SEQ ID NO: 295, and a VL.sub.B CDR3 of SEQ ID NO: 296; (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 280, a VL.sub.A CDR2 of SEQ ID NO: 281, and a VL.sub.A CDR3 of SEQ ID NO: 282; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 284, a VH.sub.B CDR2 of SEQ ID NO: 285, and a VH.sub.B CDR3 of SEQ ID NO: 286; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 289, a VH.sub.A CDR2 of SEQ ID NO: 290, and a VH.sub.A CDR3 of SEQ ID NO: 291; wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 294, a VL.sub.B CDR2 of SEQ ID NO: 295, and a VL.sub.B CDR3 of SEQ ID NO: 296;
25. The bispecific binding protein of claim 23, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 279, and a VH.sub.B having the sequence of SEQ ID NO: 283, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 288, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 293.
26. The bispecific binding protein of claim 23, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 278; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 287; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 292.
27. The bispecific binding protein of claim 7, wherein the binding protein is capable of binding the same epitope of DLL4, and the same epitope of VEGF as that of bispecific binding protein FIT017a, wherein the bispecific binding protein FIT017b comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 297; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 306; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 311.
28. The bispecific binding protein of claim 27, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 299, a VL.sub.A CDR2 of SEQ ID NO: 300, and a VL.sub.A CDR3 of SEQ ID NO: 301; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 303, a VH.sub.B CDR2 of SEQ ID NO: 304, and a VH.sub.B CDR3 of SEQ ID NO: 305; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 308, a VH.sub.A CDR2 of SEQ ID NO: 309, and a VH.sub.A CDR3 of SEQ ID NO: 310; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 313, a VL.sub.B CDR2 of SEQ ID NO: 314, and a VL.sub.B CDR3 of SEQ ID NO: 315; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 299, a VL.sub.A CDR2 of SEQ ID NO: 300, and a VL.sub.A CDR3 of SEQ ID NO: 301; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 303, a VH.sub.B CDR2 of SEQ ID NO: 304, and a VH.sub.B CDR3 of SEQ ID NO: 305; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 308, a VH.sub.A CDR2 of SEQ ID NO: 309, and a VH.sub.A CDR3 of SEQ ID NO: 310; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 313, a VL.sub.B CDR2 of SEQ ID NO: 314, and a VL.sub.B CDR3 of SEQ ID NO: 315.
29. The bispecific binding protein of claim 27, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 298, and a VH.sub.B having the sequence of SEQ ID NO: 302, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 307, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 312.
30. The bispecific binding protein of claim 27, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 297; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 306; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 311.
31. The bispecific binding protein of claim 8, wherein the binding protein is capable of binding the same epitope of Her3, and the same epitope of EGFR as that of bispecific binding protein FIT019a, wherein the bispecific binding protein FIT019a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 335; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 344; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 349.
32. The bispecific binding protein of claim 31, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 337, a VL.sub.A CDR2 of SEQ ID NO: 338, and a VL.sub.A CDR3 of SEQ ID NO: 339; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 341, a VH.sub.B CDR2 of SEQ ID NO: 342, and a VH.sub.B CDR3 of SEQ ID NO: 343; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 346, a VH.sub.A CDR2 of SEQ ID NO: 347, and a VH.sub.A CDR3 of SEQ ID NO: 348; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 351, a VL.sub.B CDR2 of SEQ ID NO: 352, and a VL.sub.B CDR3 of SEQ ID NO: 353; (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 337, a VL.sub.A CDR2 of SEQ ID NO: 338, and a VL.sub.A CDR3 of SEQ ID NO: 339; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 341, a VH.sub.B CDR2 of SEQ ID NO: 342, and a VH.sub.B CDR3 of SEQ ID NO: 343; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 346, a VH.sub.A CDR2 of SEQ ID NO: 347, and a VH.sub.A CDR3 of SEQ ID NO: 348; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 351, a VL.sub.B CDR2 of SEQ ID NO: 352, and a VL.sub.B CDR3 of SEQ ID NO: 353.
33. The bispecific binding protein of claim 31, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 336, and a VH.sub.B having the sequence of SEQ ID NO: 340, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 345, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 350.
34. The bispecific binding protein of claim 31, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 335; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 344; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 349.
35. The bispecific binding protein of claim 8, wherein the binding protein is capable of binding the same epitope of Her3, and the same epitope of EGFR as that of bispecific binding protein FIT019b, wherein the bispecific binding protein FIT019b comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 354; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 363; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 368.
36. The bispecific binding protein of claim 35, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 356, a VL.sub.A CDR2 of SEQ ID NO: 357, and a VL.sub.A CDR3 of SEQ ID NO: 358; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 360, a VH.sub.B CDR2 of SEQ ID NO: 361, and a VH.sub.B CDR3 of SEQ ID NO: 362; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 365, a VH.sub.A CDR2 of SEQ ID NO: 366, and a VH.sub.A CDR3 of SEQ ID NO: 367; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 370, a VL.sub.B CDR2 of SEQ ID NO: 371, and a VL.sub.B CDR3 of SEQ ID NO: 372; (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 356, a VL.sub.A CDR2 of SEQ ID NO: 357, and a VL.sub.A CDR3 of SEQ ID NO: 358; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 360, a VH.sub.B CDR2 of SEQ ID NO: 361, and a VH.sub.B CDR3 of SEQ ID NO: 362; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 365, a VH.sub.A CDR2 of SEQ ID NO: 366, and a VH.sub.A CDR3 of SEQ ID NO: 367; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 370, a VL.sub.B CDR2 of SEQ ID NO: 371, and a VL.sub.B CDR3 of SEQ ID NO: 372.
37. The bispecific binding protein of claim 35, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 355, and a VH.sub.B having the sequence of SEQ ID NO: 359, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 364, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 369.
38. The bispecific binding protein of claim 35, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 354; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 363; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 368.
39. The bispecific binding protein of claim 9, wherein the binding protein is capable of binding the same epitopes of PD-1, and the same epitope of PD-L1 as that of bispecific binding protein FIT020a or FIT020b, wherein the bispecific binding protein FIT020a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 120; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 121; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 122, and wherein the bispecific binding protein FIT020b comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 373; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 382; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 387.
40. The bispecific binding protein of claim 39, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises (a) a VL.sub.A CDR1 of SEQ ID NO: 389, a VL.sub.A CDR2 of SEQ ID NO: 390, and a VL.sub.A CDR3 of SEQ ID NO: 391; or (b) a VL.sub.A CDR1 of SEQ ID NO: 375, a VL.sub.A CDR2 of SEQ ID NO: 376, and a VL.sub.A CDR3 of SEQ ID NO: 377; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises (a) a VH.sub.B CDR1 of SEQ ID NO: 384, a VH.sub.B CDR2 of SEQ ID NO: 385, and a VH.sub.B CDR3 of SEQ ID NO: 386; or (b) a VH.sub.B CDR1 of SEQ ID NO: 379, a VH.sub.B CDR2 of SEQ ID NO: 380, and a VH.sub.B CDR3 of SEQ ID NO: 381; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises (a) a VH.sub.A CDR1 of SEQ ID NO: 379, a VH.sub.A CDR2 of SEQ ID NO: 380, and a VH.sub.A CDR3 of SEQ ID NO: 381, or (b) a VH.sub.A CDR1 of SEQ ID NO: 384, a VH.sub.A CDR2 of SEQ ID NO: 385, and a VH.sub.A CDR3 of SEQ ID NO: 386; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises (a) a VL.sub.B CDR1 of SEQ ID NO: 375, a VL.sub.B CDR2 of SEQ ID NO: 376, and a VL.sub.B CDR3 of SEQ ID NO: 377, or (b) a VL.sub.B CDR1 of SEQ ID NO: 389, a VL.sub.B CDR2 of SEQ ID NO: 390, and a VL.sub.B CDR3 of SEQ ID NO: 391; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein (a) the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 389, a VL.sub.A CDR2 of SEQ ID NO: 390, and a VL.sub.A CDR3 of SEQ ID NO: 391; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 384, a VH.sub.B CDR2 of SEQ ID NO: 385, and a VH.sub.B CDR3 of SEQ ID NO: 386; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 379, a VH.sub.A CDR2 of SEQ ID NO: 380, and a VH.sub.A CDR3 of SEQ ID NO: 381; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 375, a VL.sub.B CDR2 of SEQ ID NO: 376, and a VL.sub.B CDR3 of SEQ ID NO: 377; or (b) the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 375, a VL.sub.A CDR2 of SEQ ID NO: 376, and a VL.sub.A CDR3 of SEQ ID NO: 377; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 379, a VH.sub.B CDR2 of SEQ ID NO: 380, and a VH.sub.B CDR3 of SEQ ID NO: 381; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 384, a VH.sub.A CDR2 of SEQ ID NO: 385, and a VH.sub.A CDR3 of SEQ ID NO: 386; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 389, a VL.sub.B CDR2 of SEQ ID NO: 390, and a VL.sub.B CDR3 of SEQ ID NO: 391.
41. The bispecific binding protein of claim 39, wherein the binding protein comprises (a) a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 388, and a VH.sub.B having the sequence of SEQ ID NO: 383, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 378, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 374, or (b) a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 374, and a VH.sub.B having the sequence of SEQ ID NO: 378, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 383, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 388.
42. The bispecific binding protein of claim 39, wherein the binding protein comprises (a) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 120; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 121; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 122, or (b) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 373; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 382; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 387.
43. The bispecific binding protein of claim 10, wherein the binding protein is capable of binding the same epitope of Her3, and the same epitope of PD-1 as that of bispecific binding protein FIT022a, wherein the bispecific binding protein FIT022a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 411; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 420; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 425.
44. The bispecific binding protein of claim 43, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 413, a VL.sub.A CDR2 of SEQ ID NO: 414, and a VL.sub.A CDR3 of SEQ ID NO: 415; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 417, a VH.sub.B CDR2 of SEQ ID NO: 418, and a VH.sub.B CDR3 of SEQ ID NO: 419; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 422, a VH.sub.A CDR2 of SEQ ID NO: 423, and a VH.sub.A CDR3 of SEQ ID NO: 424; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 427, a VL.sub.B CDR2 of SEQ ID NO: 428, and a VL.sub.B CDR3 of SEQ ID NO: 429; (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 413, a VL.sub.A CDR2 of SEQ ID NO: 414, and a VL.sub.A CDR3 of SEQ ID NO: 415; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 417, a VH.sub.B CDR2 of SEQ ID NO: 418, and a VH.sub.B CDR3 of SEQ ID NO: 419; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 422, a VH.sub.A CDR2 of SEQ ID NO: 423, and a VH.sub.A CDR3 of SEQ ID NO: 424; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 427, a VL.sub.B CDR2 of SEQ ID NO: 428, and a VL.sub.B CDR3 of SEQ ID NO: 429.
45. The bispecific binding protein of claim 43, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 412, and a VH.sub.B having the sequence of SEQ ID NO: 416, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 421, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 426.
46. The bispecific binding protein of claim 43, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 411; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 420; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 425.
47. The bispecific binding protein of claim 11, wherein the binding protein is capable of binding the same epitope of c-Met, and the same epitope of PD-L1 as that of bispecific binding protein FIT023a, wherein the bispecific binding protein FIT023a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 430; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 439; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 444.
48. The bispecific binding protein of claim 47, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 432, a VL.sub.A CDR2 of SEQ ID NO: 433, and a VL.sub.A CDR3 of SEQ ID NO: 434; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 436, a VH.sub.B CDR2 of SEQ ID NO: 437, and a VH.sub.B CDR3 of SEQ ID NO: 438; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 441, a VH.sub.A CDR2 of SEQ ID NO: 442, and a VH.sub.A CDR3 of SEQ ID NO: 443; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 446, a VL.sub.B CDR2 of SEQ ID NO: 447, and a VL.sub.B CDR3 of SEQ ID NO: 448; (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 432, a VL.sub.A CDR2 of SEQ ID NO: 433, and a VL.sub.A CDR3 of SEQ ID NO: 434; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 436, a VH.sub.B CDR2 of SEQ ID NO: 437, and a VH.sub.B CDR3 of SEQ ID NO: 438; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 441, a VH.sub.A CDR2 of SEQ ID NO: 442, and a VH.sub.A CDR3 of SEQ ID NO: 443; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 446, a VL.sub.B CDR2 of SEQ ID NO: 447, and a VL.sub.B CDR3 of SEQ ID NO: 448;
49. The bispecific binding protein of claim 47, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 431, and a VH.sub.B having the sequence of SEQ ID NO: 435, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 440, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 445.
50. The bispecific binding protein of claim 47, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 430; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 439; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 444.
51. The bispecific binding protein of claim 12, wherein the binding protein is capable of binding the same epitope of BTLA, and the same epitope of PD-1 as that of bispecific binding protein FIT024a, wherein the bispecific binding protein FIT024a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 449; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 458; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 463.
52. The bispecific binding protein of claim 51, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 451, a VL.sub.A CDR2 of SEQ ID NO: 452, and a VL.sub.A CDR3 of SEQ ID NO: 453; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 455, a VH.sub.B CDR2 of SEQ ID NO: 456, and a VH.sub.B CDR3 of SEQ ID NO: 457; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 460, a VH.sub.A CDR2 of SEQ ID NO: 461, and a VH.sub.A CDR3 of SEQ ID NO: 462; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 465, a VL.sub.B CDR2 of SEQ ID NO: 466, and a VL.sub.B CDR3 of SEQ ID NO: 467; (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 451, a VL.sub.A CDR2 of SEQ ID NO: 452, and a VL.sub.A CDR3 of SEQ ID NO: 453; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 455, a VH.sub.B CDR2 of SEQ ID NO: 456, and a VH.sub.B CDR3 of SEQ ID NO: 457; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 460, a VH.sub.A CDR2 of SEQ ID NO: 461, and a VH.sub.A CDR3 of SEQ ID NO: 462; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 465, a VL.sub.B CDR2 of SEQ ID NO: 466, and a VL.sub.B CDR3 of SEQ ID NO: 467.
53. The bispecific binding protein of claim 51, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 450, and a VH.sub.B having the sequence of SEQ ID NO: 454, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 459, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 464.
54. The bispecific binding protein of claim 51, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 449; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 458; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 463.
55. The bispecific binding protein of claim 12, wherein the binding protein is capable of binding the same epitope of BTLA, and the same epitope of PD-1 as that of bispecific binding protein FIT024b, wherein the bispecific binding protein FIT024b comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 468; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 477; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 482.
56. The bispecific binding protein of claim 55, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 470, a VL.sub.A CDR2 of SEQ ID NO: 471, and a VL.sub.A CDR3 of SEQ ID NO: 472; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 474, a VH.sub.B CDR2 of SEQ ID NO: 475, and a VH.sub.B CDR3 of SEQ ID NO: 476; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 479, a VH.sub.A CDR2 of SEQ ID NO: 480, and a VH.sub.A CDR3 of SEQ ID NO: 481; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 484, a VL.sub.B CDR2 of SEQ ID NO: 485, and a VL.sub.B CDR3 of SEQ ID NO: 486; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 470, a VL.sub.A CDR2 of SEQ ID NO: 471, and a VL.sub.A CDR3 of SEQ ID NO: 472; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 474, a VH.sub.B CDR2 of SEQ ID NO: 475, and a VH.sub.B CDR3 of SEQ ID NO: 476; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 479, a VH.sub.A CDR2 of SEQ ID NO: 480, and a VH.sub.A CDR3 of SEQ ID NO: 481; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 484, a VL.sub.B CDR2 of SEQ ID NO: 485, and a VL.sub.B CDR3 of SEQ ID NO: 486.
57. The bispecific binding protein of claim 55, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 469, and a VH.sub.B having the sequence of SEQ ID NO: 473, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 478, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 483.
58. The bispecific binding protein of claim 55, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 468; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 477; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 482.
59. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding the same epitope of Factor IXa, and the same epitope of Factor X as that of bispecific binding protein FIT014a, wherein the bispecific binding protein FIT014a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 259; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 268; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 273.
60. The bispecific binding protein of claim 59, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 261, a VL.sub.A CDR2 of SEQ ID NO: 262, and a VL.sub.A CDR3 of SEQ ID NO: 263; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 265, a VH.sub.B CDR2 of SEQ ID NO: 266, and a VH.sub.B CDR3 of SEQ ID NO: 267; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 270, a VH.sub.A CDR2 of SEQ ID NO: 271, and a VH.sub.A CDR3 of SEQ ID NO: 272; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 275, a VL.sub.B CDR2 of SEQ ID NO: 276, and a VL.sub.B CDR3 of SEQ ID NO: 277; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 261, a VL.sub.A CDR2 of SEQ ID NO: 262, and a VL.sub.A CDR3 of SEQ ID NO: 263; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 265, a VH.sub.B CDR2 of SEQ ID NO: 266, and a VH.sub.B CDR3 of SEQ ID NO: 267; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 270, a VH.sub.A CDR2 of SEQ ID NO: 271, and a VH.sub.A CDR3 of SEQ ID NO: 272; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 275, a VL.sub.B CDR2 of SEQ ID NO: 276, and a VL.sub.B CDR3 of SEQ ID NO: 277.
61. The bispecific binding protein of claim 59, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 260, and a VH.sub.B having the sequence of SEQ ID NO: 264, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 269, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 274.
62. The bispecific binding protein of claim 59, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 259; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 268; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 273.
63. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding the same epitope of CD20, and the same epitope of CD3 as that of bispecific binding protein FIT018a, wherein the bispecific binding protein FIT018a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 316; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 325; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 330.
64. The bispecific binding protein of claim 63, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 318, a VL.sub.A CDR2 of SEQ ID NO: 319, and a VL.sub.A CDR3 of SEQ ID NO: 320; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 322, a VH.sub.B CDR2 of SEQ ID NO: 323, and a VH.sub.B CDR3 of SEQ ID NO: 324; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 327, a VH.sub.A CDR2 of SEQ ID NO: 328, and a VH.sub.A CDR3 of SEQ ID NO: 329; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 332, a VL.sub.B CDR2 of SEQ ID NO: 333, and a VL.sub.B CDR3 of SEQ ID NO: 334; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 318, a VL.sub.A CDR2 of SEQ ID NO: 319, and a VL.sub.A CDR3 of SEQ ID NO: 320; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 322, a VH.sub.B CDR2 of SEQ ID NO: 323, and a VH.sub.B CDR3 of SEQ ID NO: 324; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 327, a VH.sub.A CDR2 of SEQ ID NO: 328, and a VH.sub.A CDR3 of SEQ ID NO: 329; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 332, a VL.sub.B CDR2 of SEQ ID NO: 333, and a VL.sub.B CDR3 of SEQ ID NO: 334.
65. The bispecific binding protein of claim 63, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 317, and a VH.sub.B having the sequence of SEQ ID NO: 321, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 326, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 331.
66. The bispecific binding protein of claim 63, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 316; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 325; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 330.
67. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding the same epitope of CTLA4, and the same epitope of PD-1 as that of bispecific binding protein NBS3, wherein the bispecific binding protein NBS3 comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 126; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 135; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 140.
68. The bispecific binding protein of claim 67, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 128, a VL.sub.A CDR2 of SEQ ID NO: 129, and a VL.sub.A CDR3 of SEQ ID NO: 130; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 132, a VH.sub.B CDR2 of SEQ ID NO: 133, and a VH.sub.B CDR3 of SEQ ID NO: 134; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 137, a VH.sub.A CDR2 of SEQ ID NO: 138, and a VH.sub.A CDR3 of SEQ ID NO: 139; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 142, a VL.sub.B CDR2 of SEQ ID NO: 143, and a VL.sub.B CDR3 of SEQ ID NO: 144; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 128, a VL.sub.A CDR2 of SEQ ID NO: 129, and a VL.sub.A CDR3 of SEQ ID NO: 130; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 132, a VH.sub.B CDR2 of SEQ ID NO: 133, and a VH.sub.B CDR3 of SEQ ID NO: 134; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 137, a VH.sub.A CDR2 of SEQ ID NO: 138, and a VH.sub.A CDR3 of SEQ ID NO: 139; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.BCDR1 of SEQ ID NO: 142, a VL.sub.B CDR2 of SEQ ID NO: 143, and a VL.sub.B CDR3 of SEQ ID NO: 144.
69. The bispecific binding protein of claim 67, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 127, and a VH.sub.B having the sequence of SEQ ID NO: 131, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 136, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 141.
70. The bispecific binding protein of claim 67, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 126; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 135; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 140.
71. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding the same epitope of CTLA4, and the same epitope of PD-1 as that of bispecific binding protein NBS3R, wherein the bispecific binding protein NBS3R comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 145; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 154; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 159.
72. The bispecific binding protein of claim 71, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 147, a VL.sub.A CDR2 of SEQ ID NO: 148, and a VL.sub.A CDR3 of SEQ ID NO: 149; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 151, a VH.sub.B CDR2 of SEQ ID NO: 152, and a VH.sub.B CDR3 of SEQ ID NO: 153; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 156, a VH.sub.A CDR2 of SEQ ID NO: 157, and a VH.sub.A CDR3 of SEQ ID NO: 158; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 161, a VL.sub.B CDR2 of SEQ ID NO: 162, and a VL.sub.B CDR3 of SEQ ID NO: 163; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 147, a VL.sub.A CDR2 of SEQ ID NO: 148, and a VL.sub.A CDR3 of SEQ ID NO: 149; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 151, a VH.sub.B CDR2 of SEQ ID NO: 152, and a VH.sub.B CDR3 of SEQ ID NO: 153; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 156, a VH.sub.A CDR2 of SEQ ID NO: 157, and a VH.sub.A CDR3 of SEQ ID NO: 158; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 161, a VL.sub.B CDR2 of SEQ ID NO: 162, and a VL.sub.B CDR3 of SEQ ID NO: 163.
73. The bispecific binding protein of claim 71, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 146, and a VH.sub.B having the sequence of SEQ ID NO: 150, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 155, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 160.
74. The bispecific binding protein of claim 71, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 145; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 154; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 159.
75. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding the same epitope of CTLA4, and the same epitope of PD-1 as that of bispecific binding protein NBS3-C, wherein the bispecific binding protein NBS3-C comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 164; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 173; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 178.
76. The bispecific binding protein of claim 75, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 166, a VL.sub.A CDR2 of SEQ ID NO: 167, and a VL.sub.A CDR3 of SEQ ID NO: 168; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 170, a VH.sub.B CDR2 of SEQ ID NO: 171, and a VH.sub.B CDR3 of SEQ ID NO: 172; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 175, a VH.sub.A CDR2 of SEQ ID NO: 176, and a VH.sub.A CDR3 of SEQ ID NO: 177; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 180, a VL.sub.B CDR2 of SEQ ID NO: 181, and a VL.sub.B CDR3 of SEQ ID NO: 182; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 166, a VL.sub.A CDR2 of SEQ ID NO: 167, and a VL.sub.A CDR3 of SEQ ID NO: 168; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 170, a VH.sub.B CDR2 of SEQ ID NO: 171, and a VH.sub.B CDR3 of SEQ ID NO: 172; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 175, a VH.sub.A CDR2 of SEQ ID NO: 176, and a VH.sub.A CDR3 of SEQ ID NO: 177; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 180, a VL.sub.B CDR2 of SEQ ID NO: 181, and a VL.sub.B CDR3 of SEQ ID NO: 182.
77. The bispecific binding protein of claim 75, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 165, and a VH.sub.B having the sequence of SEQ ID NO: 169, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 174, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 179.
78. The bispecific binding protein of claim 71, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 164; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 173; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 178.
79. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding the same epitope of CTLA4, and the same epitope of PD-1 as that of bispecific binding protein NBS3R-C, wherein the bispecific binding protein NBS3R-C comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 183; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 192; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 197.
80. The bispecific binding protein of claim 79, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 185, a VL.sub.A CDR2 of SEQ ID NO: 186, and a VL.sub.A CDR3 of SEQ ID NO: 187; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 189, a VH.sub.B CDR2 of SEQ ID NO: 190, and a VH.sub.B CDR3 of SEQ ID NO: 191; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 194, a VH.sub.A CDR2 of SEQ ID NO: 195, and a VH.sub.A CDR3 of SEQ ID NO: 196; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 199, a VL.sub.B CDR2 of SEQ ID NO: 200, and a VL.sub.B CDR3 of SEQ ID NO: 201; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 185, a VL.sub.A CDR2 of SEQ ID NO: 186, and a VL.sub.A CDR3 of SEQ ID NO: 187; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 189, a VH.sub.B CDR2 of SEQ ID NO: 190, and a VH.sub.B CDR3 of SEQ ID NO: 191; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 194, a VH.sub.A CDR2 of SEQ ID NO: 195, and a VH.sub.A CDR3 of SEQ ID NO: 196; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 199, a VL.sub.B CDR2 of SEQ ID NO: 200, and a VL.sub.B CDR3 of SEQ ID NO: 201.
81. The bispecific binding protein of claim 75, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 184, and a VH.sub.B having the sequence of SEQ ID NO: 188, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 193, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 198.
82. The bispecific binding protein of claim 71, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 183; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 192; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 197.
83. A bispecific binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two antigens, wherein the first polypeptide chain comprises (1) VL.sub.A, CL, VH.sub.B, and CH1 or (2) VH.sub.B, CH1, VL.sub.A, and CL, wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CH1 is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen, wherein the binding protein is capable of binding the same epitope of CD20, and the same epitope of CD22 as that of bispecific binding protein FIT021b, wherein the bispecific binding protein FIT021b comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 392; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 401; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 406.
84. The bispecific binding protein of claim 83, selected from the group consisting of: (i) a binding protein comprising a VL.sub.A on the first polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 394, a VL.sub.A CDR2 of SEQ ID NO: 395, and a VL.sub.A CDR3 of SEQ ID NO: 396; (ii) a binding protein comprising a VH.sub.B on the first polypeptide, wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 398, a VH.sub.B CDR2 of SEQ ID NO: 399, and a VH.sub.B CDR3 of SEQ ID NO: 400; (iii) a binding protein comprising a VH.sub.A on the second polypeptide, wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 403, a VH.sub.A CDR2 of SEQ ID NO: 404, and a VH.sub.A CDR3 of SEQ ID NO: 405; (iv) a binding protein comprising a VL.sub.B on the third polypeptide, wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 408, a VL.sub.B CDR2 of SEQ ID NO: 409, and a VL.sub.B CDR3 of SEQ ID NO: 410; and (v) a binding protein comprising a VL.sub.A and VH.sub.B on the first polypeptide, a VH.sub.A on the second polypeptide, and a VL.sub.B on the third polypeptide, wherein the VL.sub.A of the first polypeptide comprises a VL.sub.A CDR1 of SEQ ID NO: 394, a VL.sub.A CDR2 of SEQ ID NO: 395, and a VL.sub.A CDR3 of SEQ ID NO: 396; wherein the VH.sub.B of the first polypeptide comprises a VH.sub.B CDR1 of SEQ ID NO: 398, a VH.sub.B CDR2 of SEQ ID NO: 399, and a VH.sub.B CDR3 of SEQ ID NO: 400; wherein the VH.sub.A of the second polypeptide comprises a VH.sub.A CDR1 of SEQ ID NO: 403, a VH.sub.A CDR2 of SEQ ID NO: 404, and a VH.sub.A CDR3 of SEQ ID NO: 405; and wherein the VL.sub.B of the third polypeptide comprises a VL.sub.B CDR1 of SEQ ID NO: 408, a VL.sub.B CDR2 of SEQ ID NO: 409, and a VL.sub.B CDR3 of SEQ ID NO: 410.
85. The bispecific binding protein of claim 83, wherein the binding protein comprises a first polypeptide chain comprising a VL.sub.A having the sequence of SEQ ID NO: 393, and a VH.sub.B having the sequence of SEQ ID NO: 397, wherein the binding protein comprises a second polypeptide chain comprising a VH.sub.A having the sequence of SEQ ID NO: 402, and wherein the binding protein comprises a third polypeptide chain comprising a VL.sub.B having the sequence of SEQ ID NO: 407.
86. The bispecific binding protein of claim 87, wherein the binding protein comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 392; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 401; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 406.
87. The binding protein of any one of claim 1 to claim 86, wherein the binding protein further comprises an Fc.
88. The binding protein of claim 87, wherein Fc is the Fc of human IgG1.
89. The binding protein of claim 87, wherein the Fc is according to SEQ ID NO: 20.
90. The binding protein of any one of claim 1 to claim 86, further comprising at least one polypeptide linker.
91. The binding protein of any one of claim 1 to claim 86, wherein the first polypeptide chain comprises, from amino to carboxyl terminus, VL.sub.A-CL-VH.sub.B-CH1-Fc, the second polypeptide chain comprises, from amino to carboxyl terminus, VH.sub.A-CH1, and the third polypeptide chain comprises, from amino to carboxyl terminus, VL.sub.B-CL.
92. The binding protein of any one of claim 1 to claim 86, wherein the first polypeptide chain comprises, from amino to carboxyl terminus, VH.sub.B-CH1-VL.sub.A-CL-Fc, the second polypeptide chain comprises, from amino to carboxyl terminus, VH.sub.A-CH1, and the third polypeptide chain comprises, from amino to carboxyl terminus, VL.sub.B-CL.
93. The binding protein of any one of claim 1 to claim 86, wherein the herein the CL of the first polypeptide chain is fused directly to VH.sub.B, or via an amino acid or an oligopeptide linker.
94. The binding protein of any one of claim 1 to claim 86, wherein the herein the CH1 of the first polypeptide chain is fused directly to VL.sub.A, or via an amino acid or an oligopeptide linker.
95. A binding protein having one or more amino acid conservative substitutions in the binding protein of any one of claim 1 to claim 86, which has equivalent activity as the corresponding binding protein in claim 1 to claim 86 without the amino acid conservative substitutions.
96. A pharmaceutical composition comprising the binding protein of any one of the preceding claims and one or more pharmaceutically acceptable carrier.
97. A method of treating or preventing a condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of claim 96.
98. Use of the binding protein according to any one of claim 1 to claim 86 in the manufacture of a medicament for treatment or prevention of a condition.
99. The use of claim 98, wherein the condition is an inflammatory disease, autoimmune disease, neurodegenerative disease, cancer, or spinal cord injury.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0256] The present invention relates to multivalent and multispecific binding proteins, methods of making the binding proteins, and to their uses in the prevention and/or treatment of acute and chronic inflammatory diseases and disorders, cancers, and other diseases. This invention pertains to multivalent and/or multispecific binding proteins capable of binding two or more antigens. Specifically, the invention relates to Fabs-in-tandem immunoglobulins (FIT-Ig), and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such FIT-Igs. Methods of using the FIT-Igs of the invention to detect specific antigens, either in vitro or in vivo are also encompassed by the invention.
[0257] The novel family of binding proteins provided herein are capable of binding two or more antigens, e.g., with high affinity. Specifically, the present invention provides an approach to construct a bispecific binding protein using 2 parental monoclonal antibodies: mAb A, which binds to antigen a; and mAb B, which binds to antigen b.
[0258] In one aspect, the present invention provides a binding protein comprising a variable light chain specific for a first antigen or epitope, a first light chain constant domain, a variable heavy chain specific for a second antigen or epitope, a first heavy chain CH1, a variable heavy chain specific for the first antigen or epitope, a second heavy chain CH1, a variable heavy chain specific for the second antigen or epitope, and a second light chain constant domain. In one embodiment, the binding protein further comprises an Fc region. The binding protein may further comprise one or more amino acid or polypeptide linker linking two or more of the components of the binding protein. For example, the binding protein may comprise a polypeptide linker linking the light chain variable region to the light chain constant region.
[0259] In one embodiment, the present disclosure provides a binding protein comprising a polypeptide chain comprising VL.sub.A-CL-(X1)n-VH.sub.B-CH1-(X2)n, wherein VL.sub.A is the light chain variable domain of mAb A, CL is a light chain constant domain, X1 represents an amino acid or an oligopeptide linker, VH.sub.B is the heavy chain variable domain of mAb B, CH1 is the first constant domain of the heavy chain, X2 represents an Fc region or a different dimerization domain, and n is 0 or 1.
[0260] In one embodiment, the invention provides a binding protein comprising three different polypeptide chains (
[0261] In another embodiment, the invention provides a binding protein comprising three different polypeptide chains with the overall molecular design similar to the previous embodiment except the order of the variable domains are reversed. In the embodiment the first polypeptide chain comprises VH.sub.B-CH1-(X1)n-VL.sub.A-CL-(X2)n, wherein VL.sub.A is a light chain variable domain of mAb A, CL is a light chain constant domain, X1 represents an amino acid or an oligopeptide linker, VH.sub.B is the heavy chain variable domain of mAb B, CH1 is the first constant domain of the heavy chain, X2 represents an Fc region or a different dimerization domain, and n is 0 or 1. The second polypeptide chain comprises VH.sub.A-CH1, wherein VH.sub.A is the heavy chain variable domain of mAb A and CH1 is the first constant domain of the heavy chain. The third polypeptide chain comprises VL.sub.B-CL, wherein VL.sub.B is the light chain variable domain of mAb B and CL is the constant domain of the light chain.
[0262] In another embodiment the invention provides a binding protein comprising two different polypeptide chains (
[0263] In another embodiment the invention provides a binding protein comprising two polypeptide chains with the overall molecular design similar to the previous embodiment except the order of the variable domains are reversed. In this embodiment the first polypeptide chain comprises VH.sub.B-CH1-(X1)n-VL.sub.A-CL-(X2)n, wherein VL.sub.A is a light chain variable domain of mAb A, CL is a light chain constant domain, X1 represents an amino acid or an oligopeptide linker, VH.sub.B is the heavy chain variable domain of mAb B, CH1 is the first constant domain of the heavy chain, X2 represents an Fc region or a different dimerization domain, and n is 0 or 1. The second polypeptide chain comprises VL.sub.B-CL-(X3)n-VH.sub.A-CH1, wherein VH.sub.A is the heavy chain variable domain of mAb A, CH1 is the first constant domain of the heavy chain, X3 represents an amino acid or an oligopeptide linker, n is 0 or 1, VL.sub.B is the light chain variable domain of mAb B, and CL is the constant domain of the light chain.
[0264] In one embodiment, the VH and VL domains in the binding protein are selected from the group consisting of murine heavy/light chain variable domains, fully human heavy/light chain variable domains, CDR grafted heavy/light chain variable domains, humanized heavy/light chain variable domains, and mixtures thereof. In a preferred embodiment VH.sub.A/VL.sub.A and VH.sub.B/VL.sub.B are capable of binding the same antigen. In another embodiment VH.sub.A/VL.sub.A and VH.sub.B/VL.sub.B are capable of binding different antigens.
[0265] In one embodiment, the first polypeptide chain comprises VL.sub.A-CL-VH.sub.B-CH1-Fc, and the CL and VH.sub.B of the first polypeptide chain are directly fused together. In another embodiment, the CL and VH.sub.B are linked by an amino acid or an oligopeptide linker. In another embodiment, the first polypeptide chain comprises VH.sub.B-CH1-VL.sub.A-CL-Fc, and the CH1 and VL.sub.A are directly fused together. In another embodiment, the CH1 and VL.sub.A are linked by an amino acid or an oligopeptide linker. In a further embodiment, the oligo- or poly-peptide linker comprises 1 or more amino acids of any reasonable sequence that provides flexibility. Preferably the linker is selected from the group consisting of G, GS, SG, GGS, GSG, SGG, GGG, GGGS (SEQ ID NO: 489), SGGG (SEQ ID NO: 490), GGGGS (SEQ ID NO: 491), GGGGSGS (SEQ ID NO: 492), GGGGSGGS (SEQ ID NO: 493), GGGGSGGGGS (SEQ ID NO: 494), GGGGSGGGGSGGGGS (SEQ ID NO: 495), AKTTPKLEEGEFSEAR (SEQ ID NO: 496), AKTTPKLEEGEFSEARV (SEQ ID NO: 497), AKTTPKLGG (SEQ ID NO: 498), SAKTTPKLGG (SEQ ID NO: 499), SAKTTP (SEQ ID NO: 500), RADAAP (SEQ ID NO: 501), RADAAPTVS (SEQ ID NO: 502), RADAAAAGGPGS (SEQ ID NO: 503), RADAAAA(G.sub.4S).sub.4 (SEQ ID NO: 504), SAKTTPKLEEGEFSEARV (SEQ ID NO: 505), ADAAP (SEQ ID NO: 506), ADAAPTVSIFPP (SEQ ID NO: 507), TVAAP (SEQ ID NO: 508), TVAAPSVFIFPP (SEQ ID NO: 509), QPKAAP (SEQ ID NO: 510), QPKAAPSVTLFPP (SEQ ID NO: 511), AKTTPP (SEQ ID NO: 512), AKTTPPSVTPLAP (SEQ ID NO: 513), AKTTAPSVYPLAP (SEQ ID NO: 514), ASTKGP (SEQ ID NO: 515), ASTKGPSVFPLAP (SEQ ID NO: 516), GENKVEYAPALMALS (SEQ ID NO: 517), GPAKELTPLKEAKVS (SEQ ID NO: 518), GHEAAAVMQVQYPAS (SEQ ID NO: 519), and AKTTAP (SEQ ID NO: 80). In one embodiment, the amino acid sequence of the linker may be selected from the group consisting of SEQ ID NOs. 26, 28, and 49-86. In one embodiment, the linker is GSG (SEQ ID NO: 26) or GGGGSGS (SEQ ID NO: 28). The linkers can also be in vivo cleavable peptide linkers, protease (such as MMPs) sensitive linkers, disulfide bond-based linkers that can be cleaved by reduction, etc., as previously described (Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges, edited by Stefan R. Schmidt), or any cleavable linkers known in the art. Such cleavable linkers can be used to release the top Fab in vivo for various purposes, in order to improve tissue/cell penetration and distribution, to enhance binding to targets, to reduce potential side effect, as well as to modulate in vivo functional and physical half-life of the 2 different Fab regions. In one embodiment, the binding protein comprises an Fc region. As used herein, the term “Fc region” refers to the C-terminal region of an IgG heavy chain. An example of the amino acid sequence containing the human IgG1 Fc region is SEQ ID NO: 20. The Fc region of an IgG comprises two constant domains, CH2 and CH3.
[0266] In one embodiment, the Fc region is a variant Fc region. In one embodiment, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions, or insertions, relative to the parent Fc region. In a further embodiment, amino acid modifications of the Fc region alter the effector function activity relative to the parent Fc region activity. For example, in one embodiment, the variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding. In another embodiment, amino acid modifications of the Fc region may alter (i.e., increase or decrease) the affinity of the variant Fc region for an FcγR relative to the parent Fc region. For example, the variant Fc region may alter the affinity for FcγRI, FcγRII, FcγRIII.
[0267] In one preferred embodiment, the binding proteins provided herein are capable of binding one or more targets. In one embodiment, the target is selected from the group consisting of cytokines, cell surface proteins, enzymes and receptors. Preferably the binding protein is capable of modulating a biological function of one or more targets. More preferably the binding protein is capable of neutralizing one or more targets.
[0268] In one embodiment, the binding protein of the invention is capable of binding cytokines selected from the group consisting of lymphokines, monokines, and polypeptide hormones. In a further embodiment, the binding protein is capable of binding pairs of cytokines selected from the group consisting of IL-1α and IL-1β; IL-12 and IL-18, TNFα and IL-23, TNFα and IL-13; TNF and IL-18; TNF and IL-12; TNF and IL-1beta; TNF and MIF; TNF and IL-6, TNF and IL-6 Receptor, TNF and IL-17; IL-17 and IL-20; IL-17 and IL-23; TNF and IL-15; TNF and VEGF; VEGFR and EGFR; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAM8; and TNFα and PGE4, IL-13 and PED2, TNF and PEG2.
[0269] In another embodiment, the binding protein of the invention is capable of binding pairs of targets selected from the group consisting of CD137 and CD20, CD137 and EGFR, CD137 and Her-2, CD137 and PD-1, CD137 and PDL-1, VEGF and PD-L1, Lag-3 and TIM-3, OX40 and PD-1, TIM-3 and PD-1, TIM-3 and PDL-1, EGFR and DLL-4, VEGF and EGFR, HGF and VEGF, VEGF and VEGF (same or a different epitope), VEGF and Ang2, EGFR and cMet, PDGF and VEGF, VEGF and DLL-4, OX40 and PD-L1, ICOS and PD-1, ICOS and PD-L1, Lag-3 and PD-1, Lag-3 and PD-L1, Lag-3 and CTLA-4, ICOS and CTLA-4, CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD3 and CD33; CD3 and CD133; CD38 & CD138; CD38 and CD20; CD20 and CD22; CD38 and CD40; CD40 and CD20; CD47 and CD20, CD-8 and IL-6; CSPGs and RGM A; CTLA-4 and BTNO2; CTLA-4 and PD-1; IGF1 and IGF2; IGF1/2 and Erb2B; IGF-1R and EGFR; EGFR and CD13; IGF-1R and ErbB3; EGFR-2 and IGFR; Her2 and Her2 (same or a different epitope); Factor IXa and Factor X, VEGFR-2 and Met; VEGF-A and Angiopoietin-2 (Ang-2); IL-12 and TWEAK; IL-13 and IL-1beta; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; PDL-1 and CTLA-4; PD-1 and CTLA-4, PD-1 and TIM-3; RGM A and RGM B; Te38 and TNFα; TNFα and Blys; TNFα and CD-22; TNFα and CTLA-4 domain; TNFα and GP130; TNFα and IL-12p40; TNFα and RANK ligand; EGFR and PD-L1; EGFR and cMet; Her3 and IGF-IR; DLL-4 and VEGF; PD-1 and PD-L1; Her3 and PD-1, Her3 and EGFR, cMet and PD-L1, and BTLA and PD-1.
[0270] In some embodiments, the binding proteins contain variable regions or CDRs derived from CD20 antibodies including, but not limited to, ofatumumab, rituximab, iodine i 131 tositumomab, obinutuzumab, ibritumomab, and those described in U.S. Pat. Nos. 9,228,008, 8,206,711, 7,682,612, 8,562,992, 7,799,900, 7,422,739, 7,850,962, 8,097,713, 8,057,793, 8,592,156, 6,652,852, 6,893,625, 6,120,767, 8,084,582, 8,778,339, 9,184,781, 7,381,560, 8,101,179, 9,382,327, 7,151,164, 7,435,803, 8,529,902, 9,416,187, 7,812,116, 8,329,181, 8,034,902, 9,289,479, 9,234,045, 4,987,084, 9,173,961, 9,175,086, 6,410,319, each of which is incorporated by reference in its entirety.
[0271] In some embodiments, the binding proteins contain variable regions or CDRs derived from CD3 antibodies including, but not limited to, muromonab-CD3, otelixizumab, teplizumab and visilizumab, and those described in U.S. Pat. Nos. 8,569,450, 7,635,472,5585097, 6706265, 5834597, 9056906, 9486475, 7728114, 8551478, 9226962, 9192665, 9505849, 8394926, 6306575, 5795727, 8840888, 5627040, 9249217, 8663634, 6491917, 5877299, each of which is incorporated by reference in its entirety.
[0272] In some embodiments, the binding proteins contain variable regions or CDRs derived from CTLA-4 antibodies including, but not limited to, ipilimumab and those described in U.S. Pat. Nos. 5,434,131, 5,968,510, 5,844,095, 7,572,772, 6,090,914, 7,311,910, 5,885,796, 5,885,579, 5,770,197, 5,851,795, 5,977,318, 7,161,058, 6,875,904, 7,504,554, 7,034,121, 6,719,972, 7,592,007, each of which is incorporated by reference in its entirety.
[0273] In some embodiments, the binding proteins contain variable regions or CDRs derived from PD-1 antibodies including, but are not limited to, pembrolizumab, nivolumab, atezolizumab, and those described in U.S. Pat. Nos. 8,741,295, 7,029,674, 7,722,868, 9,243,052, 8,927,697, 9,181,342, 8,552,154, 9,102,727, 9,220,776, 9,084,776, 8,008,449, 9,387,247, 9,492,540, 8,779,105, 9,358,289, 9,492,539, 9,205,148, 8,900,587, 8,952,136, 8,460,886, 7,414,171, 8,287,856, 8,580,247, 7,488,802, 7,521,051, 8,088,905, 7,709,214, 8,617,546, 9,381,244, 8,993,731, 8,574,872, 7,432,059, 8,216,996, 9,499,603, 9,102,728, 9,212,224, each of which is incorporated by reference in its entirety.
[0274] In some embodiments, the binding proteins contain variable regions or CDRs derived from PD-L1 antibodies including, but not limited to, durvalumab, avelumab, and those described in U.S. Pat. Nos. 8,741,295, 9,102,725, 8,168,179, 8,952,136, 8,552,154, 8,617,546, 9,212,224, 8,217,149, 8,383,796, each of which is incorporated by reference in its entirety.
[0275] In some embodiments, the binding proteins contain variable regions or CDRs derived from EGFR antibodies including, but not limited to, gefitinib, erlotinib, lapatinib, cetuximab, panitumumab, vandetanib, necitumumab, osimertinib, and those described in U.S. Pat. Nos. 7,723,484, 9,044,460, 9,226,964, 8,658,175, 7,618,631, 8,748,175, 9,499,622, 9,527,913, 9,493,568, 8,580,263, 7,514,240, 9,314,536, 9,051,370, 9,233,171, 9,029,513, 8,592,152, 8,597,652, 9,327,035, 8,628,773, 9,023,356, 9,132,192, 8,637,026, 9,283,276, 9,540,440, 9,545,442, 8,758,756, 9,120,853, 7,981,605, 8,546,107, 7,598,350, 5,212,290, 8,017,321, 7,589,180, 9,260,524, 8,790,649, 9,125,896, 9,238,690, 8,071,093, each of which is incorporated by reference in its entirety.
[0276] In some embodiments, the binding proteins contain variable regions or CDRs derived from TIM3 (CD366) antibodies including, but not limited to, 4C4G3, 7D3, B8.2C12, F38-2E2, and those described in U.S. Pat. Nos. 8,841,418, 8,552,156, 9,556,270, each of which is incorporated by reference in its entirety.
[0277] In some embodiments, the binding proteins contain variable regions or CDRs derived from cMet antibodies including, but not limited to, h1332, 71-8000, ab74217, and those described in U.S. Pat. Nos. 8,673,302, 9,120,852, 7,476,724, 7,892,550, 9,249,221, 9,535,055, 9,487,589, 8,329,173, 9,101,610, 8,101,727, 9,068,011, 9,260,531, 9,296,817, 8,481,689, 8,546,544, 8,563,696, 8,871,909, 8,889,832, 8,871,910, 9,107,907, 8,747,850, 9,469,691, 8,765,128, 8,729,249, 8,741,290, 8,637,027, 8,900,582, 9,192,666, 9,201,074, 9,505,843, 8,821,869, 8,163,280, 7,498,420, 8,562,985, 8,545,839, 9,213,031, 9,213,032, 8,217,148, 8,398,974, 9,394,367, 9,364,556, 8,623,359, 9,011,865, 9,375,425, 9,233,155, 9,169,329, 9,150,655, each of which is incorporated by reference in its entirety.
[0278] In some embodiments, the binding proteins contain variable regions or CDRs derived from Factor IXa antibodies including, but not limited to, ab97619, ab128048, ab128038, and those described in U.S. Pat. Nos. 7,279,161, 7,033,590, 4,786,726, 6,624,295, 7,049,411, 7,297,336, each of which is incorporated by reference in its entirety.
[0279] In some embodiments, the binding proteins contain variable regions or CDRs derived from Factor X antibodies including, but not limited to, PA5-22059, ab97632, B122M.
[0280] In some embodiments, the binding proteins contain variable regions or CDRs derived from Her3 (ErbB3) antibodies including, but not limited to, duligotumab, elgemtumab, lumretuzumab, patritumab, seribantumab, and those described in U.S. Pat. Nos. 9,346,883, 9,321,839, 8,859,737, 8,362,215, 8,828,388, 9,220,775, 9,217,039, 9,527,913, 9,085,622, 9,192,663, 8,735,551, 9,011,851, 7,846,440, 9,284,380, 8,791,244, 8,691,225, 9,487,588, 8,961,966, 9,034,328, 5,968,511, 9,346,889, 9,217,039, 9,346,890, each of which is incorporated by reference in its entirety.
[0281] In some embodiments, the binding proteins contain variable regions or CDRs derived from IGF-1R (CD221) antibodies including, but not limited to cixutumumab, dalotuzumab, figitumumab, ganitumab, robatumumab, teprotumumab, and those described in U.S. Pat. Nos. 7,572,897, 7,579,157, 7,968,093, 7,638,605, 7,329,745, 7,037,498, 7,982,024, 8,642,037, 7,700,742, 9,234,041, 7,815,907, 8,945,871, 8,361,461, 9,056,907, 8,168,410, 7,241,444, 7,914,784, 9,150,644, 7,985,842, 7,538,195, 8,268,617, 8,034,904, 8,344,112, 7,553,485, 8,101,180, 8,105,598, 7,824,681, 8,124,079, 8,420,085, 7,854,930, each of which is incorporated by reference in its entirety.
[0282] In some embodiments, the binding proteins contain variable regions or CDRs derived from DLL4 antibodies including, but not limited to demcizumab, enoticumab, navicixizumab, and those described in U.S. Pat. Nos. 9,469,689, 9,115,195, 8,623,358, 9,132,190, 9,469,688, 9,403,904, 8,663,636, 8,192,738, 750,124, each of which is incorporated by reference in its entirety.
[0283] In some embodiments, the binding proteins contain variable regions or CDRs derived from VEGF antibodies including, but not limited to Bevacizumab, Brolucizumab, Ranibizumab, and those described in U.S. Pat. Nos. 8,921,537, 7,910,098, 7,365,166, 7,060,269, 7,169,901, 6,884,879, 7,297,334, 7,375,193, 9,388,239, 8,834,883, 8,287,873, 7,998,931, 8,007,799, 7,785,803, 9,102,720, 8,486,397, 6,730,489, 6,383,484, 9,441,034, 7,097,986, 9,079,953, 8,945,552, 8,236,312, 7,740,844, 6,403,088, 9,018,357, 8,975,381, 7,691,977, 7,758,859, 8,512,699, 8,492,527, 9,353,177, 8,092,797, 7,811,785, 8,101,177, 8,592,563, 9,090,684, 8,349,322, each of which is incorporated by reference in its entirety.
[0284] In some embodiments, the binding proteins contain variable regions or CDRs derived from CD22 antibodies including, but not limited to bectumomab, epratuzumab, inotuzumab, moxetumomab, pinatuzumab, and those described in U.S. Pat. Nos. 9,181,343, 5,484,892, 9,279,019, 8,591,889, 9,499,632, 8,481,683, 7,355,012, 7,777,019, 8,809,502, 8,389,688, 7,829,086, each of which is incorporated by reference in its entirety.
[0285] In some embodiments, the binding proteins contain variable regions or CDRs derived from BTLA(CD272) antibodies including, but not limited to MIH26, AAP44003, MA5-16843, 6A5, and those described in U.S. Pat. Nos. 8,563,694, 9,346,882, 8,580,259, 8,247,537, each of which is incorporated by reference in its entirety.
[0286] In some embodiments, the binding proteins contain antibodies as described in WO2015103072, which is herein incorporated by reference in its entirety.
[0287] In one embodiment, the binding protein is capable of binding human IL-17 and human IL-20. In a further embodiment, the binding protein is capable of binding human IL-17 and human IL-20 and comprises a FIT-Ig polypeptide chain #1 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO. 15, 25, and 27; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 21; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 23. In another embodiment, the binding protein is capable of binding human IL-17 and human IL-20 and comprises FIT-Ig polypeptide chain #1 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO. 15, 25, and 27; and a polypeptide chain #4 that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO. 29, 30, and 31.
[0288] In one embodiment, the binding protein is capable of binding human CD3 and human CD20. In a further embodiment, the binding protein comprises a FIT-Ig polypeptide chain #1 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO. 41, 48 and 316; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 44 or SEQ ID NO. 325; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 46 or SEQ ID NO. 330.
[0289] In one embodiment, the binding protein is capable of binding human IL-17 and human TNF. In a further embodiment, the binding protein is capable of binding human IL-17 and human TNF and comprises a FIT-Ig polypeptide chain #1 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 87; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 89; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 91.
[0290] In one embodiment, the binding protein is capable of binding human CTLA-4 and human PD-1. In a further embodiment, the binding protein is capable of binding human CTLA-4 and human PD-1 and comprises a FIT-Ig polypeptide chain #1 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 92, SEQ ID NO. 126, SEQ ID NO. 145, SEQ ID NO. 164, or SEQ ID NO. 183, a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 95, SEQ ID NO. 135, SEQ ID NO. 154, SEQ ID NO. 173, or SEQ ID NO. 192; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 97, SEQ ID NO. 140, SEQ ID NO. 159, SEQ ID NO. 178, or SEQ ID NO. 197.
[0291] In one embodiment, the binding protein is capable of binding EGFR and PD-L1. In a further embodiment, the binding protein is capable of binding EGFR and PD-L1 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 99, SEQ ID NO. 202 or SEQ ID NO. 221; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 100, SEQ ID NO. 211, or SEQ ID NO. 230; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 101, SEQ ID NO. 216, or SEQ ID NO. 235.
[0292] In one embodiment, the binding protein is capable of binding cMet and EGFR. In a further embodiment, the binding protein is capable of binding cMET and EGFR and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 102 or SEQ ID NO. 240; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 103 or SEQ ID NO. 249; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 104 or SEQ ID NO. 254.
[0293] In one embodiment, the binding protein is capable of binding Factor IXa and Factor X. In a further embodiment, the binding protein is capable of binding Factor IXa and Factor X and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 105, or SEQ ID NO. 259; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 106 or SEQ ID NO. 268; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 107 or SEQ ID NO. 273.
[0294] In one embodiment, the binding protein is capable of binding Her3 and IGF-1R. In a further embodiment, the binding protein is capable of binding Her3 and IGF-1R and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 108, SEQ ID NO. 278; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 109 or SEQ ID NO. 287; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 110 or SEQ ID NO. 292.
[0295] In one embodiment, the binding protein is capable of binding DLL-4 and VEGF. In a further embodiment, the binding protein is capable of binding DLL-4 and VEGF and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 111, or SEQ ID NO. 297; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 112, or SEQ ID NO. 306; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 113 or SEQ ID NO. 311.
[0296] In one embodiment, the binding protein is capable of binding CD20 and CD3. In a further embodiment, the binding protein is capable of binding CD20 and CD3 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 114 or SEQ ID NO. 316; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 115 or SEQ ID NO. 325; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 116 or SEQ ID NO. 330.
[0297] In one embodiment, the binding protein is capable of binding Her3 and EGFR. In a further embodiment, the binding protein is capable of binding Her3 and EGFR and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 117, SEQ ID NO. 335, or SEQ ID NO. 354; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 118, SEQ ID NO. 344, or SEQ ID NO. 363; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 119, SEQ ID NO. 349, or SEQ ID NO. 368.
[0298] In one embodiment, the binding protein is capable of binding PD-1 and PD-L1. In a further embodiment, the binding protein is capable of binding PD-1 and PD-L1 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 120 or SEQ ID NO. 373; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 121 or SEQ ID NO. 382; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 122 or SEQ ID NO. 387.
[0299] In one embodiment, the binding protein is capable of binding Her3 and PD-1. In a further embodiment, the binding protein is capable of binding Her3 and PD-1 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 123 or SEQ ID NO. 411; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 124 or SEQ ID NO. 420; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 125 or SEQ ID NO. 425.
[0300] In one embodiment, the binding protein is capable of binding cMet and PD-L1. In a further embodiment, the binding protein is capable of binding cMet and PD-L1 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 430; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 439; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 444.
[0301] In one embodiment, the binding protein is capable of binding BTLA and PD-1. In a further embodiment, the binding protein is capable of binding BTLA and PD-1 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 449 or SEQ ID NO. 468; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 458 or SEQ ID NO. 477; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 463 or SEQ ID NO. 482.
[0302] In one embodiment, the binding protein is capable of binding CD20 and CD22. In a further embodiment, the binding protein is capable of binding CD20 and CD22 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 392; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 401; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 406.
[0303] Biologically active variants or functional variants of the exemplary binding proteins described herein are also a part of the present invention. As used herein, the phrase “a biologically active variant” or “functional variant” with respect to a protein refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence, while still maintains substantial biological activity of the reference sequence. The variant can have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. The following table shows exemplary conservative amino acid substitutions. In some embodiments, the variant has one or more amino acid substitutions, wherein one or more or all substitutions are acidic amino acid, such as Aspartic acid, Asparagine, Glutamc acid, or Glutamine.
TABLE-US-00001 Very Highly - Highly Conserved Original Conserved Substitutions (from the Conserved Substitutions Residue Substitutions Blosum90 Matrix) (from the Blosum65 Matrix) Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, Val Arg Lys Gln, His, Lys Asn, Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His, Lys, Ser, Thr Arg, Asp, Gln, Glu, His, Lys, Ser, Thr Asp Glu Asn, Glu Asn, Gln, Glu, Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, His, Lys, Met Arg, Asn, Asp, Glu, His, Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg, Asn, Asp, Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn, Gln, Tyr Arg, Asn, Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu, Met, Phe, Val Leu Ile; Val Ile, Met, Phe, Val Ile, Met, Phe, Val Lys Arg; Gln; Glu Arg, Asn, Gln, Glu Arg, Asn, Gln, Glu, Ser, Met Leu; Ile Gln, Ile, Leu, Val Gln, Ile, Leu, Phe, Val Phe Met; Leu; Tyr Leu, Trp, Tyr Ile, Leu, Met, Trp, Tyr Ser Thr Ala, Asn, Thr Ala, Asn, Asp, Gln, Glu, Gly, Lys, Thr Thr Ser Ala, Asn, Ser Ala, Asn, Ser, Val Trp Tyr Phe, Tyr Phe, Tyr Tyr Trp; Phe His, Phe, Trp His, Phe, Trp Val Ile; Leu Ile, Leu, Met Ala, Ile, Leu, Met, Thr
[0304] Alternatively, a variant can have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations can also include amino acid deletion or insertion, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software.
[0305] Binding proteins that are capable of binding the same epitopes on a given group of targets as that of an exemplary bispecific binding protein described herein are also a part of the present invention. The epitopes can be linear epitopes, conformational epitopes, or a mixture thereof. In some embodiments, such same epitopes can be identified by a suitable epitope mapping technique, including but not limited to, X-ray co-crystallography, array-based oligo-peptide scanning, site-directed mutagenesis, high throughput mutagenesis mapping, bacteriophage surface display, and hydrogen-deuterium exchange. Additional methods are described in U.S. Pat. Nos. 5,955,264, 65,796,676, 6,984,488, and 8,802,375, each of which is incorporated by reference in its entirety for all purposes.
[0306] In another embodiment, the binding protein of the invention is capable of binding one or two cytokines, cytokine-related proteins, and cytokine receptors selected from the group consisting of BMP1, BMP2, BMP313 (GDF10), BMP4, BMP6, BMP8, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (aFGF), FGF2 (bFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFNG, IFNW1, FIL1, FIL1 (EPSILON), FIL1 (ZETA), IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, FGER1, FGFR2, FGFR3, EGFR, ROR1, 2B4, KIR, CD137, CD27, OX40, CD40L, A2aR, CD48, B7-1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CD70, CD40, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, FIGF (VEGFD), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.
[0307] The binding protein of the invention is capable of binding one or more chemokines, chemokine receptors, and chemokine-related proteins selected from the group consisting of CCL1 (I-309), CCL2 (MCP-1/MCAF), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCL11 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL20 (MIP-3a), CCL21 (SLC/exodus-2), CCL22 (MDC/STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK/ILC), CCL28, CXCL1 (GRO1), CXCL2 (GRO2), CXCL3 (GRO3), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL10 (IP 10), CXCL11 (I-TAC), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1), SCYE1, XCL1 (lymphotactin), XCL2 (SCM-1b), BLR1 (MDR15), CCBP2 (D6/JAB61), CCR1 (CKR1/HM145), CCR2 (mcp-1RB/RA), CCR3 (CKR3/CMKBR3), CCR4, CCR5 (CMKBR5/ChemR13), CCR6 (CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBIl), CCR8 (CMKBR8/TER1/CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), XCR1 (GPR5/CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4, GPR2 (CCR10), GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6 (TYMSTR/STRL33/Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R (GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HIF1A, IL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL.
[0308] In another embodiment, a binding protein of the invention is capable of binding cell surface protein such as, for example, integrins. In another embodiment, the binding protein of the invention is capable of binding enzymes selected from the group consisting of kinases and proteases. In yet another embodiment, the binding protein of the invention is capable of binding receptors selected from the group consisting of lymphokine receptors, monokine receptors, and polypeptide hormone receptors.
[0309] In one embodiment, the binding protein is multivalent. In another embodiment, the binding protein is multispecific. The multivalent and or multispecific binding proteins described above have desirable properties particularly from a therapeutic standpoint. For instance, the multivalent and or multispecific binding protein may (1) be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind; (2) be an agonist antibody; and/or (3) induce cell death and/or apoptosis of a cell expressing an antigen which the multivalent antibody is capable of binding to. The “parent antibody” which provides at least one antigen binding specificity of the multivalent and or multispecific binding proteins may be one which is internalized (and/or catabolized) by a cell expressing an antigen to which the antibody binds; and/or may be an agonist, cell death-inducing, and/or apoptosis-inducing antibody, and the multivalent and or multispecific binding protein as described herein may display improvement(s) in one or more of these properties. Moreover, the parent antibody may lack any one or more of these properties, but may be endowed with them when constructed as a multivalent binding protein as herein described.
[0310] In another embodiment a binding protein of the invention has an on rate constant (Kon) to one or more targets selected from the group consisting of: at least about 10.sup.2M.sup.−1s.sup.−1; at least about 10.sup.3M.sup.−1s.sup.−1; at least about 10.sup.4M.sup.−1s.sup.−1; at least about 10.sup.5M.sup.−1s.sup.−1; and at least about 10.sup.6M.sup.−1s.sup.−1 (inclusive of all values therebetween), as measured by surface plasmon resonance. Preferably, the binding protein of the invention has an on rate constant (Kon) to one or more targets between 10.sup.2M.sup.−1s.sup.−1 to 10.sup.3M.sup.−1s.sup.−1; between 10.sup.3M.sup.−1s.sup.−1 to 10.sup.4M.sup.−1s.sup.−1; between 10.sup.4M.sup.−1s.sup.−1 to 10.sup.5M.sup.−1s.sup.−1; or between 10.sup.5M.sup.−1s.sup.−1 to 10.sup.6M.sup.−1s.sup.−1 (inclusive of all values therebetween), as measured by surface plasmon resonance.
[0311] In another embodiment a binding protein has an off rate constant (Koff) for one or more targets selected from the group consisting of: at most about 10.sup.−3 s.sup.−1; at most about 10.sup.−4 s.sup.−1; at most about 10.sup.−5 s.sup.−1; and at most about 10.sup.−6 s.sup.−1, as measured by surface plasmon resonance (inclusive of all values therebetween). Preferably, the binding protein of the invention has an off rate constant (Koff) to one or more targets of 10.sup.−3 s.sup.−1 to 10.sup.−4 s.sup.−1; of 10.sup.−4 s.sup.−1 to 10.sup.−5 s.sup.−1; or of 10.sup.−5 s.sup.1 to 10.sup.−6 s.sup.−1, as measured by surface plasmon resonance (inclusive of all values therebetween).
[0312] In another embodiment a binding protein has a dissociation constant (K.sub.D) to one or more targets selected from the group consisting of: at most about 10.sup.−7 M; at most about 10.sup.−8 M; at most about 10.sup.−9 M; at most about 10.sup.−10 M; at most about 10.sup.−11 M; at most about 10.sup.−12 M; and at most 10.sup.−13 M (inclusive of all values therebetween). Preferably, the binding protein of the invention has a dissociation constant (K.sub.D) to IL-12 or IL-23 of 10.sup.−7 M to 10.sup.−8 M; of 10.sup.−8 M to 10.sup.−9 M; of 10.sup.−9 M to 10.sup.−10 M; of 10.sup.−10 to 10.sup.−11 M; of 10.sup.−11 M to 10.sup.−12 M; or of 10.sup.−12 M to 10.sup.−13 M (inclusive of all values therebetween).
[0313] In another embodiment a binding protein of the invention has a monomer % of at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 80%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, or more, or 100% (inclusive of all values therebetween) in a one-step Protein A purification using Size-exclusion chromatography (SEC)-HPLC.
[0314] In another embodiment a binding protein of the invention has an expression level at least about 0.01 mg/L, 0.05 mg/L, 0.1 mg/L, about 0.2 mg/L, about 0.3 mg/L, about 0.4 mg/L, about 0.5 mg/L, about 0.6 mg/L, about 0.7 mg/L, about 0.8 mg/L, about 0.9 mg/L, about 1.0 mg/L, about 2 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg/L, about 7 mg/L, about 8 mg/L, about 9 mg/L, about 10 mg/L, about 11 mg/L, about 12 mg/L, about 13 mg/L, about 14 mg/L, about 15 mg/L, about 16 mg/L, about 17 mg/L, about 18 mg/L, about 19 mg/L, about 20 mg/L, about 21 mg/L, about 22 mg/L, about 23 mg/L, about 24 mg/L, about 25 mg/L, about 26 mg/L, about 27 mg/L, about 28 mg/L, about 29 mg/L, about 30 mg/L, about 40 mg/L, about 50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg/L, about 90 mg/L, about 100 mg/L, about 200 mg/L, about 300 mg/L, about 400 mg/L, about 500 mg/L, about 600 mg/L, about 700 mg/L, about 800 mg/L, about 900 mg/L, about 1000 mg/L (inclusive of all values therebetween) or more, under optimized conditions. In some embodiments, the binding protein of the invention is expressed in 293E cells or in any other cells suitable for the purpose.
[0315] In another embodiment a binding protein of the invention has an Tm1 transition temperature as measured by Differential scanning calorimetry (DSC) which is at least about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., about 75° C., about 76° C., about 77° C., about 78° C., about 79° C., about 80° C., about 81° C., about 82° C., about 83° C., about 84° C., about 85° C., about 86° C., about 87° C., about 88° C., about 89° C., about 90° C., about 95° C., about 99° C. (inclusive of all values therebetween) or more.
[0316] A binding protein of the invention has great stability in freeze-thaw test. In some embodiments, when a binding protein sample of the invention is thawed and incubated at 4° C., 25° C. and 40° C. for 1 day, 3 days or 7 days, reduction of intact binding protein due to aggregates is less than about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.1%, about 0.05%, about 0.01% (inclusive of all values therebetween), or less, as measured by SEC-HPLC.
[0317] In another embodiment, when a binding protein of the invention is administered intravenously, it has one or more of the following PK parameters (1) an Apparent total body clearance of the drug from plasma (CL, mL/day/kg) of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 (inclusive of all values therebetween) or more; (2) an Apparent volume of distribution at steady state (Vss, mL/kg) of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 (inclusive of all values therebetween), or more; (3) an Apparent volume of the central or plasma compartment in a two-compartment model (V1, mL/kg) of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 (inclusive of all values therebetween), or more; (4) an initial or disposition half-life (Alpha t½, day) of about 0.01, 0.05, 0.1, 0.2 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 (inclusive of all values therebetween) or more; (5) a Terminal elimination half-life (Beta t½, day) of about 0.01, 0.05, 0.1, 0.2 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 (inclusive of all values therebetween) or more; (6) an area under the plasma concentration-time curve (AUC, day×μg/mL) of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 (inclusive of all values therebetween) or more; and (6) a Mean residence time (MRT, day) of about 0.01, 0.05, 0.1, 0.2 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 (inclusive of all values therebetween) or more. In some embodiments, the above parameters are associated with a dosage of about 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg (inclusive of all values therebetween) or more.
[0318] In another embodiment, when a binding protein of the invention is administered subcutaneously, it has one or more of the following PK parameters (1) a Time to reach maximum (peak) plasma concentration following drug administration (Tmax, day) of about 0.05, 0.1, 0.2 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 (inclusive of all values therebetween) or more; (2) a Maximum (peak) plasma drug concentration (Cmax, μg/ml) of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500 (inclusive of all values therebetween) or more; (3) an Elimination half-life (Terminal t.sub.1/2, day) of about 0.01, 0.05, 0.1, 0.2 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 (inclusive of all values therebetween) or more; (4) an Area under the plasma concentration-time curve from time zero to time of last measurable concentration (AUClast, day×μg/ml) of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 (inclusive of all values therebetween) or more; (5) an Area under the plasma concentration-time curve from time zero to infinity (AUC.sub.inf, day×μg/ml) of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 (inclusive of all values therebetween) or more; (6) a Formation clearance of drug to metabolite (CL/F, mL/day/kg) of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 (inclusive of all values therebetween) or more; (7) a Bioavailability (F, %) of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 (inclusive of all values therebetween), or more. In some embodiments, the above parameters are associated with a dosage of about 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg (inclusive of all values therebetween) or more.
[0319] In another embodiment, the binding protein described above is a conjugate further comprising an agent selected from the group consisting of an immunoadhesion molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent. In one embodiment, the imaging agent is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. In a further embodiment, the imaging agent is a radiolabel selected from the group consisting of: .sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, and .sup.153Sm. In one embodiment, the therapeutic or cytotoxic agent is selected from the group consisting of an immunosuppressive agent, an immuno-stimulatory agent, an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, a toxin, and an apoptotic agent. In one embodiment, the binding protein is conjugated directly to the agent. In another embodiment, the binding protein is conjugated to the agent via a linker. Suitable linkers include, but are not limited to, amino acid and polypeptide linkers disclosed herein. Linkers may be cleavable or non-cleavable.
[0320] In another embodiment the binding protein described above is a crystallized binding protein and exists as a crystal. Preferably the crystal is a carrier-free pharmaceutical controlled release crystal. More preferably the crystallized binding protein has a greater half life in vivo than the soluble counterpart of said binding protein. Most preferably the crystallized binding protein retains biological activity.
[0321] In another embodiment the binding protein described above is glycosylated. Preferably, the glycosylation is a human glycosylation pattern.
[0322] One aspect of the invention pertains to an isolated nucleic acid encoding any one of the binding protein disclosed above. A further embodiment provides a vector comprising the isolated nucleic acid disclosed above wherein said vector is selected from the group consisting of pcDNA; pTT (Durocher et al., Nucleic Acids Research 2002, Vol 30, No. 2); pTT3 (pTT with additional multiple cloning site; pEFBOS (Mizushima, S. and Nagata, S., (1990) Nucleic acids Research Vol 18, No. 17); pBV; pJV; pcDNA3.1 TOPO, pEF6 TOPO and pBJ. The multi-specific binding proteins and methods of making the same are provided. The binding protein can be generated using various techniques. Expression vectors, host cells and methods of generating the binding proteins are provided in this disclosure.
[0323] The antigen-binding variable domains of the binding proteins of this disclosure can be obtained from parent binding proteins, including polyclonal Abs, monoclonal Abs, and or receptors capable of binding antigens of interest. These parent binding proteins may be naturally occurring or may be generated by recombinant technology. The person of ordinary skill in the art is well familiar with many methods for producing antibodies and/or isolated receptors, including, but not limited to using hybridoma techniques, selected lymphocyte antibody method (SLAM), use of a phage, yeast, or RNA-protein fusion display or other library, immunizing a non-human animal comprising at least some of the human immunoglobulin locus, and preparation of chimeric, CDR-grafted, and humanized antibodies. See, e.g., US Patent Publication No. 20090311253 A1. Variable domains may also be prepared using affinity maturation techniques. The binding variable domains of the binding proteins can also be obtained from isolated receptor molecules obtained by extraction procedures known in the art (e.g., using solvents, detergents, and/or affinity purifications), or determined by biophysical methods known in the art (e.g., X-ray crystallography, NMR, interferometry, and/or computer modeling).
[0324] An embodiment is provided comprising selecting parent binding proteins with at least one or more properties desired in the binding protein molecule. In an embodiment, the desired property is one or more of those used to characterize antibody parameters, such as, for example, antigen specificity, affinity to antigen, potency, biological function, epitope recognition, stability, solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross reactivity, or orthologous antigen binding. See, e.g., US Patent Publication No. 20090311253.
[0325] The multi-specific antibodies may also be designed such that one or more of the antigen binding domain are rendered non-functional. The variable domains may be obtained using recombinant DNA techniques from parent binding proteins generated by any one of the methods described herein. In an embodiment, a variable domain is a murine heavy or light chain variable domain. In another embodiment, a variable domain is a CDR grafted or a humanized variable heavy or light chain domain. In an embodiment, a variable domain is a human heavy or light chain variable domain.
[0326] In an embodiment, one or more constant domains are linked to the variable domains using recombinant DNA techniques. In an embodiment, a sequence comprising one or more heavy chain variable domains is linked to a heavy chain constant domain and a sequence comprising one or more light chain variable domains is linked to a light chain constant domain. In an embodiment, the constant domains are human heavy chain constant domains and human light chain constant domains, respectively. In an embodiment, the heavy chain is further linked to an Fc region. The Fc region may be a native sequence Fc region or a variant Fc region. In another embodiment, the Fc region is a human Fc region. In another embodiment, the Fc region includes Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.
[0327] Additionally, the binding proteins provided herein can be employed for tissue-specific delivery (target a tissue marker and a disease mediator for enhanced local PK thus higher efficacy and/or lower toxicity), including intracellular delivery (targeting an internalizing receptor and an intracellular molecule), delivering to inside brain (targeting transferrin receptor and a CNS disease mediator for crossing the blood-brain barrier). The binding proteins can also serve as a carrier protein to deliver an antigen to a specific location via binding to a non-neutralizing epitope of that antigen and also to increase the half-life of the antigen. Furthermore, the binding proteins can be designed to either be physically linked to medical devices implanted into patients or target these medical devices (see Burke et al. (2006) Advanced Drug Deliv. Rev. 58(3): 437-446; Hildebrand et al. (2006) Surface and Coatings Technol. 200(22-23): 6318-6324; Drug/device combinations for local drug therapies and infection prophylaxis, Wu (2006) Biomaterials 27(11):2450-2467; Mediation of the cytokine network in the implantation of orthopedic devices, Marques (2005) Biodegradable Systems in Tissue Engineer. Regen. Med. 377-397). Directing appropriate types of cell to the site of medical implant may promote healing and restoring normal tissue function. Alternatively, inhibition of mediators (including but not limited to cytokines), released upon device implantation by a receptor antibody fusion protein coupled to or target to a device is also provided.
[0328] In one aspect, a host cell is transformed with the vector disclosed above. In one embodiment, the host cell is a prokaryotic cell. In a further embodiment, the host cell is Escherichia coli. In another embodiment, the host cell is an eukaryotic cell. In a further embodiment, the eukaryotic cell is selected from the group consisting of protist cell, animal cell, plant cell and fungal cell. In one embodiment, the host cell is a mammalian cell including, but not limited to, 293, COS, NS0, and CHO and; or a fungal cell such as Saccharomyces cerevisiae; or an insect cell such as Sf9.
[0329] Another aspect of the invention provides a method of producing a binding protein disclosed above comprising culturing any one of the host cells also disclosed above in a culture medium under conditions sufficient to produce the binding protein. Preferably 50%-75% of the binding protein produced by this method is a dual specific tetravalent binding protein. More preferably 75%-90% of the binding protein produced by this method is a dual specific tetravalent binding protein. Most preferably 90%-95% of the binding protein produced is a dual specific tetravalent binding protein.
[0330] Another embodiment provides a binding protein produced according to the method disclosed above.
[0331] One embodiment provides a composition for the release of a binding protein wherein the composition comprises a formulation which in turn comprises a crystallized binding protein, as disclosed above and an ingredient; and at least one polymeric carrier. Preferably the polymeric carrier is a polymer selected from one or more of the group consisting of: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polyeaccharides, blends and copolymers thereof. Preferably the ingredient is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-p-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol. Another embodiment provides a method for treating a mammal comprising the step of administering to the mammal an effective amount of the composition disclosed above.
[0332] The invention also provides a pharmaceutical composition comprising a binding protein, as disclosed above and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, phosphate buffer or saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In some cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[0333] In a further embodiment the pharmaceutical composition comprises at least one additional therapeutic agent for treating a disorder. In one embodiment, the additional agent is selected from the group consisting of: therapeutic agents, imaging agents, cytotoxic agent, angiogenesis inhibitors (including but not limited to anti-VEGF antibodies or VEGF-trap); kinase inhibitors (including but not limited to KDR and TIE-2 inhibitors); co-stimulation molecule blockers (including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig, anti-PD-1, anti-CD20); adhesion molecule blockers (including but not limited to anti-LFA-1 Abs, anti-E/L selectin Abs, small molecule inhibitors); anti-cytokine antibody or functional fragment thereof (including but not limited to anti-IL-18, anti-TNF, anti-TL-6/cytokine receptor antibodies); methotrexate; cyclosporin; rapamycin; FK506; detectable label or reporter; a TNF antagonist; an antirheumatic; a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or analog, a cytokine, and a cytokine antagonist.
[0334] In another aspect, the invention provides a method for treating a human subject suffering from a disorder in which the target, or targets, capable of being bound by the binding protein disclosed above is detrimental, comprising administering to the human subject a binding protein disclosed above such that the activity of the target or targets in the human subject is inhibited and treatment or preventions of the disorder is achieved. In one embodiment, the disease or disorder is an inflammatory condition, autoimmune disease, or cancer. In one embodiment, the disease or disorder is selected from the group comprising arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft versus host disease, organ transplant rejection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease, sporadic, polyglandular deficiency type I and polyglandular deficiency type II, Schmidt's syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, chlamydia, yersinia and salmonella associated arthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, post-inflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjögren's disease associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjörgren's syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, choleosatatis, idiosyncratic liver disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group B streptococci (GBS) infection, mental disorders (e.g., depression and schizophrenia), Th2 Type and Th1 Type mediated diseases, acute and chronic pain (different forms of pain), and cancers such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), Abetalipoprotemia, Acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti cd3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aordic and peripheral aneuryisms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage transplant rejection, cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chronic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated disorders, Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, dermatologic conditions, diabetes, diabetes mellitus, diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's Syndrome in middle age, drug-induced movement disorders induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, epstein-barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hematophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallerrorden-Spatz disease, hashimoto's thyroiditis, hay fever, heart transplant rejection, hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis (A), His bundle arrythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic pulmonary fibrosis, antibody mediated cytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammation of the aorta, influenza a, ionizing radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, Legionella, leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver transplant rejection, lymphederma, malaria, malignamt Lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic, migraine headache, mitochondrial multi-system disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple systems degenerations (Mencel Dejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis, Mycobacterium avium intracellulare, Mycobacterium tuberculosis, myelodyplastic syndrome, myocardial infarction, myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscular atrophies, neutropenic fever, non-Hodgkin's lymphoma, occlusion of the abdominal aorta and its branches, occlusive arterial disorders, okt3 therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, peripheral atherlosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, Pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleo Palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynoud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, Senile Dementia of Lewy body type, seronegative arthropathies, shock, sickle cell anemia, skin allograft rejection, skin changes syndrome, small bowel transplant rejection, solid tumors, specific arrythmias, spinal ataxia, spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, Subacute sclerosing panencephalitis, Syncope, syphilis of the cardiovascular system, systemic anaphalaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type III hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose veins, vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal infections, vital encephalitis/aseptic meningitis, vital-associated hemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or tissue.
[0335] In another aspect the invention provides a method of treating a patient suffering from a disorder comprising the step of administering any one of the binding proteins disclosed above before, concurrent, or after the administration of a second agent, as discussed above. In a preferred embodiment the second agent is selected from the group consisting of budenoside, epidermal growth factor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates, 6-mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists, anti-IL-1β monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors, pyridinyl-imidazole compounds, antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-23, EMAP-II, GM-CSF, FGF, and PDGF, antibodies of CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands, methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen, corticosteroids, prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-1β converting enzyme inhibitors, TNFα converting enzyme inhibitors, T-cell signalling inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor, sIL-IRI, sIL-IRII, sTL-6R, antiinflammatory cytokines, IL-4, IL-1β, IL-11, IL-13 and TGFβ.
[0336] In one embodiment, the pharmaceutical compositions disclosed above are administered to the subject by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.
[0337] One aspect of the invention provides at least one anti-idiotype antibody to at least one binding protein of the present invention. The anti-idiotype antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule such as, but not limited to, at least one complementarily determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or; any portion thereof, that can be incorporated into a binding protein of the present invention.
[0338] In another embodiment the binding proteins of the invention are capable of binding one or more targets selected from the group consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAIl; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC); CCL28; CCL3 (MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2 (mcp-1RB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBIl); CCR8 (CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD47, CD48, CD52; CD69; CD70, CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CD137, CD138, B7-1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A (p21Wap1/Cip1); CDKN1B (p27Kip1); CDKN1C; CDKN2A (p161NK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); cMet; CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA-4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 (IP-10); CXCL11 (I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DES; DKFZp451J0118; DLL-4; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON); FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2 (CCR10); GPR31; GPR44; GPR81 (FKSG80); GRCC10 (C10); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HIP1; histamine and histamine receptors; Her3; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-α; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; IFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; ILlA; IL1B; IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2 IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8; IL8RA; IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); LAMA5; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIB1; midkine; MIF; MIP-2; MKI67 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1 (mucin); MYC; MYD88; NCK2; neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; PCSK9; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PD-1; PD-L1; alpha4beta7, OX40, GITR, TIM-3, Lag-3, B7-H3, B7-H4, GDF8, CGRP, Lingo-1, Factor IXa, Factor X, ICOS, GARP, BTLA, CD160, ROR1, 2B4, KIR, CD27, OX40, CD40L, A2aR, PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13; RGS3; RNF110 (ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCP10; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2; TGFB3; TGFB1; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-11B ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase Iia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VL.sub.A-4; XCL1 (lymphotactin); XCL2 (SCM-1b); XCR1 (GPR5/CCXCR1); YY1; and ZFPM2.
[0339] Given their ability to bind to two or more antigens, the binding proteins of the present invention can be used to detect the antigens (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. The FIT-Ig is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm.
[0340] The binding proteins of the invention, in one embodiment, are capable of neutralizing the activity of the antigens both in vitro and in vivo. Accordingly, such FIT-Igs can be used to inhibit antigen activity, e.g., in a cell culture containing the antigens, in human subjects or in other mammalian subjects having the antigens with which a binding protein of the invention cross-reacts. In another embodiment, the invention provides a method for reducing antigen activity in a subject suffering from a disease or disorder in which the antigen activity is detrimental. A binding protein of the invention can be administered to a human subject for therapeutic purposes.
[0341] As used herein, the term “a disorder in which antigen activity is detrimental” is intended to include diseases and other disorders in which the presence of the antigen in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which antigen activity is detrimental is a disorder in which reduction of antigen activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of the antigen in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of antigen in serum, plasma, synovial fluid, etc. of the subject). Non-limiting examples of disorders that can be treated with the binding proteins of the invention include those disorders discussed below and in the section pertaining to pharmaceutical compositions of the antibodies of the invention.
[0342] The FIT-Igs of the invention may bind one antigen or multiple antigens. Such antigens include, but are not limited to, the targets listed in the following databases, which databases are incorporated herein by reference. These target databases include, but are not limited to, the following listings: [0343] Therapeutic targets (http://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp); [0344] Cytokines and cytokine receptors (http://www.cytokinewebfacts.com/, http://www.copewithcytokines.de/cope.cgi, and [0345] http://cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/cytokine.medic.kumamoto-u.ac.jp/CFC/indexR.html); [0346] Chemokines (http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html); [0347] Chemokine receptors and GPCRs (http://csp.medic.kumamoto-u.ac.jp/CSP/Receptor.html, http://www.gpcr.org/7tm/); [0348] Olfactory Receptors (http://senselab.med.yale.edu/senselab/ORDB/default.asp); [0349] Receptors (http://www.iuphar-db.org/iuphar-rd/list/index.htm); [0350] Cancer targets (http://cged.hgc.jp/cgi-bin/input.cgi); [0351] Secreted proteins as potential antibody targets (http://spd.cbi.pku.edu.cn/); [0352] Protein kinases (http://spd.cbi.pku.edu.cn/), and [0353] Human CD markers (http://content.labvelocity.com/tools/6/1226/CD_table_final_locked.pdf) and (Zola H, 2005 CD molecules 2005: human cell differentiation molecules Blood, 106:3123-6).
[0354] FIT-Igs are useful as therapeutic agents to simultaneously block two different targets to enhance efficacy/safety and/or increase patient coverage. Such targets may include soluble targets (e.g., IL-13 and TNF) and cell surface receptor targets (e.g., VEGFR and EGFR). It can also be used to induce redirected cytotoxicity between tumor cells and T cells (Her2 and CD3) for cancer therapy, or between autoreactive cell and effector cells for autoimmune/transplantation, or between any target cell and effector cell to eliminate disease-causing cells in any given disease.
[0355] In addition, FIT-Ig can be used to trigger receptor clustering and activation when it is designed to target two different epitopes on the same receptor. This may have benefit in making agonistic and antagonistic anti-GPCR therapeutics. In this case, FIT-Ig can be used to target two different epitopes on one cell for clustering/signaling (two cell surface molecules) or signaling (on one molecule). Similarly, a FIT-Ig molecule can be designed to trigger CTLA-4 ligation, and a negative signal by targeting two different epitopes (or 2 copies of the same epitope) of CTLA-4 extracellular domain, leading to down regulation of the immune response. CTLA-4 is a clinically validated target for therapeutic treatment of a number of immunological disorders. CTLA-4/B7 interactions negatively regulate T cell activation by attenuating cell cycle progression, IL-2 production, and proliferation of T cells following activation, and CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by agonistic antibody engagement of CTLA-4 has been unsuccessful since CTLA-4 activation requires ligation. The molecular interaction of CTLA-4/B7 is in “skewed zipper” arrays, as demonstrated by crystal structural analysis (Stamper 2001 Nature 410:608). However none of the currently available CTLA-4 binding reagents have ligation properties, including anti-CTLA-4 monoclonal antibodies. There have been several attempts to address this issue. In one case, a cell member-bound single chain antibody was generated, and significantly inhibited allogeneic rejection in mice (Hwang 2002 JI 169:633). In a separate case, artificial APC surface-linked single-chain antibody to CTLA-4 was generated and demonstrated to attenuate T cell responses (Griffin 2000 JI 164:4433). In both cases, CTLA-4 ligation was achieved by closely localized member-bound antibodies in artificial systems. While these experiments provide proof-of-concept for immune down-regulation by triggering CTLA-4 negative signaling, the reagents used in these reports are not suitable for therapeutic use. To this end, CTLA-4 ligation may be achieved by using a FIT-Ig molecule, which target two different epitopes (or 2 copies of the same epitope) of CTLA-4 extracellular domain. The rationale is that the distance spanning two binding sites of an IgG, approximately 150-170A, is too large for active ligation of CTLA-4 (30-50 Å between 2 CTLA-4 homodimer). However the distance between the two binding sites on FIT-Ig (one arm) is much shorter, also in the range of 30-50 Å, allowing proper ligation of CTLA-4.
[0356] Similarly, FIT-Ig can target two different members of a cell surface receptor complex (e.g. IL-12R alpha and beta). Furthermore, FIT-Ig can target CR1 and a soluble protein/pathogen to drive rapid clearance of the target soluble protein/pathogen.
[0357] Additionally, FIT-Igs of the invention can be employed for tissue-specific delivery (target a tissue marker and a disease mediator for enhanced local PK thus higher efficacy and/or lower toxicity), including intracellular delivery (targeting an internalizing receptor and a intracellular molecule), delivering to inside brain (targeting transferrin receptor and a CNS disease mediator for crossing the blood-brain barrier). FIT-Ig can also serve as a carrier protein to deliver an antigen to a specific location via biding to a non-neutralizing epitope of that antigen and also to increase the half-life of the antigen. Furthermore, FIT-Ig can be designed to either be physically linked to medical devices implanted into patients or target these medical devices (Burke, Sandra E.; Kuntz, Richard E.; Schwartz, Lewis B. Zotarolimus (ABT-578) eluting stents. Advanced Drug Delivery Reviews (2006), 58(3), 437-446.; Surface coatings for biological activation and functionalization of medical devices. Hildebrand, H. F.; Blanchemain, N.; Mayer, G.; Chai, F.; Lefebvre, M.; Boschin, F. Surface and Coatings Technology (2006), 200(22-23), 6318-6324.; Drug/device combinations for local drug therapies and infection prophylaxis. Wu, Peng; Grainger, David W. Biomaterials (2006), 27(11), 2450-2467.; Mediation of the cytokine network in the implantation of orthopedic devices. Marques, A. P.; Hunt, J. A.; Reis, Rui L. Biodegradable Systems in Tissue Engineering and Regenerative Medicine (2005), 377-397; Page: 52
[0358] Mediation of the cytokine network in the implantation of orthopedic devices. Marques, A. P.; Hunt, J. A.; Reis, Rui L. Biodegradable Systems in Tissue Engineering and Regenerative Medicine (2005), 377-397.) Briefly, directing appropriate types of cell to the site of medical implant may promote healing and restoring normal tissue function. Alternatively, inhibition of mediators (including but not limited to cytokines), released upon device implantation by a FIT-Ig coupled to or target to a device is also provided. For example, Stents have been used for years in interventional cardiology to clear blocked arteries and to improve the flow of blood to the heart muscle. However, traditional bare metal stents have been known to cause restenosis (re-narrowing of the artery in a treated area) in some patients and can lead to blood clots. Recently, an anti-CD34 antibody coated stent has been described which reduced restenosis and prevents blood clots from occurring by capturing endothelial progenitor cells (EPC) circulating throughout the blood. Endothelial cells are cells that line blood vessels, allowing blood to flow smoothly. The EPCs adhere to the hard surface of the stent forming a smooth layer that not only promotes healing but prevents restenosis and blood clots, complications previously associated with the use of stents (Aoji et al. 2005 J Am Coll Cardiol. 45(10):1574-9). In addition to improving outcomes for patients requiring stents, there are also implications for patients requiring cardiovascular bypass surgery. For example, a prosthetic vascular conduit (artificial artery) coated with anti-EPC antibodies would eliminate the need to use arteries from patients legs or arms for bypass surgery grafts. This would reduce surgery and anesthesia times, which in turn will reduce coronary surgery deaths. FIT-Ig are designed in such a way that it binds to a cell surface marker (such as CD34) as well as a protein (or an epitope of any kind, including but not limited to lipids and polysaccharides) that has been coated on the implanted device to facilitate the cell recruitment. Such approaches can also be applied to other medical implants in general. Alternatively, FIT-Igs can be coated on medical devices and upon implantation and releasing all FITs from the device (or any other need which may require additional fresh FIT-Ig, including aging and denaturation of the already loaded FIT-Ig) the device could be reloaded by systemic administration of fresh FIT-Ig to the patient, where the FIT-Ig is designed to binds to a target of interest (a cytokine, a cell surface marker (such as CD34) etc.) with one set of binding sites and to a target coated on the device (including a protein, an epitope of any kind, including but not limited to lipids, polysaccharides and polymers) with the other. This technology has the advantage of extending the usefulness of coated implants.
[0359] FIT-Ig molecules of the invention are also useful as therapeutic molecules to treat various diseases. Such FIT-Ig molecules may bind one or more targets involved in a specific disease. Examples of such targets in various diseases are described below.
[0360] Many proteins have been implicated in general autoimmune and inflammatory responses, including C5, CCL1 (I-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (I-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1 (endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5, IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA-4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFB1, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144). FIT-Igs capable of binding one or more of the targets listed above are also contemplated.
[0361] Allergic asthma is characterized by the presence of eosinophilia, goblet cell metaplasia, epithelial cell alterations, airway hyperreactivity (AHR), and Th2 and Th1 cytokine expression, as well as elevated serum IgE levels. It is now widely accepted that airway inflammation is the key factor underlying the pathogenesis of asthma, involving a complex interplay of inflammatory cells such as T cells, B cells, eosinophils, mast cells and macrophages, and of their secreted mediators including cytokines and chemokines. Corticosteroids are the most important anti-inflammatory treatment for asthma today, however their mechanism of action is non-specific and safety concerns exist, especially in the juvenile patient population. The development of more specific and targeted therapies is therefore warranted. There is increasing evidence that IL-13 in mice mimics many of the features of asthma, including AHR, mucus hypersecretion and airway fibrosis, independently of eosinophilic inflammation (Finotto et al., International Immunology (2005), 17(8), 993-1007; Padilla et al., Journal of Immunology (2005), 174(12), 8097-8105).
[0362] IL-13 has been implicated as having a pivotal role in causing pathological responses associated with asthma. The development of anti-IL-13 monoclonal antibody therapy to reduce the effects of IL-13 in the lung is an exciting new approach that offers considerable promise as a novel treatment for asthma. However other mediators of differential immunological pathways are also involved in asthma pathogenesis, and blocking these mediators, in addition to IL-13, may offer additional therapeutic benefit. Such target pairs include, but are not limited to, IL-13 and a pro-inflammatory cytokine, such as tumor necrosis factor-α (TNF-α). TNF-α may amplify the inflammatory response in asthma and may be linked to disease severity (McDonnell, et al., Progress in Respiratory Research (2001), 31 (New Drugs for Asthma, Allergy and COPD), 247-250.). This suggests that blocking both IL-13 and TNF-α may have beneficial effects, particularly in severe airway disease. In a preferred embodiment the FIT-Ig of the invention binds the targets IL-13 and TNFα and is used for treating asthma.
[0363] Animal models such as OVA-induced asthma mouse model, where both inflammation and AHR can be assessed, are known in the art and may be used to determine the ability of various FIT-Ig molecules to treat asthma. Animal models for studying asthma are disclosed in Coffman, et al., Journal of Experimental Medicine (2005), 201(12), 1875-1879; Lloyd, et al., Advances in Immunology (2001), 77, 263-295; Boyce et al., Journal of Experimental Medicine (2005), 201(12), 1869-1873; and Snibson, et al., Journal of the British Society for Allergy and Clinical Immunology (2005), 35(2), 146-52. In addition to routine safety assessments of these target pairs specific tests for the degree of immunosuppression may be warranted and helpful in selecting the best target pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43; Descotes, et al., Developments in biological standardization (1992), 77 99-102; Hart et al., Journal of Allergy and Clinical Immunology (2001), 108(2), 250-257).
[0364] Based on the rationale disclosed above and using the same evaluation model for efficacy and safety other pairs of targets that FIT-Ig molecules can bind and be useful to treat asthma may be determined. Preferably such targets include, but are not limited to, IL-13 and IL-1beta, since IL-1beta is also implicated in inflammatory response in asthma; IL-13 and cytokines and chemokines that are involved in inflammation, such as IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-0; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13 and ADAM8. The present invention also contemplates FIT-Igs capable of binding one or more targets involved in asthma selected from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG, histamine and histamine receptors, IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R1, TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STAT6, TBX21, TGFB1, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase.
[0365] Rheumatoid arthritis (RA), a systemic disease, is characterized by a chronic inflammatory reaction in the synovium of joints and is associated with degeneration of cartilage and erosion of juxta-articular bone. Many pro-inflammatory cytokines including TNF, chemokines, and growth factors are expressed in diseased joints. Systemic administration of anti-TNF antibody or sTNFR fusion protein to mouse models of RA was shown to be anti-inflammatory and joint protective. Clinical investigations in which the activity of TNF in RA patients was blocked with intravenously administered infliximab (Harriman G, Harper L K, Schaible T F. 1999 Summary of clinical trials in rheumatoid arthritis using infliximab, an anti-TNFalpha treatment. Ann Rheum Dis 58 Suppl 1:I61-4.), a chimeric anti-TNF monoclonal antibody (mAB), has provided evidence that TNF regulates IL-6, IL-8, MCP-1, and VEGF production, recruitment of immune and inflammatory cells into joints, angiogenesis, and reduction of blood levels of matrix metalloproteinases-1 and -3. A better understanding of the inflammatory pathway in rheumatoid arthritis has led to identification of other therapeutic targets involved in rheumatoid arthritis. Promising treatments such as interleukin-6 antagonists (MRA), CTLA4Ig (abatacept, Genovese Mc et al 2005 Abatacept for rheumatoid arthritis refractory to tumor necrosis factor alpha inhibition. N Engl J Med. 353:1114-23.), and anti-B cell therapy (rituximab, Okamoto H, Kamatani N. 2004 Rituximab for rheumatoid arthritis. N Engl J Med. 351:1909) have already been tested in randomized controlled trials over the past year. Other cytokines have been identified and have been shown to be of benefit in animal models, including interleukin-15, interleukin-17, and interleukin-18, and clinical trials of these agents are currently under way. Dual-specific antibody therapy, combining anti-TNF and another mediator, has great potential in enhancing clinical efficacy and/or patient coverage. For example, blocking both TNF and VEGF can potentially eradicate inflammation and angiogenesis, both of which are involved in pathophysiology of RA. Blocking other pairs of targets involved in RA including, but not limited to, TNF and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1beta; TNF and MIF; TNF and IL-17; and TNF and IL-15 with specific FIT-Ig Igs is also contemplated. In addition to routine safety assessments of these target pairs, specific tests for the degree of immunosuppression may be warranted and helpful in selecting the best target pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43; Descotes, et al., Developments in biological standardization (1992), 77 99-102; Hart et al., Journal of Allergy and Clinical Immunology (2001), 108(2), 250-257). Whether a FIT-Ig Ig molecule will be useful for the treatment of rheumatoid arthritis can be assessed using pre-clinical animal RA models such as the collagen-induced arthritis mouse model. Other useful models are also well known in the art (see Brand D D., Comp Med. (2005) 55(2):114-22).
[0366] The immunopathogenic hallmark of systemic lupus erythematosus (SLE) is the polyclonal B cell activation, which leads to hyperglobulinemia, autoantibody production and immune complex formation. The fundamental abnormality appears to be the failure of T cells to suppress the forbidden B cell clones due to generalized T cell dysregulation. In addition, B and T-cell interaction is facilitated by several cytokines such as IL-1β as well as co-stimulatory molecules such as CD40 and CD40L, B7 and CD28 and CTLA-4, which initiate the second signal. These interactions together with impaired phagocytic clearance of immune complexes and apoptotic material, perpetuate the immune response with resultant tissue injury. The following targets may be involved in SLE and can potentially be used for FIT-Ig approach for therapeutic intervention: B cell targeted therapies: CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81, IFNB1, IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, TL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA, and NT5E.; co-stimulatory signals: CTLA-4 or B7.1/B7.2; inhibition of B cell survival: BlyS, BAFF; Complement inactivation: C5; Cytokine modulation: the key principle is that the net biologic response in any tissue is the result of a balance between local levels of proinflammatory or anti-inflammatory cytokines (see Sfikakis P P et al 2005 Curr Opin Rheumatol 17:550-7). SLE is considered to be a Th-2 driven disease with documented elevations in serum IL-4, IL-6, IL-10. FIT-Ig Igs capable of binding one or more targets selected from the group consisting of IL-4, IL-6, IL-1β, IFN-a, and TNF-a are also contemplated. Combination of targets discussed above will enhance therapeutic efficacy for SLE which can be tested in a number of lupus preclinical models (see Peng S L (2004) Methods Mol Med.; 102:227-72).
[0367] Multiple sclerosis (MS) is a complex human autoimmune-type disease with a predominantly unknown etiology. Immunologic destruction of myelin basic protein (MBP) throughout the nervous system is the major pathology of multiple sclerosis. MS is a disease of complex pathologies, which involves infiltration by CD4+ and CD8+ T cells and of response within the central nervous system. Expression in the CNS of cytokines, reactive nitrogen species and costimulator molecules have all been described in MS. Of major consideration are immunological mechanisms that contribute to the development of autoimmunity. In particular, antigen expression, cytokine and leukocyte interactions, and regulatory T-cells, which help balance/modulate other T-cells such as Th1 and Th2 cells, are important areas for therapeutic target identification.
[0368] IL-12 is a proinflammatory cytokine that is produced by APC and promotes differentiation of Th1 effector cells. IL-12 is produced in the developing lesions of patients with MS as well as in EAE-affected animals. Previously it was shown that interference in IL-12 pathways effectively prevents EAE in rodents, and that in vivo neutralization of IL-12p40 using a anti-IL-12 mAb has beneficial effects in the myelin-induced EAE model in common marmosets.
[0369] TWEAK is a member of the TNF family, constitutively expressed in the central nervous system (CNS), with pro-inflammatory, proliferative or apoptotic effects depending upon cell types. Its receptor, Fn14, is expressed in CNS by endothelial cells, reactive astrocytes and neurons. TWEAK and Fn14 mRNA expression increased in spinal cord during experimental autoimmune encephalomyelitis (EAE). Anti-TWEAK antibody treatment in myelin oligodendrocyte glycoprotein (MOG) induced EAE in C57BL/6 mice resulted in a reduction of disease severity and leukocyte infiltration when mice were treated after the priming phase.
[0370] One aspect of the invention pertains to FIT-Ig Ig molecules capable of binding one or more, preferably two, targets selected from the group consisting of IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VL.sub.A-4, TNF, CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. A preferred embodiment includes a dual-specific anti-IL-12/TWEAK FIT-Ig Ig as a therapeutic agent beneficial for the treatment of MS. Several animal models for assessing the usefulness of the FIT-Ig molecules to treat MS are known in the art (see Steinman L, et al., (2005) Trends Immunol. 26(11):565-71; Lublin F D., et al., (1985) Springer Semin Immunopathol. 8(3):197-208; Genain C P, et al., (1997) J Mol Med. 75(3):187-97; Tuohy V K, et al., (1999) J Exp Med. 189(7):1033-42; Owens T, et al., (1995) Neurol Clin. 13(1):51-73; and 't Hart B A, et al., (2005) J Immunol 175(7):4761-8. In addition to routine safety assessments of these target pairs specific tests for the degree of immunosuppression may be warranted and helpful in selecting the best target pairs (see Luster et al., Toxicology (1994), 92(1-3), 229-43; Descotes, et al., Developments in biological standardization (1992), 77 99-102; Jones R. 2000 Rovelizumab (ICOS Corp). IDrugs. 3(4):442-6).
[0371] The pathophysiology of sepsis is initiated by the outer membrane components of both gram-negative organisms (lipopolysaccharide [LPS], lipid A, endotoxin) and gram-positive organisms (lipoteichoic acid, peptidoglycan). These outer membrane components are able to bind to the CD14 receptor on the surface of monocytes. By virtue of the recently described toll-like receptors, a signal is then transmitted to the cell, leading to the eventual production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-I). Overwhelming inflammatory and immune responses are essential features of septic shock and play a central part in the pathogenesis of tissue damage, multiple organ failure, and death induced by sepsis. Cytokines, especially tumor necrosis factor (TNF) and interleukin (IL)-1, have been shown to be critical mediators of septic shock. These cytokines have a direct toxic effect on tissues; they also activate phospholipase A2. These and other effects lead to increased concentrations of platelet-activating factor, promotion of nitric oxide synthase activity, promotion of tissue infiltration by neutrophils, and promotion of neutrophil activity.
[0372] The treatment of sepsis and septic shock remains a clinical conundrum, and recent prospective trials with biological response modifiers (i.e. anti-TNF, anti-MIF) aimed at the inflammatory response have shown only modest clinical benefit. Recently, interest has shifted toward therapies aimed at reversing the accompanying periods of immune suppression. Studies in experimental animals and critically ill patients have demonstrated that increased apoptosis of lymphoid organs and some parenchymal tissues contribute to this immune suppression, anergy, and organ system dysfunction. During sepsis syndromes, lymphocyte apoptosis can be triggered by the absence of IL-2 or by the release of glucocorticoids, granzymes, or the so-called ‘death’ cytokines: tumor necrosis factor alpha or Fas ligand. Apoptosis proceeds via auto-activation of cytosolic and/or mitochondrial caspases, which can be influenced by the pro- and anti-apoptotic members of the Bcl-2 family. In experimental animals, not only can treatment with inhibitors of apoptosis prevent lymphoid cell apoptosis; it may also improve outcome. Although clinical trials with anti-apoptotic agents remain distant due in large part to technical difficulties associated with their administration and tissue targeting, inhibition of lymphocyte apoptosis represents an attractive therapeutic target for the septic patient. Likewise, a dual-specific agent targeting both inflammatory mediator and a apoptotic mediator, may have added benefit. One aspect of the invention pertains to FIT-Ig Igs capable of binding one or more targets involved in sepsis, preferably two targets, selected from the group consisting TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4, IL10, IL1B, NFKB1, PROC, TNFRSF1A, CSF3, IL10, IL1B, IL6, ADORA2A, CCR3, IL10, IL1B, IL1RN, MIF, NFKB1, PTAFR, TLR2, TLR4, GPR44, HMOX1, midkine, IRAK1, NFKB2, SERPINA1, SERPINE1, and TREM1. The efficacy of such FIT-Ig Igs for sepsis can be assessed in preclinical animal models known in the art (see Buras J A, et al., (2005) Nat Rev Drug Discov. 4(10):854-65 and Calandra T, et al., (2000) Nat Med. 6(2):164-70).
[0373] Chronic neurodegenerative diseases are usually age-dependent diseases characterized by progressive loss of neuronal functions (neuronal cell death, demyelination), loss of mobility and loss of memory. Emerging knowledge of the mechanisms underlying chronic neurodegenerative diseases (e.g. Alzheimer's disease) show a complex etiology and a variety of factors have been recognized to contribute to their development and progression e.g. age, glycemic status, amyloid production and multimerization, accumulation of advanced glycation-end products (AGE) which bind to their receptor RAGE (receptor for AGE), increased brain oxidative stress, decreased cerebral blood flow, neuroinflammation including release of inflammatory cytokines and chemokines, neuronal dysfunction and microglial activation. Thus these chronic neurodegenerative diseases represent a complex interaction between multiple cell types and mediators. Treatment strategies for such diseases are limited and mostly constitute either blocking inflammatory processes with non-specific anti-inflammatory agents (e.g., corticosteroids, COX inhibitors) or agents to prevent neuron loss and/or synaptic functions. These treatments fail to stop disease progression. Recent studies suggest that more targeted therapies such as antibodies to soluble A-β peptide (including the A-b oligomeric forms) can not only help stop disease progression but may help maintain memory as well. These preliminary observations suggest that specific therapies targeting more than one disease mediator (e.g. A-β and a pro-inflammatory cytokine such as TNF) may provide even better therapeutic efficacy for chronic neurodegenerative diseases than observed with targeting a single disease mechanism (e.g. soluble A-Palone) (see C. E. Shepherd, et al, Neurobiol Aging. 2005 Oct. 24; Nelson R B., Curr Pharm Des. 2005; 11:3335; William L. Klein.; Neurochem Int. 2002; 41:345; Michelle C Janelsins, et al., J Neuroinflammation. 2005; 2:23; Soloman B., Curr Alzheimer Res. 2004; 1:149; Igor Klyubin, et al., Nat Med. 2005; 11:556-61; Arancio O, et al., EMBO Journal (2004) 1-10; Bornemann K D, et al., Am J Pathol. 2001; 158:63; Deane R, et al., Nat Med. 2003; 9:907-13; and Eliezer Masliah, et al., Neuron. 2005; 46:857).
[0374] The FIT-Ig molecules of the invention can bind one or more targets involved in Chronic neurodegenerative diseases such as Alzheimer's. Such targets include, but are not limited to, any mediator, soluble or cell surface, implicated in AD pathogenesis e.g. AGE (S100 A, amphoterin), pro-inflammatory cytokines (e.g. IL-1), chemokines (e.g. MCP 1), molecules that inhibit nerve regeneration (e.g. Nogo, RGM A), molecules that enhance neurite growth (neurotrophins). The efficacy of FIT-Ig molecules can be validated in pre-clinical animal models such as the transgenic mice that over-express amyloid precursor protein or RAGE and develop Alzheimer's disease-like symptoms. In addition, FIT-Ig molecules can be constructed and tested for efficacy in the animal models and the best therapeutic FIT-Ig can be selected for testing in human patients. FIT-Ig molecules can also be employed for treatment of other neurodegenerative diseases such as Parkinson's disease. Alpha-Synuclein is involved in Parkinson's pathology. A FIT-Ig capable of targeting alpha-synuclein and inflammatory mediators such as TNF, IL-1, MCP-1 can prove effective therapy for Parkinson's disease and are contemplated in the invention.
[0375] Despite an increase in knowledge of the pathologic mechanisms, spinal cord injury (SCI) is still a devastating condition and represents a medical indication characterized by a high medical need. Most spinal cord injuries are contusion or compression injuries and the primary injury is usually followed by secondary injury mechanisms (inflammatory mediators e.g. cytokines and chemokines) that worsen the initial injury and result in significant enlargement of the lesion area, sometimes more than 10-fold. These primary and secondary mechanisms in SCI are very similar to those in brain injury caused by other means e.g. stroke. No satisfying treatment exists and high dose bolus injection of methylprednisolone (MP) is the only used therapy within a narrow time window of 8 h post injury. This treatment, however, is only intended to prevent secondary injury without causing any significant functional recovery. It is heavily criticized for the lack of unequivocal efficacy and severe adverse effects, like immunosuppression with subsequent infections and severe histopathological muscle alterations. No other drugs, biologics or small molecules, stimulating the endogenous regenerative potential are approved, but promising treatment principles and drug candidates have shown efficacy in animal models of SCI in recent years. To a large extent the lack of functional recovery in human SCI is caused by factors inhibiting neurite growth, at lesion sites, in scar tissue, in myelin as well as on injury-associated cells. Such factors are the myelin-associated proteins NogoA, OMgp and MAG, RGM A, the scar-associated CSPG (Chondroitin Sulfate Proteoglycans) and inhibitory factors on reactive astrocytes (some semaphorins and ephrins). However, at the lesion site not only growth inhibitory molecules are found but also neurite growth stimulating factors like neurotrophins, laminin, L1 and others. This ensemble of neurite growth inhibitory and growth promoting molecules may explain that blocking single factors, like NogoA or RGM A, resulted in significant functional recovery in rodent SCI models, because a reduction of the inhibitory influences could shift the balance from growth inhibition to growth promotion. However, recoveries observed with blocking a single neurite outgrowth inhibitory molecule were not complete. To achieve faster and more pronounced recoveries either blocking two neurite outgrowth inhibitory molecules e.g. Nogo and RGM A, or blocking an neurite outgrowth inhibitory molecule and enhancing functions of a neurite outgrowth enhancing molecule e.g. Nogo and neurotrophins, or blocking a neurite outgrowth inhibitory molecule e.g. Nogo and a pro-inflammatory molecule e.g. TNF, may be desirable (see McGee A W, et al., Trends Neurosci. 2003; 26:193; Marco Domeniconi, et al., J Neurol Sci. 2005; 233:43; Milan Makwanal, et al., FEBS J. 2005; 272:2628; Barry J. Dickson, Science. 2002; 298:1959; Felicia Yu Hsuan Teng, et al., J Neurosci Res. 2005; 79:273; Tara Karnezis, et al., Nature Neuroscience 2004; 7, 736; Gang Xu, et al., J. Neurochem. 2004; 91; 1018).
[0376] Other FIT-Igs contemplated are those capable of binding target pairs such as NgR and RGM A; NogoA and RGM A; MAG and RGM A; OMGp and RGM A; RGM A and RGM B; CSPGs and RGM A; aggrecan, midkine, neurocan, versican, phosphacan, Te38 and TNF-α; AB globulomer-specific antibodies combined with antibodies promoting dendrite & axon sprouting. Dendrite pathology is a very early sign of AD and it is known that NOGO A restricts dendrite growth. One can combine such type of ab with any of the SCI-candidate (myelin-proteins) Ab. Other FIT-Ig targets may include any combination of NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG or Omgp. Additionally, targets may also include any mediator, soluble or cell surface, implicated in inhibition of neurite e.g Nogo, Ompg, MAG, RGM A, semaphorins, ephrins, soluble A-b, pro-inflammatory cytokines (e.g. IL-1), chemokines (e.g. MIP 1a), molecules that inhibit nerve regeneration. The efficacy of anti-nogo/anti-RGM A or similar FIT-Ig molecules can be validated in pre-clinical animal models of spinal cord injury. In addition, these FIT-Ig molecules can be constructed and tested for efficacy in the animal models and the best therapeutic FIT-Ig can be selected for testing in human patients. In addition, FIT-Ig molecules can be constructed that target two distinct ligand binding sites on a single receptor e.g. Nogo receptor which binds three ligand Nogo, Ompg, and MAG and RAGE that binds A-b and S100 A. Furthermore, neurite outgrowth inhibitors e.g. nogo and nogo receptor, also play a role in preventing nerve regeneration in immunological diseases like multiple sclerosis. Inhibition of nogo-nogo receptor interaction has been shown to enhance recovery in animal models of multiple sclerosis. Therefore, FIT-Ig molecules that can block the function of one immune mediator eg a cytokine like IL-12 and a neurite outgrowth inhibitor molecule eg nogo or RGM may offer faster and greater efficacy than blocking either an immune or an neurite outgrowth inhibitor molecule alone.
[0377] Monoclonal antibody therapy has emerged as an important therapeutic modality for cancer (von Mehren M, et al 2003 Monoclonal antibody therapy for cancer. Annu Rev Med.; 54:343-69). Antibodies may exert antitumor effects by inducing apoptosis, redirected cytotoxicity, interfering with ligand-receptor interactions, or preventing the expression of proteins that are critical to the neoplastic phenotype. In addition, antibodies can target components of the tumor microenvironment, perturbing vital structures such as the formation of tumor-associated vasculature. Antibodies can also target receptors whose ligands are growth factors, such as the epidermal growth factor receptor. The antibody thus inhibits natural ligands that stimulate cell growth from binding to targeted tumor cells. Alternatively, antibodies may induce an anti-idiotype network, complement-mediated cytotoxicity, or antibody-dependent cellular cytotoxicity (ADCC). The use of dual-specific antibody that targets two separate tumor mediators will likely give additional benefit compared to a mono-specific therapy. FIT-Ig Igs capable of binding the following pairs of targets to treat oncological disease are also contemplated: IGF1 and IGF2; IGF1/2 and Erb2B; VEGFR and EGFR; CD20 and CD3, CD138 and CD20, CD38 and CD20, CD38 & CD138, CD40 and CD20, CD138 and CD40, CD38 and CD40. Other target combinations include one or more members of the EGF/erb-2/erb-3 family. Other targets (one or more) involved in oncological diseases that FIT-Ig Igs may bind include, but are not limited to those selected from the group consisting of: CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, EGF, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1, IGF1R, IL2, VEGF, BCL2, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1, IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1, IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NROB1, NROB2, NR1D2, NR1H2, NR1H4, NR1I2, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1, BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2, IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3, CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164, COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB2IP, DES, DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6, ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR, LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3, BAIl, COL4A3, IL8, LAMA5, NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4, PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B (p27Kip1), CDKN2A (p161NK4a), COL6A1, CTNNB1 (b-catenin), CTSB (cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3, GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, CTLA-4, OX40, GITR, TIM-3, Lag-3, B7-H3, B7-H4, GDF8, CGRP, Lingo-1, ICOS, GARP, BTLA, CD160, ROR1, SERPINB5 (maspin), SERPINE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1), CLDN7 (claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin), KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type II keratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4, and TNFAIP2 (B94).
[0378] In an embodiment, diseases that can be treated or diagnosed with the compositions and methods provided herein include, but are not limited to, primary and metastatic cancers, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract (including cervix, uterus, and ovaries as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as well as Kaposi's sarcoma), tumors of the brain, nerves, eyes, and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, and meningiomas), solid tumors arising from hematopoietic malignancies such as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin's lymphomas).
[0379] In an embodiment, the antibodies provided herein or antigen-binding portions thereof, are used to treat cancer or in the prevention of metastases from the tumors described herein either when used alone or in combination with radiotherapy and/or other chemotherapeutic agents.
[0380] According to another embodiment of the invention, the human immune effector cell is a member of the human lymphoid cell lineage. In this embodiment, the effector cell may advantageously be a human T cell, a human B cell or a human natural killer (NK) cell. Advantageously, such cells will have either a cytotoxic or an apoptotic effect on the target cell. Especially advantageously, the human lymphoid cell is a cytotoxic T cell which, when activated, exerts a cytotoxic effect on the target cell. According to this embodiment, then, the recruited activity of the human effector cell is this cell's cytotoxic activity.
[0381] According to a preferred embodiment, activation of the cytotoxic T cell may occur via binding of the CD3 antigen as effector antigen on the surface of the cytotoxic T cell by a bispecific antibody of this embodiment of the invention. The human CD3 antigen is present on both helper T cells and cytotoxic T cells. Human CD3 denotes an antigen which is expressed on T cells as part of the multimolecular T cell complex and which comprises three different chains: CD3-epsilon, CD3-delta and CD3-gamma.
[0382] The activation of the cytotoxic potential of T cells is a complex phenomenon which requires the interplay of multiple proteins. The T cell receptor (“TCR”) protein is a membrane bound disulfide-linked heterodimer consisting of two different glycoprotein subunits. The TCR recognizes and binds foreign peptidic antigen which itself has been bound by a member of the highly diverse class of major histocompatibility complex (“MHC”) proteins and has been presented, bound to the MHC, on the surface of antigen presenting cells (“APCs”).
[0383] Although the variable TCR binds foreign antigen as outlined above, signaling to the T cell that this binding has taken place depends on the presence of other, invariant, signaling proteins associated with the TCR. These signaling proteins in associated form are collectively referred to as the CD3 complex, here collectively referred to as the CD3 antigen.
[0384] The activation of T cell cytotoxicity, then, normally depends first on the binding of the TCR with an MHC protein, itself bound to foreign antigen, located on a separate cell. Only when this initial TCR-MHC binding has taken place can the CD3-dependent signaling cascade responsible for T cell clonal expansion and, ultimately, T cell cytotoxicity ensue.
[0385] However, binding of the human CD3 antigen by the first or second portion of a bispecific antibody of the invention activates T cells to exert a cytotoxic effect on other cells in the absence of independent TCR-MHC binding. This means that T cells may be cytotoxically activated in a clonally independent fashion, i.e., in a manner which is independent of the specific TCR clone carried by the T cell. This allows an activation of the entire T cell compartment rather than only specific T cells of a certain clonal identity.
[0386] In light of the foregoing discussion, then, an especially preferred embodiment of the invention provides a bispecific antibody in which the effector antigen is the human CD3 antigen. The bispecific antibody according to this embodiment of the invention may have a total of either two or three antibody variable domains.
[0387] According to further embodiments of the invention, other lymphoid cell-associated effector antigens bound by a bispecific antibody of the invention may be the human CD16 antigen, the human NKG2D antigen, the human NKp46 antigen, the human CD2 antigen, the human CD28 antigen or the human CD25 antigen.
[0388] According to another embodiment of the invention, the human effector cell is a member of the human myeloid lineage. Advantageously, the effector cell may be a human monocyte, a human neutrophilic granulocyte or a human dendritic cell. Advantageously, such cells will have either a cytotoxic or an apoptotic effect on the target cell. Advantageous antigens within this embodiment which may be bound by a bispecific antibody of the invention may be the human CD64 antigen or the human CD89 antigen.
[0389] According to another embodiment of the invention, the target antigen is an antigen which is uniquely expressed on a target cell or effector cell in a disease condition, but which remains either non-expressed, expressed at a low level or non-accessible in a healthy condition. Examples of such target antigens which might be specifically bound by a bispecific antibody of the invention may advantageously be selected from EpCAM, CCR5, CD19, HER-2 neu, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, PhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue-1, Plasma Cell Antigen, (membrane-bound) IgE, Melanoma Chondroitin Sulfate Proteoglycan (MCSP), CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6; desmoglein 4, E-cadherin neoepitope, Fetal Acetylcholine Receptor, CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory Substance (MIS) Receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS and CD63.
[0390] According to a specific embodiment, the target antigen specifically bound by a bispecific antibody may be a cancer-related antigen, e.g., an antigen related to a malignant condition. Such an antigen is either expressed or accessible on a malignant cell, whereas the antigen is either not present, not significantly present, or is not accessible on a non-malignant cell. As such, a bispecific antibody according to this embodiment of the invention is a bispecific antibody which recruits the activity of a human immune effector cell against the malignant target cell bearing the target antigen, or rendering the target antigen accessible.
[0391] Gene Therapy: In a specific embodiment, nucleic acid sequences encoding a binding protein provided herein or another prophylactic or therapeutic agent provided herein are administered to treat, prevent, manage, or ameliorate a disorder or one or more symptoms thereof by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment, the nucleic acids produce their encoded antibody or prophylactic or therapeutic agent provided herein that mediates a prophylactic or therapeutic effect.
[0392] Any of the methods for gene therapy available in the art can be used in the methods provided herein. For general reviews of the methods of gene therapy, see Goldspiel et al. (1993) Clin. Pharmacy 12:488-505; Wu and Wu (1991) Biotherapy 3:87-95; Tolstoshev (1993) Ann Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993) Science 260:926-932; Morgan and Anderson (1993) Ann Rev. Biochem. 62:191-217; and May (1993) TIBTECH 11(5):155-215. Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &Sons, N Y (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). Detailed description of various methods of gene therapy is disclosed in US Patent Publication No. US20050042664.
[0393] Diagnostics: The disclosure herein also provides diagnostic applications including, but not limited to, diagnostic assay methods, diagnostic kits containing one or more binding proteins, and adaptation of the methods and kits for use in automated and/or semi-automated systems. The methods, kits, and adaptations provided may be employed in the detection, monitoring, and/or treatment of a disease or disorder in an individual. This is further elucidated below.
[0394] A. Method of Assay: The present disclosure also provides a method for determining the presence, amount or concentration of an analyte, or fragment thereof, in a test sample using at least one binding protein as described herein. Any suitable assay as is known in the art can be used in the method. Examples include, but are not limited to, immunoassays and/or methods employing mass spectrometry. Immunoassays provided by the present disclosure may include sandwich immunoassays, radioimmunoassay (RIA), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), competitive-inhibition immunoassays, fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), bioluminescence resonance energy transfer (BRET), and homogenous chemiluminescent assays, among others. A chemiluminescent microparticle immunoassay, in particular one employing the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park, Ill.), is an example of an immunoassay. Methods employing mass spectrometry are provided by the present disclosure and include, but are not limited to MALDI (matrix-assisted laser desorption/ionization) or by SELDI (surface-enhanced laser desorption/ionization).
[0395] Methods for collecting, handling, processing, and analyzing biological test samples using immunoassays and mass spectrometry would be well-known to one skilled in the art, are provided for in the practice of the present disclosure (US 2009-0311253 A1).
[0396] B. Kit: A kit for assaying a test sample for the presence, amount or concentration of an analyte, or fragment thereof, in a test sample is also provided. The kit comprises at least one component for assaying the test sample for the analyte, or fragment thereof, and instructions for assaying the test sample for the analyte, or fragment thereof. The at least one component for assaying the test sample for the analyte, or fragment thereof, can include a composition comprising a binding protein, as disclosed herein, and/or an anti-analyte binding protein (or a fragment, a variant, or a fragment of a variant thereof), which is optionally immobilized on a solid phase. Optionally, the kit may comprise a calibrator or control, which may comprise isolated or purified analyte. The kit can comprise at least one component for assaying the test sample for an analyte by immunoassay and/or mass spectrometry. The kit components, including the analyte, binding protein, and/or anti-analyte binding protein, or fragments thereof, may be optionally labeled using any art-known detectable label. The materials and methods for the creation provided for in the practice of the present disclosure would be known to one skilled in the art (US 2009-0311253 A1).
[0397] C. Adaptation of Kit and Method: The kit (or components thereof), as well as the method of determining the presence, amount or concentration of an analyte in a test sample by an assay, such as an immunoassay as described herein, can be adapted for use in a variety of automated and semi-automated systems (including those wherein the solid phase comprises a microparticle), as described, for example, in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as commercially marketed, for example, by Abbott Laboratories (Abbott Park, Ill.) as ARCHITECT®. Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM®, IMx® (see, for example, U.S. Pat. No. 5,294,404, PRISM®, EIA (bead), and Quantum™ II, as well as other platforms. Additionally, the assays, kits and kit components can be employed in other formats, for example, on electrochemical or other hand-held or point-of-care assay systems. The present disclosure is, for example, applicable to the commercial Abbott Point of Care (i-STAT®, Abbott Laboratories) electrochemical immunoassay system that performs sandwich immunoassays. Immunosensors and their methods of manufacture and operation in single-use test devices are described, for example in, U.S. Pat. Nos. 5,063,081, 7,419,821, and 7,682,833; and US Publication Nos. 20040018577, 20060160164 and US 20090311253. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein are obvious and may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.
[0398] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.
EXAMPLES
Example 1. Construction, Expression, Purification, and Analysis of Anti-IL17/IL-20 Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0399] To demonstrate the FIT-Ig technology, we have generated a group of anti-IL-17/IL-20 FIT-Ig molecules: FIT1-Ig, FIT2-Ig, and FIT3-Ig, all of which contains 3 different polypeptides, as shown in
[0400] We also generated a group of anti-IL-17/IL-20 FIT-Ig molecules: FIT4-Ig, FIT5-Ig, and FIT6-Ig, each of which contains 2 different polypeptides, as shown in
Example 1.1: Molecular Cloning of Anti-IL-17/IL-20 FIT-Ig Molecules
[0401] For construct #1 cloning, LY light chain was amplified by PCR using forward primers annealing on light chain signal sequence and reverse primers annealing on C-terminus of the light chain. 15D2 heavy chain was amplified by PCR using forward primers annealing on N-terminus of 15D2 VH and reverse primers annealing on C-terminus of CH. These 2 PCR fragments were gel purified and combined by overlapping PCR using signal peptide and CH primer pair. The combined PCR product was cloned into a 293 expression vector, which already contained the human Fc sequence.
TABLE-US-00002 TABLE 1 Anti-IL-17/IL-20 FIT-Ig molecules and DNA constructs. FIT-Ig Construct Construct Construct Construct molecule #1 Linker #2 #3 #4 FIT1-Ig VL.sub.17-CL-VH.sub.20-CH1-Fc No linker VH.sub.17-CH1 VL.sub.20-CL FIT2-Ig VL.sub.17-CL-linker-VH.sub.20- GSG VH.sub.17-CH1 VL.sub.20-CL CH1-Fc FIT3-Ig VL.sub.17-CL-linker-VH.sub.20- GGGGSGS VH.sub.17-CH1 VL.sub.20-CL CH1-Fc FIT4-Ig VL.sub.17-CL-VH.sub.20-CH1-Fc No linker VH.sub.17-CH1-VL.sub.20-CL FIT5-Ig VL.sub.17-CL-linker-VH.sub.20- GSG VH.sub.17-CH1-linker-VL.sub.20- CH1-Fc CL FIT6-Ig VL.sub.17-CL-linker-VH.sub.20- GGGGSGS VH.sub.17-CH1-linker-VL.sub.20- CH1-Fc CL
[0402] For construct #2 cloning, LY VH-CH1 was amplified by PCR using forward primers annealing on heavy chain signal peptide and reverse primer annealing on C-terminal of CH1. The PCR product was gel purified before cloning into 293 expression vector.
[0403] For construct #3, 15D32 light chain was amplified by PCR using forward primer annealing on N-terminal of light chain signal peptide and reverse primer annealing on the end of CL. The PCR product was gel purified before cloning into 293 expression vector.
[0404] For construct #4 cloning, LY VH-CH1 was amplified by PCR using forward primer annealing on N-terminus of heavy chain signal peptide and reverse primer annealing on the end of CH1. 151D2 VL was amplified using primers annealing on the end of 151D2 VL. Both PCR products were gel purified and combined by overlap PCR. The combined PCR product was gel purified and cloned in 293 expression vector. Table 2 shows sequences of PCR primers used for above molecular cloning.
TABLE-US-00003 TABLE 2 PCR primers used for molecular construction of anti-IL-17/anti-CD20 FIT-Igs P1: 5′ CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAG 3′ SEQ ID NO. 1 P2: 5′GCTGGACCTGAGAGCCTGAACCGCCACCACCACACTCTCCCCTGTTGAAGC 3′ SEQ ID NO. 2 P3: 5′ GGTGGTGGCGGTTCAGGCTCTCAGGTCCAGCTTGTGCAATCTGGCGCCGAGG3′ SEQ ID NO. 3 P4: 5′ GTCTGCGGCCGCTCATTTACCCGGAGACAGGGAGAG 3′ SEQ ID NO. 4 P5: 5′ TAAGCGTACGGTGGCTGCACCATCTGTCTTC 3′ SEQ ID NO. 5 P6: 5′ SEQ ID NO. 6 CGGCGCCAGATTGCACAAGCTGGACCTGGCCTGAACCACACTCTCCCCTGTTGAAGCTC 3′ P7: 5′ SEQ ID NO. 7 GCTGGACCTGAGAGCCTGAACCGCCACCACCACACTCTCCCCTGTTGAAGC3′ P8: 5′ SEQ ID NO. 8 GGTGGTGGCGGTTCAGGCTCTCAGGTCCAGCTTGTGCAATCTGGCGCCGAGG3′ P9: 5′ SEQ ID NO. 9 TACCTCGGCGCCAGATTGCACAAGCTGGACCTGACACTCTCCCCTGTTGAAGCTCTTTG 3′ P10: 5′ SEQ ID NO. CATGACACCTTAACAGAGGCCCCAGGTCGTTTTACCTCGGCGCCAGATTGCACAAG3′ 10 P11: 5′ CAATAAGCTTTACATGACACCTTAACAGAGGCCCCAG3′ SEQ ID NO. 11 P12: 5′ TCGAGCGGCCGCTCAACAAGATTTGGGCTCAACTTTCTTG3′ SEQ ID NO. 12 P13: 5′GCTGCTGCTGTGGTTCCCCGGCTCGCGATGCGCTATACAGTTGACACAGTC3′ SEQ ID NO. 13 P14: 5′ SEQ ID NO. GAAGATGAAGACAGATGGTGCAGCCACCGTACGCTTGATCTCTACCTTTGTTC 3′ 14
[0405] The final sequences of hIL-17/hIL-20 FIT1-Ig, FIT2-Ig, FIT3-Ig, FIT4-Ig, FIT5-Ig, and FIT6-Ig are listed in Table 3.
TABLE-US-00004 TABLE 3 Amino acid sequences of anti-IL-17/IL-20 FIT-Ig molecules Sequence Sequence Protein Protein region Identifier 12345678901234567890 Anti-IL-17/IL-20 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY FIT1-Ig NO.: 15 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR POLYPEPTIDE #1 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECQVQLVQSGAEVKRPGASVKVSCKASGY TFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQDRV SITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPH DYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY NO.: 16 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Linker None 15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAP NO.: 18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTV TVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.: 20 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK Anti-IL-17/IL-20 SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP FIT1-Ig NO.: 21 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME POLYPEPTIDE #2 LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSC LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.: 22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Anti-IL-17/IL-20 SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG FIT1-Ig NO.: 23 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED POLYPEPTIDE #3 FATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG NO.: 24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQFNSYPLTFGGGTKVEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Anti-IL-17/IL-20 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY FIT2-Ig NO.: 25 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR POLYPEPTIDE #1 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGSGQVQLVQSGAEVKRPGASVKVSCKA SGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQ DRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGES SPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY NO.: 16 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Linker SEQ ID GSG NO.: 26 15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAP NO.: 18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTV TVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.: 20 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK Anti-IL-17/IL-20 SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP FIT2-Ig NO.: 21 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME POLYPEPTIDE #2 LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSC LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.: 22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Anti-IL-17/IL-20 SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG FIT2-Ig NO.: 23 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED POLYPEPTIDE #3 FATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG NO.: 24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQFNSYPLTFGGGTKVEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Anti-IL-17/IL-20 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY FIT3-Ig NO.: 27 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR POLYPEPTIDE #1 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGGGGSGSQVQLVQSGAEVKRPGASVKV SCKASGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYS QNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLW FGESSPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY NO.: 16 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Linker SEQ ID GGGGSGS NO.: 28 15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAP NO.: 18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTV TVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.: 20 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK Anti-IL-17/IL- SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP 20 FIT3-Ig NO.: 21 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME POLYPEPTIDE LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTK #2 GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSC LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.: 22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Anti-IL-17/IL- SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG 20 FIT3-Ig NO.: 23 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED POLYPEPTIDE FATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDE #3 QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG NO.: 24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQFNSYPLTFGGGTKVEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Anti-IL-17/IL-20 FIT SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY 4-Ig POLYPEPTIDE NO.: 15 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR #1 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECQVQLVQSGAEVKRPGASVKVSCKASGY TFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQDRV SITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPH DYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY NO.: 16 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Linker None 15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAP NO.: 18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTV TVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.: 20 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK Anti-IL-17/IL-20 SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP FIT4-Ig NO.: 29 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME POLYPEPTIDE #4 LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCAIQLTQSPSSLSASVGDRVTITCR ASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.: 22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Linker none 15D2VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG NO.: 24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQFNSYPLTFGGGTKVEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Anti-IL-17/IL-20 FIT SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY 5-Ig POLYPEPTIDE NO.: 25 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR #1 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGSGQVQLVQSGAEVKRPGASVKVSCKA SGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQ DRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGES SPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY NO.: 16 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Linker SEQ ID GSG NO.: 26 15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAP NO.: 18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTV TVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.: 20 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK Anti-IL-17/IL-20 SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP FIT5-Ig NO.: 30 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME POLYPEPTIDE #4 LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGSGAIQLTQSPSSLSASVGDRVTI TCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.: 22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Linker SEQ ID GSG NO.: 26 15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG NO.: 24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQFNSYPLTFGGGTKVEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Anti-IL-17/IL-20 FIT SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY 6-Ig POLYPEPTIDE NO.: 27 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR #1 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGGGGSGSQVQLVQSGAEVKRPGASVKV SCKASGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYS QNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLW FGESSPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY NO.: 16 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC Linker GGGGSGS 15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTNDIIHWVRQAP NO.: 18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTV TVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.: 20 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK Anti-IL-17/IL-20 SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP FIT6-Ig NO.: 31 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME POLYPEPTIDE #4 LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGSAIQLTQSPSSLSASVGD RVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFG GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.: 22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.: 19 NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC Linker SEQ ID GGGGSGS NO.: 28 15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG NO.: 24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQFNSYPLTFGGGTKVEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW NO.: 17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC
Example 1.2: Expression, Purification, and Analysis of Anti-IL-17/IL-20 FIT-Ig Proteins
[0406] All DNA constructs of each FIT-Ig were subcloned into pBOS based vectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 of each FIT-Ig (1, 2, and 3), or construct #1 and #4 of each FIT-Ig (4, 5, and 6) were transiently co-expressed using Polyethyleneimine (PEI) in 293E cells. Briefly, DNA in FreeStyle™ 293 Expression Medium was mixed with the PEI with the final concentration of DNA to PEI ratio of 1:2, incubated for 15 min (no more than 20 min) at room temperature, and then added to the 293E cells (1.0-1.2×10.sup.6/ml, cell viability >95%) at 60 μg DNA/120 ml culture. After 6-24 hours culture in shaker, peptone was added to the transfected cells at a final concentration of 500, with shaking at 125 rpm/min., at 37° C., 8% CO.sub.2. On the 6th-7th day, supernatant was harvested by centrifugation and filtration, and FIT-Ig protein was purified using protein A chromatography (Pierce, Rockford, Ill.) according to the manufacturer's instructions. The proteins were analyzed by SDS-PAGE and their concentrations determined by A280 and BCA (Pierce, Rockford, Ill.).
[0407] For the expression of FIT1-Ig, FIT2-Ig, and FIT3-Ig, different DNA molar ratios of the 3 constructs were used, including construct #1:#2:#3=1:1:1, construct #1:#2:#3=1:1.5:1.5, and construct #1:#2:#3=1:3:3 (Table 4). FIT-Ig proteins were purified by protein A chromatography. The purification yield (7-16 mg/L) was consistent with hIgG quantification of the expression medium for each protein. The composition and purity of the purified FIT-Igs were analyzed by SDS-PAGE in both reduced and non-reduced conditions. In non-reduced conditions, FIT-Ig migrated as a single band of approximately 250 KDa. In reducing conditions, each of the FIT-Ig proteins yielded two bands, one higher MW band is construct #1 of approximately 75 KDa, and one lower MW band corresponds to both construct #2 and #3 overlapped at approximately 25 KDa. The SDS-PAGE showed that each FIT-Ig is expressed as a single species, and the 3 polypeptide chains are efficiently paired to form an IgG-like molecule. The sizes of the chains as well as the full-length protein of FIT-Ig molecules are consistent with their calculated molecular mass based on amino acid sequences.
TABLE-US-00005 TABLE 4 Expression and SEC analysis of hIL-17/IL-20 FIT-Ig proteins DNA ratio: Expression level % Peak monomeric FIT-Ig protein Construct 1:2:3 (mg/L) fraction by SEC FIT1-Ig 1:1:1 15.16 92.07 1:1.5:1.5 14.73 95.49 1:3:3 9.87 97.92 FIT2-Ig 1:1:1 15.59 90.92 1:1.5:1.5 12.61 94.73 1:3:3 7.03 97.29 FIT3-Ig 1:1:1 15.59 91.47 1:1.5:1.5 15.16 94.08 1:3:3 7.75 97.57
[0408] To further study the physical properties of FIT-Ig in solution, size exclusion chromatography (SEC) was used to analyze each protein. For SEC analysis of the FIT-Ig, purified FIT-Ig, in PBS, was applied on a TSKgel SuperSW3000, 300×4.6 mm column (TOSOH). An HPLC instrument, Model U3000 (DIONEX) was used for SEC. All proteins were determined using UV detection at 280 nm and 214 nm. The elution was isocratic at a flow rate of 0.25 mL/min. All 3 FIT-Ig proteins exhibited a single major peak, demonstrating physical homogeneity as monomeric proteins (Table 4). The ratio of construct #1:#2:#3=1:3:3 showed a better monomeric profile by SEC for all 3 FIT-Ig proteins (Table 4).
[0409] Table 4 also shows that the expression levels of all the FIT-Ig proteins are comparable to that of the regular mAbs, indicating that the FIT-Ig can be expressed efficiently in mammalian cells. For the expression of FIT4-Ig, FIT5-Ig, and FIT6-Ig, the DNA ration of construct #1:#4=1:1, and the expression level were in the range of 1-10 mg/L, and the % Peak monomeric fraction as determined by SEC was in the range of 58-76%. Based on this particular mAb combination (LY and 15D2), the 3-polypeptide FIT-Ig constructs (FIT1-Ig, FIT2-Ig, and FIT3-Ig) showed better expression profile than that of the 2-polypeptide FIT-Ig constructs (FIT4-Ig, FIT5-Ig, and FIT6-Ig), therefore FIT1-Ig, FIT2-Ig, and FIT3-Ig were further analyzed for functional properties
Example 1.3 Determination of Antigen Binding Affinity of Anti-IL-17/IL-20 FIT-Igs
[0410] The kinetics of FIT-Ig binding to rhIL-17 and rhIL-20 was determined by surface plasmon resonance (Table 5) with a Biacore X100 instrument (Biacore AB, Uppsala, Sweden) using HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25° C. Briefly, goat anti-human IgG Fcγ fragment specific polyclonal antibody (Pierce Biotechnology Inc, Rockford, Ill.) was directly immobilized across a CM5 research grade biosensor chip using a standard amine coupling kit according to manufacturer's instructions. Purified FIT-Ig samples were diluted in HEPES-buffered saline for capture across goat anti-human IgG Fc specific reaction surfaces and injected over reaction matrices at a flow rate of 5 μl/min. The association and dissociation rate constants, kon (M-1s-1) and koff (s-1) were determined under a continuous flow rate of 30 μL/min. Rate constants were derived by making kinetic binding measurements at ten different antigen concentrations ranging from 1.25 to 1000 nM. The equilibrium dissociation constant (M) of the reaction between FIT-Ig and the target proteins was then calculated from the kinetic rate constants by the following formula: KD=koff/kon. Aliquots of antigen samples were also simultaneously injected over a blank reference and reaction CM surface to record and subtract any nonspecific binding background to eliminate the majority of the refractive index change and injection noise. Surfaces were regenerated with two subsequent 25 ml injections of 10 mM Glycine (pH 1.5) at a flow rate of 10 μL/min. The anti-Fc antibody immobilized surfaces were completely regenerated and retained their full capture capacity over twelve cycles.
TABLE-US-00006 TABLE 5 Functional characterizations of anti-IL-17/IL-20 FIT-Ig molecules Binding Kinetics by Biacore Neutralization mAb or k.sub.on k.sub.off K.sub.d Potency FIT-Ig Antigen (M.sup.−1 s.sup.−1) (s.sup.−1) (M) IC.sub.50 (pM) LY hIL-17 8.24E+5 1.80E−5 2.18E−11 101 FIT1-Ig hIL-17 1.07E+7 3.88E−5 3.64E−12 102 FIT2-Ig hIL-17 9.24E+6 1.53E−5 1.65E−12 137 FIT3-Ig hIL-17 8.71E+6 9.58E−6 1.10E−12 146 15D2 hIL-20 1.70E+6 8.30E−5 5.00E−11 50 FIT1-Ig hIL-20 1.40E+6 3.82E−5 2.73E−11 54 FIT2-Ig hIL-20 1.80E+6 3.50E−5 1.95E−11 50 FIT3-Ig hIL-20 1.40E+6 3.82E−5 2.73E−11 72
[0411] The Biacore analysis indicated the overall binding parameters of the three FIT-Igs to hIL-17 and hIL-20 were similar, with the affinities of the FIT-Igs being very close to that of the parental mAb LY and 15D2, and there was no lose of binding affinities for either antigen binding domains (Table 5).
[0412] In addition, tetravalent dual-specific antigen binding of FIT-Ig was also analyzed by Biacore. FIT1-Ig was first captured via a goat anti-human Fc antibody on the Biacore sensor chip, and the first antigen was injected and a binding signal observed. As the FIT1-Ig was saturated by the first antigen, the second antigen was then injected and the second signal observed. This was done either by first injecting IL-17 then IL-20 (
[0413] The expression profile and dual-binding properties of FIT-Ig clearly demonstrated that, within the FIT-Ig molecule, both VL-CL paired correctly with their corresponding VH-CH1 to form 2 functional binding domains, and expressed as a single monomeric, tetravalent, and bispecific full length FIT-Ig protein. This is in contrast to the multivalent antibody type of molecules (Miller and Presta, U.S. Pat. No. 8,722,859), which displayed tetravalent but mono-specific binding activities to one target antigen.
Example 1.4 Determination of Biological Activity of Anti-IL-17/IL-20 FIT-Ig
[0414] The biological activity of FIT-Ig to neutralize IL-17 function was measured using GROα, bioassay. Briefly, Hs27 cells were seeded at 10000 cells/50 μL/well into 96 well plates. FIT-Ig or anti-IL-17 control antibody (25 μL) were added in duplicate wells, with starting concentration at 2.5 nM followed by 1:2 serial dilutions until 5 pM. IL-17A (25 μL) was then added to each well. The final concentration of IL-17A was 0.3 nM. Cells were incubated at 37° C. for 17 h before cell culture supernatant were collected. Concentrations of GRO-α, in cell culture supernatants were measured by human CACL1/GRO alpha Quantikine kit according to the manufacturer's protocol (R&D systems).
[0415] The biological activity of FIT-Ig to neutralize IL-20 function was measured using IL-20R.sup.+ BAF3 cell proliferation assay. Briefly, 25 μL of recombinant human IL-20 at 0.8 nM was added to each well of 96-well plates (the final concentration of IL-20 is 0.2 nM). Anti-IL20 antibody or FIT-Ig or other control antibody were diluted to 400 nM (working concentration was 100 nM) followed by 5-fold serial dilutions and were added to 96-well assay plates (25 μL per well). BaF3 cells stably transfected with IL-20 receptor were then added to each well at concentration of 10000 cell/well in volume of 50 μL RPMI 1640 plus 10% FBS, Hygromycin B at the concentration of 800 μg/ml, G418 at the concentration of 800 μg/ml. After 48-hr incubation, 100 μL CellTiter-Glo Luminescent buffer were added to each well. Contents were mixed for 2 minutes on an orbital shaker to induce cell lysis and plates were incubated at room temperature for 10 minutes to stabilize luminescent signal. Luminescence was recorded by SpectraMax M5.
[0416] As shown in Table 5, all FIT-Igs were able to neutralize both hIL-20 and hIL-17, with affinities similar to that of the paternal antibodies. Based on functional analysis using both Biacore and cell-based neutralization assays, it appears that all 3 FIT-Igs fully retain the activities of the parental mAbs. There was no significant functional difference among the three FIT-Igs, indicating that the linker was optional, and that FIT-Ig construct provided sufficient flexibility and special dimension to allow dual binding in the absence of a peptide spacer between the 2 Fab binding regions. This is in contrast to DVD-Ig type of molecules, where a linker between the 2 variable domains on each of the 2 polypeptide chain is required for retaining activities of the lower (2.sup.nd) variable domain.
Example 1.5 Stability Study of Anti-IL-17/IL-20 FIT-Ig
[0417] FIT1-Ig protein samples in citrate buffer (pH=6.0) were individually incubated at constant 4° C., 25° C. and 40° C. for 1 day, 3 days or 7 days; Similarly, FIT1-Ig protein samples were freeze-thawed once, twice or three times. The fractions of intact full monomeric protein of all samples was detected by SEC-HPLC, with 10 μg of each protein sample injected into Ultimate 3000 HPLC equipping Superdex200 5/150 GL at flow rate 0.3 mL/min for 15 min, and data was recorded and analyzed using Chromeleon software supplied by the manufacturer. Table 6 shows that FIT1-Ig and FIT3-Ig remained full intact monomeric molecule under these thermo-challenged conditions.
TABLE-US-00007 TABLE 6 Stability analysis of FIT-Ig by measuring % full monomeric fractions by SEC Temp. (° C.) Time (day) FIT1-Ig FIT3-Ig 4 0 (Starting) 98.74 98.60 1 98.09 97.78 3 97.81 97.45 7 97.63 97.65 25 1 99.00 98.26 3 99.00 98.01 7 98.86 98.53 40 1 98.95 98.50 3 98.94 98.35 7 98.82 98.37 1X freeze-thaw 98.89 98.21 2X freeze-thaw 95.37 98.21 3X freeze-thaw 95.24 98.35
Example 1.6 Solubility Study of Anti-IL-17/IL-20 FIT-Ig
[0418] The solubility of FIT1-Ig was analyzed by measuring sign of precipitation in the presence of increasing concentration of PEG6000 (PEG6000 was purchased from Shanghai lingfeng chemical reagent co. Ltd). Briefly, solubility of protein in the presence of PEG6000 was obtained as a function of PEG6000 concentration (0, 5%, 10%, 15%, 20%, 25% and 30%). The solubility studies were conducted at a temperature of 25° C. at a solution pH of 6.0. Briefly, protein was precipitated by mixing appropriate quantities of buffered stock solutions of the protein, PEG and the buffer to get the desired concentration of the components. The final volume was made up to 200 μl and the concentration of protein was set at 1.0 mg/mL. The final solutions were mixed well and equilibrated for 16 h. After equilibration, the solutions were centrifuged at 13000 rpm for 10 min to separate the protein precipitate. Protein solubility was measured at 280 nm using Spectra Max Plus384 (Molecular Device) and obtained from the absorbance of the supernatant, and calculating the concentration based on standard curve of protein concentration (
Example 1.7 Pharmacokinetic Study of Anti-IL-17/IL-20 FIT-Ig
[0419] Pharmacokinetic properties of FIT1-Ig were assessed in male Sprague-Dawley (SD) rats. FIT-Ig proteins were administered to male SD rats at a single intravenous dose of 5 mg/kg via a jugular cannula or subcutaneously under the dorsal skin. Serum samples were collected at different time points over a period of 28 days with sampling at 0, 5, 15, and 30 min; 1, 2, 4, 8, and 24 hr; and 2, 4, 7, 10, 14, 21, and 28 day serial bleeding via tail vein, and analyzed by human IL-17 capture and/or human IL-20 capture ELISAs. Briefly, ELISA plates were coated with goat anti-biotin antibody (5 μg/ml, 4° C., overnight), blocked with Superblock (Pierce), and incubated with biotinylated human IL-17 (IL-17 capture ELISA) or IL-20 (IL-20 capture ELISA) at 50 ng/ml in 10% Superblock TTBS at room temperature for 2 h. Serum samples were serially diluted (0.5% serum, 10% Superblock in TTBS) and incubated on the plate for 30 min at room temperature. Detection was carried out with HRP-labeled goat anti human antibody and concentrations were determined with the help of standard curves using the four parameter logistic fit. Several animals, especially in the subcutaneous group, showed a sudden drop in FIT-Ig concentrations following day 10, probably due to developing an anti-human response. These animals were eliminated from the final calculations. Values for the pharmacokinetic parameters were determined by non-compartmental model using WinNonlin software (Pharsight Corporation, Mountain View, Calif.).
[0420] The rat PK study, FIT1-Ig serum concentrations were very similar when determined by the two different ELISA methods, indicating that the molecule was intact, and capable of binding both antigens in vivo. Upon IV dosing, FIT1-Ig exhibited a bi-phasic pharmacokinetic profile, consisting of a distribution phase followed by an elimination phase, similar to the PK profile of conventional IgG molecules. The pharmacokinetic parameters calculated based on the two different analytical methods were very similar and are shown in Table 7. Clearance of FIT-Ig was low (.sup.˜12 mL/day/kg), with low volumes of distribution (Vss.sup.˜130 mL/kg) resulting in a long half-life (T½>10 days). Following subcutaneous administration, FIT-Ig absorbed slowly, with maximum serum concentrations of approximately 26.9 μg/ml reached at 4 days post-dose. The terminal half-life was about 11 days and the subcutaneous bioavailability was close to 100%. As demonstrated by these results, the properties of FIT1-Ig are very similar to a conventional IgG molecule in vivo, indicating a potential for therapeutic applications using comparable dosing regimens.
[0421] The pharmacokinetics study of FIT-Ig has demonstrated a surprising breakthrough in the field of multi-specific Ig-like biologics development. The rat pharmacokinetic system is commonly used in the pharmaceutical industry for preclinical evaluation of therapeutic mAbs, and it well predicts the pharmacokinetic profile of mAbs in humans. The long half-life and low clearance of FIT-Ig will enable its therapeutic utility for chronic indications with less frequent dosing, similar to a therapeutic mAb. In addition, FIT-Ig, being 100-kDa larger than an IgG, seemed to penetrate efficiently into the tissues based on its IgG-like volume of distribution parameter from the PK study.
TABLE-US-00008 TABLE 7 Pharmacokinetics analysis of FIT1-Ig in SD Rats IV PK parameters CL Vss Beta t.sub.1/2 AUC MRT Unit mL/day/kg mL/kg Day Day × μg/mL Day IL-17 ELISA 12.2 131 10.8 411 10.7 IL-20 ELISA 11.9 128 10.8 421 10.7 SC PK parameters T.sub.max C.sub.max t.sub.1/2 AUC.sub.INF CL/F F Unit Day ug/mL Day Day × ug/mL mL/day/kg % IL-17 ELISA 4.00 26.9 11.0 406 12.4 103.5 IL-20 ELISA 4.00 23.1 10.4 350 14.3 86.4
Example 1.8 Stable CHO Cell Line Development Studies of FIT-Ig
[0422] It has been observed that FIT-Ig was efficiently expressed in transiently-transfected 293E cells. In order to further determine the manufacturing feasibility of FIT-Ig, stable transfections were carried out in both CHO-DG44 and CHO-S cell lines, and subsequent clone selections as well as productivity analysis were performed. Briefly, CHO cells were transfected by electroporation with 8×10.sup.6 cells in 400 μl transfection solution plus 20 ug DNA (for CHO DG44 cells) or 25 μg DNA (for CHO-S cells) subcloned in Freedom pCHO vector (Life Technologies). The stable cell line selection was done using routine procedures. Briefly, for CHO-DG44 selection, upon transfection, stable pool was selected (−HT/2P/400G, where P is μg/mL Puromycin, G is g/mL G418), and protein production was analyzed by IgG ELISA. Top pools were selected and proceed to amplification for several rounds with increasing concentration of MTX (50, 100, 200 and 500 nM), followed by analysis of protein production by IgG ELISA. The top pools were then selected for subcloning. For CHO-S cell selection, the first phase selection was performed in medium containing 10P/400G/100M (M is nM MTX), followed by analysis of protein production. Then the top pools were selected and proceed to 2.sup.nd phase selection in either 30P/400G/500M or 50P/400G/1000M, followed by protein production measurement by ELISA. The top pools were then selected for subcloning. For protein productivity analysis, fully recovered cell pools (viability >90%) were seeded at 5×10.sup.5 viable cells/mL (CHO DG44) or 3×10.sup.5 viable cells/mL (CHO-S) using 30 mL fresh medium (CD FortiCHO™ medium supplemented with 6 mM L-glutamine) in 125-mL shake flasks. The cells were incubated on a shaking platform at 37° C., 80% relative humidity, 8% CO2, and 130 rpm. Sample cultures daily or at regular intervals (e.g., on day 0, 3, 5, 7, 10, 12, and 14) to determine the cell density, viability, and productivity until culture viability drops below 50% or day 14 of culture is reached. After sampling, feed the cultures with glucose as needed.
[0423] The overall process of FIT1-Ig CHO stable cell line development showed features similar to that of a monoclonal antibody development in CHO cells. For example, during DG44 pool analysis under 2P/400G, the VCD continued to increase until day 10-12 up to about 1.3E7, whereas cell viability remained above 80% up to day 13-14, and the productivity reached almost 40 mg/mL on day 14. Upon amplification at 5P/400G/50M, productivity reached above 50 mg/mL on day 14. For CHO-S cell selection, the titer reached above 200 mg/mL during the phase 1 selection, and above 370 mg/mL at the phase 2 selection. These levels of productivity are similar to what have been previously observed for regular human mAb development is our laboratory, suggesting that FIT-Ig display mAb-like manufacturing feasibility for commercial applications.
Example 2: Construction, Expression, and Purification of Anti-CD3/CD20 Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0424] To demonstrate if a FIT-Ig can bind to cell surface antigens, we have generated an anti-CD3/CD20 FIT-Ig molecule FIT7-Ig and FIT8-Ig, which is the 3-polypeptide construct, as shown in
Example 2.1 Molecular Cloning of Anti-CD3/CD20 FIT-Ig
[0425] The molecular cloning method is similar as that for anti-hIL-17/hIL-20 FIT-Ig.
TABLE-US-00009 TABLE 8 Anti-CD3/CD20 FIT-Ig molecules and constructs. FIT-Ig Construct Construct molecule Construct #1 Linker #2 #3 FIT7-Ig VL.sub.CD3-CL- No linker VH.sub.CD3-CH1 VL.sub.CD20-CL VH.sub.CD20-CH1-Fc FIT8-Ig VL.sub.CD3-CL-linker- GGGGSGS VH.sub.CD3-CH1 VL.sub.CD20-CL VH.sub.CD20-CH1-Fc
[0426] Table 9 shows sequences of PCR primers used for molecular construction above.
TABLE-US-00010 TABLE 9 PCR primers used for molecular construction of anti-IL-17/IL-20 FIT-Igs SEQ ID NO. P4: GTCTGCGGCCGCTCATTTACCCGGAGACAGGGAGAG 32 P12: TCGAGCGGCCGCTCAACAAGATTTGGGCTCAACTT 33 TCTTG P20: CAGGTCCAGCTGCAGCAGTCTG 34 P22: GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCC 35 CGTCACAAAGAGCTTCAACAGGGG P23: TACGAGAAACACAAAGTCTACGCCTGCGAAGTCAC 36 CCATCAGGGCCTGAG P24: TGACGCTGAGCAAAGCAGACTACGAGAAACACAAA 37 GTCTACGCCTGCGAA P25: CTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT 38 GTGAAGTGCAGCTGGTGGAGTCTG P28: GCTGCTGCTGTGGTTCCCCGGCTCGCGATGCGAAA 39 TTGTGTTGACACAGTC P29: AAGATGAAGACAGATGGTGCAGCCACCGTACGTTT 40 AATCTCCAGTCGTGTCC
[0427] The final sequences of anti-CD3/CD20 FIT-Ig are described in Table 10.
TABLE-US-00011 TABLE 10 Amino acid sequences of anti-CD3/CD20 FIT-Ig Sequence Sequence Protein Protein region Identifier 12345678901234567890 OKT3/Ofatumumab SEQ ID QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKS FIT7-Ig POLYPEPTIDE NO.: 41 GTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGME #1 AEDAATYYCQQWSSNPFTFGSGTKLEINRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECEVQLVESGGGLVQPGRSLRLS CAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGY ADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKD IQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK OKT3 VL SEQ ID QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKS NO.: 42 GTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGME AEDAATYYCQQWSSNPFTFGSGTKLEIN CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV NO.: 17 QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC Linker none Ofatumumab VH SEQ ID EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQ NO.: 43 APGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKS LYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTT VTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV NO.: 19 SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV NO.: 20 TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK OKT3/Ofatumumab SEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ FIT7-Ig POLYPEPTIDE NO.: 44 RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSST #2 AYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSC OKT3 VH SEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ NO.: 45 RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSST AYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTV SS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV NO.: 19 SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC OKT3/Ofatumumab SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK FIT7-Ig POLYPEPTIDE NO.: 46 PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL #3 EPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC Ofatumumab VL SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK NO.: 47 PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWPITFGQGTRLEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV NO.: 17 QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC OKT3/Ofatumumab SEQ ID QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKS FIT8-Ig POLYPEPTIDE NO.: 48 GTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGME #1 AEDAATYYCQQWSSNPFTFGSGTKLEINRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECGGGGSGSEVQLVESGGGLVQP GRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISW NSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTA LYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK OKT3 VL SEQ ID QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKS NO.: 42 GTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGME AEDAATYYCQQWSSNPFTFGSGTKLEIN CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV NO.: 17 QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC Linker SEQ ID GGGGSGS NO.: 28 Ofatumumab VH EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQ APGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKS LYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTT VTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV NO.: 19 SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV NO.: 20 TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK OKT3/Ofatumumab SEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ FIT8-Ig POLYPEPTIDE NO.: 44 RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSST #2 AYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSC OKT3 VH SEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ NO.: 45 RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSST AYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTV SS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV NO.: 19 SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC OKT3/Ofatumumab SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK FIT8-Ig POLYPEPTIDE NO.: 46 PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL #3 EPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC Ofatumumab VL SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK NO.: 47 PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWPITFGQGTRLEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV NO.: 17 QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC
Example 2.2 Expression and Purification of Anti-CD3/CD20 FIT-Ig
[0428] All DNA constructs of each FIT-Ig were subcloned into pBOS based vectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 of each FIT-Ig were transiently co-expressed using Polyethyleneimine (PEI) in 293E cells. Briefly, DNA in FreeStyle™ 293 Expression Medium was mixed with the PEI with the final concentration of DNA to PEI ratio of 1:2, incubated for 15 min (no more than 20 min) at room temperature, and then added to the 293E cells (1.0-1.2×10.sup.6/ml, cell viability >95%) at 60 μg DNA/120 ml culture. After 6-24 hours culture in shaker, add peptone to the transfected cells at a final concentration of 5%, with shaking at 125 rpm/min., at 37° C., 8% CO.sub.2. On the 6th-7th day, supernatant was harvested by centrifugation and filtration, and FIT-Ig protein purified using protein A chromatography (Pierce, Rockford, Ill.) according to manufacturer's instructions. The proteins were analyzed by SDS-PAGE and their concentration determined by A280 and BCA (Pierce, Rockford, Ill.) (Table 11).
TABLE-US-00012 TABLE 11 Expression and SEC analysis of anti-CD3/CD20 FIT-Ig proteins DNA ratio: Expression % Peak monomeric FIT-Ig protein Construct 1:2:3 level (mg/L) fraction by SEC FIT7-Ig 1:3:3 21.3 99.53 FIT8-Ig 1:3:3 25.6 99.16
Example 2.3 Binding Activities of Anti-CD3/CD20 FIT-Ig Molecules
[0429] Binding of anti-CD3/CD20 FIT-Igs to both targets were analyzed by FACS, using Jurkat cells that express CD3 on the cell surface, as well as Raji cells that express CD20 on the cell surface. Briefly, 5×10.sup.5 cells were washed in ice-cold PBS and blocked with 2% FBS on ice for 1 hr. Cells were incubated with antibody, FIT-Ig (100 nM), or isotype control on ice for 1 hr and washed 3 times with PBS. Secondary antibody (goat anti-human IgG labeled with Alexa Fluor 488, Invitrogen) were added and incubated with cells on ice for 1 hr in dark followed by three times wash with PBS. Samples were analyzed in FACs calibur. The cell surface binding shows that both FIT7-Ig and FIT8-Ig were able to binding to both cell surface antigens CD3 and CD20 in a concentration dependent manner. Compared to the binding activities of the parental mAbs, FIT-Ig showed a reduced binding intensity to CD3 on Jurkat cells, but an enhanced binding intensity to CD20 on Raji cells. In all binding studies, FIT7-Ig and FIT8-Ig showed similar binding activities to both antigens, indicating the linker did not make a significant impact on its binding ability for FIT8-Ig (Table 12).
TABLE-US-00013 TABLE 12 Cell surface antigen binding studies of anti-CD3/CD20 FIT-Ig proteins Binding Intensity by FIT-Ig protein Antigen (cell line) FACS (MFI) OKT3 CD3 (Jurkat) 399 FIT7-Ig 159 FIT8-Ig 211 Ofatumumab CD20 (Raji) 181 FIT7-Ig 291 FIT8-Ig 274
Example 3: Construction, Expression, and Purification of Anti-TNF/IL-17 Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0430] Another FIT-Ig that can bind to human IL-17 and human TNFα (FIT9-Ig) was also generated using anti-IL-17 mAb clone LY, and anti-TNF mAb Golimumab, in the 3-polypeptide construct, as shown in
Example 3.1 Molecular Cloning of Anti-TNF/IL-17 FIT-Ig
[0431] The molecular cloning method is similar as that for anti-hIL-17/hIL-20 FIT-Ig.
TABLE-US-00014 TABLE 13 Anti-TNF/IL-17 FIT-Ig molecule and constructs. FIT-Ig molecule Construct #1 Linker Construct #2 Construct #3 FIT9-Ig VL.sub.TNF-CL- No linker VH.sub.TNF-CH1 VL.sub.IL-17-CL VH.sub.IL-17-CH1-Fc
TABLE-US-00015 TABLE 14 Amino acid sequences of anti-TNF/IL-17 FIT-Ig molecules SEQ ID Sequence Protein Protein region NO: 12345678901234567890 Anti-IL-TNF/IL-17 SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQK FIT9-Ig NO.: 87 PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL POLYPEPTIDE #1 EPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGECQVQLVQSGAEVKKPGSSVK VSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYGTT DYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCA RYDYFTGTGVYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K GOLIMUMAB VL SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQK NO.: 88 PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWPPFTFGPGTKVDIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV NO.: 17 QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC Linker None LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQ NO.: 22 APGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTST AYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTV SS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV NO.: 19 SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV NO.: 20 TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Anti-TNF/IL-17 SEQ ID QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQ FIT9-Ig NO.: 89 APGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSKNT POLYPEPTIDE #2 LYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC GOLIMUMAB VH SEQ ID QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQ NO.: 90 APGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWG QGTTVTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV NO.: 19 SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSC Anti-IL-TNF/IL-17 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLH FIT9-Ig NO.: 91 WYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTL POLYPEPTIDE #3 KISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLH NO.: 16 WYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV NO.: 17 QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC
Example 3.2 Expression, Purification, and Analysis of Anti-TNF/IL-17 FIT-Ig Proteins
[0432] All DNA constructs of each FIT-Ig were subcloned into pBOS based vectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 of FIT9-Ig were transiently co-expressed using Polyethyleneimine (PEI) in 293E cells as described previously and FIT9-Ig proteins were purified by protein A chromatography. The expression level was 10-23 mg/L. The purified protein was subjected to functional analysis using cell-based assays for IL-17 (production of GROα by Hs27 cells) and TNF (production of IL-8 by L929 cells). The neutralization potency of FIT9-Ig against human TNF was 11.6 pM (compared to 15.9 pM by Golimumab in the same experiment), as against human IL-17 was 122 pM (compared to 51.5 pM by LY in the same experiment). Overall FIT9-Ig maintained the biological activities of the parental mAbs.
Example 4: Construction, Expression, and Purification of Anti-CTLA-4/PD-1 Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0433] Another FIT-Ig that can bind to human CTLA-4 and human PD-1 (FIT10-Ig) was generated using anti-CTLA-4 mAb Ipilimumab, and anti-PD-1 mAb Nivolumab, in the 3-polypeptide construct, as shown in
Example 4.1 Molecular Cloning of Anti-CTLA-4/PD-1 FIT-Ig
[0434] The molecular cloning method is similar as that for anti-hIL-17/hIL-20 FIT-Ig. The final sequences of anti-CTLA-4/PD-1 FIT-Ig are described in Table 16.
TABLE-US-00016 TABLE 15 Anti-CTLA-4/PD-1 FIT-Ig molecule and constructs. FIT-Ig molecule Construct #1 Linker Construct #2 Construct #3 FIT10-Ig VL.sub.CTLA-4-CL- No linker VH.sub.CTLA-4-CH1 VL.sub.PD-1-CL VH.sub.PD-1-CH1-Fc
TABLE-US-00017 TABLE 16 Amino acid sequences of anti-CTLA-4/PD-1 FIT-Ig molecules SEQ ID Sequence Protein Protein region NO: 12345678901234567890 Anti-CTLA-4/PD-1 SEQ ID EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQ FIT10-Ig NO.: 92 APRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV POLYPEPTIDE #1 YYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGK GLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSL RAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK IPILIMUMAB VL SEQ ID EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQ NO.: 93 APRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV YYCQQYGSSPWTFGQGTKVEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV NO.: 17 DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC Linker None NIVOLUMAB VH SEQ ID QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGK NO.: 94 GLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSL RAEDTAVYYCATNDDYWGQGTLVTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS NO.: 19 GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSC Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV NO.: 20 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK Anti-CTLA-4/PD-1 SEQ ID QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGK FIT10-Ig NO.: 95 GLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSL POLYPEPTIDE #2 RAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSC IPILIMUMAB VH SEQ ID QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGK NO.: 96 GLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS CH1 SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS NO.: 19 GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSC Anti-CTLA-4/PD-1 SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA FIT10-Ig NO.: 97 PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY POLYPEPTIDE #3 YCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC NivolumabVL SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA NO.: 98 PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY YCQQSSNWPRTFGQGTKVEIK CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV NO.: 17 DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC
Example 4.2 Expression, Purification, and Functional Analysis of Anti-CTLA-4/PD-1 FIT-Ig Proteins
[0435] All DNA constructs of each FIT-Ig were subcloned into pBOS based vectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 of FIT10-Ig were transiently co-expressed using Polyethyleneimine (PEI) in 293E cells as described previously and FIT9-Ig proteins were purified by protein A chromatography to 9800 monomeric full protein. The expression levels were up to 43 mg/L. The purified protein was subjected to binding analysis using ELISA against recombinant CTLA-4Ig and PD-1. Briefly, for binding to CTLA-4, human CTLA-4Ig (R&D Systems) was immobilized on 96-well plates, followed by routine wash and blocking procedures. Then FIT-10-Ig or Ipilimumab at various concentrations were added to the plate, followed by incubation and multiple wash steps, and detected with anti-human Fab-HRP. For binding to PD-1, human PD-1 (with a his tag) (R&D Systems) was immobilized on 96-well plates, followed by routine wash and blocking procedures. Then FIT-10-Ig or Nivolumab at various concentrations were added to the plate, followed by incubation and multiple wash steps, and detected with anti-human Fc-HRP (
[0436] In addition, multiple-antigen binding study was done using OctetRed to determine if FIT10-Ig was able to bind recombinant CTLA-4 and PD-1 simultaneously. Briefly, FIT10-Ig was immobilize on AR2G sensor at concentration of 10 μg/ml, followed by binding of CTLA-4Ig and then PD-1 (or PD-1 first, then CTLA-4Ig) in assay buffer (PBS pH 7.4, 0.1% BSA, 0.02% Tween), with concentration at 80 nM. At the end of the experiment, the surface was regenerated with 10 mM glycine at pH1.5 five times (
Example 5: Construction, Expression, and Purification of Additional Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0437] FIT-Ig having specificity for EGFR and PD-L1; cMet and EGFR; Factor IXa and Factor X; Her3 and IGF-1R; DLL-4 and VEGF; CD20 and CD3; Her3 and EGFR; PD-1 and PD-L1; and Her3 and PD-1 were constructed as in the foregoing Examples. These exemplary FIT-Ig and their corresponding sequences are provided below in Table 17. Table 18 provides the expression level in 293E cells and the SEC profile for each of the FIT-Ig.
TABLE-US-00018 TABLE 17 Amino acid sequences of additional exemplary FIT-Ig Name SEQ Target Protein ID (mAb) region NO Sequence FIT12a-Ig Pani VL-hCk- 99 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTI EGFR 1B12VH-hCg1 TCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSG (panitumumab)/ SGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK PD-L1 (1B12) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECQVQLVQSGAEVKKPGSSVKVSC KTSGDTFSSYAISWVRQAPGQGLEWMGGIIPIFGRAHYAQKFQ GRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPF GMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K Pani VH-CH1h 100 MEFGLSWLFLVAILKGVQCQVQLQESGPGLVKPSETLSLTCTV SGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKS RLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWG QGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSC 1B12 VL-hCk 101 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGERATL SCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC FIT13a-Ig h1332VL-hCk- 102 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTI cMet PaniVH-hCg1 TCRASQGINTWLAWYQQKPGKAPKLLIYAASSLKSGVPSRFSG (h1332)/EGFR SGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK (panitumumab) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECQVQLQESGPGLVKPSETLSLTC TVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSL KSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDI WGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK h1332 VH- 103 MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASVKVSCKA CH1h SGYTFTSYGFSWVRQAPGQGLEWMGWISASNGNTYYAQKLQGR VTMTTDTSTSTAYMELRSLRSDDTAVYYCARVYADYADYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSC Pani VL-hCk 104 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTI TCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSG SGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC FIT14-Ig FIX VL-hCk- 105 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTI Factor IXa/ FX-VH-hCg4 TCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSG FactorX SRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECQVQLVQSGSELKKPGASVKVSC KASGYTFTDNNMDWVRQAPGQGLEWMGDINTRSGGSIYNEEFQ DRVIMTVDKSTDTAYMELSSLRSEDTATYHCARRKSYGYYLDE WGEGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSP F-IX VH-CH1h 106 MEFGLSWLFLVAILKGVQCQVQLVESGGGLVQPGGSLRLSCAA SGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYRREVKGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTGREYGGGWYF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSC F-X VL-hCk 107 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTI TCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSG SRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC FIT16a-Ig Paritu VL-hCk- 108 MDMRVPAQLLGLLLLWFPGSRCDIEMTQSPDSLAVSLGERATI Her3 figituVH-hCg1 NCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGV (paritumumab)/ PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQG IGF-1R TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA (figitumumab) KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGECEVQLLESGGGLVQPGG SLRLSCTASGFTFSSYAMNWVRQAPGKGLEWVSAISGSGGTTF YADSVKGRFTISRDNSRTTLYLQMNSLRAEDTAVYYCAKDLGW SDSYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK Patritumab- 109 MEFGLSWLFLVAILKGVQCQVQLQQWGAGLLKPSETLSLTCAV CH1h YGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRV TISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSC Figitu VL-hCk 110 MDMRVPAQLLGLLLLWFPGSRCDIQMTQFPSSLSASVGDRVTI TCRASQGIRNDLGWYQQKPGKAPKRLIYAASRLHRGVPSRFSG SGSGTEFTLTISSLQPEDFATYYCLQHNSYPCSFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC FIT17a-Ig Demci VL- 111 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSPDSLAVSLGERATI DLL-4 hCk-Bevci VH- SCRASESVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGVPD (demcizumab)/ hCg1 RFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTK VEGF VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV (bevicizumab) QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGGSL RLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYA ADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYG SSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK Demci-CH1h 112 MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASVKISCKA SGYSFTAYYIHWVKQAPGQGLEWIGYISSYNGATNYNQKFKGR VTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSC Bevci VL-hCk 113 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTI TCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC FIT18a-Ig OfatuVL-hCk- 114 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGERATL CD20 CD3mAb VH- SCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG (ofatumumab)/ hCg1mut SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK CD3 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECEVQLLESGGGLVQPGGSLKLSC AASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS VKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY VSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK Ofatu VH-CH1 115 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGRSLRLSCAA SGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGR FTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMD VWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSC CD3mAb VL- 116 MTWTPLLFLTLLLHCTGSLSELVVTQEPSLTVSPGGTVTLTCR hCL SSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSG SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWK ADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS CQVTHEGSTVEKTVAPTECS FIT19a-Ig patritu VL-hCk- 117 MDMRVPAQLLGLLLLWFPGSRCDIEMTQSPDSLAVSLGERATI Her3 PaniVH-hCg1 NCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGV (patritumab)/ PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQG EGFR TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA (panitumumab) KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGECQVQLQESGPGLVKPSE TLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNT NYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRV TGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK Patritumab- 118 MEFGLSWLFLVAILKGVQCQVQLQQWGAGLLKPSETLSLTCAV CH1 YGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRV TISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSC Pani VL-hCk 119 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDRVTI TCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSG SGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC FIT20a-Ig Nivolu VL- 120 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGERATL PD-1 hCk-1B12 VH- SCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG (nivolumab)/ hCg1Mut SGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK PD-L1 (1B12) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECQVQLVQSGAEVKKPGSSVKVSC KTSGDTFSSYAISWVRQAPGQGLEWMGGIIPIFGRAHYAQKFQ GRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPF GMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K Nivo VH-CH1 121 MEFGLSWLFLVAILKGVQCQVQLVESGGGVVQPGRSLRLDCKA SGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGR FTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSC 1B12 VL-hCk 122 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGERATL SCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC FIT22a-Ig patritu VL-hCk- 123 MDMRVPAQLLGLLLLWFPGSRCDIEMTQSPDSLAVSLGERATI Her3 Nivolu VH- NCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGV (patritumab)/ hCg1mut PDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQG PD-1 TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA (nivolumab) KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGECQVQLVESGGGVVQPGR SLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRY YADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Patritumab- 124 MEFGLSWLFLVAILKGVQCQVQLQQWGAGLLKPSETLSLTCAV CH1 YGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRV TISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSC NivoVL-hCK 125 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGERATL SCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC
TABLE-US-00019 TABLE 18 Expression level in 293E cells and SEC profile for FIT-Igs Expression Level Name in 293E cells SEC Profile Target (mAb) (mg/L) (% monomeric fraction) FIT12a-Ig 0.26 74.72 EGFR (panitumumab)/ PD-L1 (IB12) FIT13a-Ig 0.705 97.70 cMet (h1332)/ EGFR (panitumumab) FIT14-Ig 0.135 100.00 Factor IXa/ Factor X FIT16a-Ig 0.15 100.00 Her3 (paritumumab)/ IGF-1R (figitumumab) FIT17a-Ig 0.11 78.384 DLL-4 (demcizumab)/ VEGF (bevicizumab) FIT18a-Ig 0.39 100.00 CD20 (ofatumumab)/CD3 FIT19a-Ig 0.37 86.762 Her3 (patritumab)/ EGFR (panitumumab) FIT20a-Ig 0.51 73.721 PD-1 (nivolumab)/ PD-L1 (1B12) FIT22a-Ig 0.098 100.00 Her3 (patritumab)/ PD-1 (nivolumab)
[0438] The SEC profiles for each of the FIT-Ig of Tables 17 and 18 are also provided in
[0439] Functional binding data for FIT13a-Ig are provided below in Table 19. In addition, a multiple-antigen binding study was performed to determine if FIT13a-Ig was able to bind cMet and EGFR. The results of the study are shown in
TABLE-US-00020 TABLE 19 Functional binding data for FIT13a-Ig Ig Target Kon Koff KD mAb-h1332 c-met 2.61E+05 6.87E−04 2.63E−09 FIT-Ig13a 2.94E+05 7.26E−04 2.47E−09 Panitumumab hEGFR 3.61E+05 5.59E−04 1.55E−09 FIT-Ig13a 2.69E+05 4.07E−04 1.52E−09
[0440] Functional binding data for the Factor IXa binding activity of FIT14-Ig are provided below in Table 20.
TABLE-US-00021 TABLE 20 Functional binding data for FIT14-Ig Ig Target Kon Koff KD Factor IXa mAb Factor IXa 2.74E+04 3.55E−04 1.30E−08 FIT-Ig 014 3.35E+04 3.32E−04 9.90E−09 Factor X mAb Factor X FIT-Ig 14
[0441] The results of the study show that additional FIT-Ig can be constructed, expressed, and purified and will exhibit functional binding to the target proteins.
Example 6: Construction, Expression, and Purification of New Anti-CTLA-4/PD-1 Fabs-in-Tandem Immunoglobulin (FIT-Ig)
[0442] New FIT-Ig having specificity for CTLA4 and PD1 were constructed as in the foregoing Examples. These exemplary FIT-Ig and their corresponding sequences are provided below in Table 21. Table 21 provides the expression level in 293E cells and the SEC profile for each of the FIT-Ig.
TABLE-US-00022 TABLE 21 Amino acid sequences of additional exemplary FIT-Ig for CTLA4 and PD1 Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences NBS3-Ig Long chain 126 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGER CTLA4 (IpiliVL-hCk- ATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATG (ipilimumab)/ NivoluVH- IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT PD-1 (nivolumab) hCg1mut) FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECQVQ LVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGK GLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQM NSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ipili VL 127 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQK PGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSPWTFGQGTKVEIK Ipili VL - CDR1 128 RASQSVGSSYLA Ipili VL - CDR2 129 GAFSRAT Ipili VL - CDR3 130 QQYGSSPWT NivoluVH 131 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQA PGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLF LQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS Nivolu VH - CDR1 132 NSGMH Nivolu VH - CDR2 133 VIWYDGSKRYYADSVKG Nivolu VH - CDR3 134 NDDY Short Chai #1 (Ipili 135 MEFGLSWLFLVAILKGVQCQVQLVESGGGVVQPGRSLRLS VH-CH1) CAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGW LGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Ipili VH 136 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQA PGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS Ipili VH - CDR1 137 SYTMH Ipili VH - CDR2 138 FISYDGNNKYYADSVKG Ipili VH - CDR3 139 TGWLGPFDY Short Chain #2 140 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGERA (Nivolu VL-hCK) TLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Nivolu VL 141 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQSSNWPRTFGQGTKVE1K Nivolu VL - CDR1 142 RASQSVSSYLA Nivolu VL - CDR2 143 DASNRAT Nivolu VL - CDR3 144 QQSSNWPRT NBS3R - Ig Long Chain (Nivolu 145 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGER PD- VL-hCk-Ipili VH- ATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI 1(nivolumab)/ hCg1mut) PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTF CTLA4 GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN (ipilimumab) FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECQVQL VESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG LEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK Nivolu VL 146 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQSSNWPRTFGQGTKVE1K Nivolu VL - CDR1 147 RASQSVSSYLA Nivolu VL - CDR2 148 DASNRAT Nivolu VL - CDR3 149 QQSSNWPRT Ipili VH 150 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQA PGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS Ipili VH - CDR1 151 SYTMH Ipili VH - CDR2 152 FISYDGNNKYYADSVKG Ipili VH - CDR3 153 TGWLGPFDY Short Chain #1 154 MEFGLSWLFLVAILKGVQCQVQLVESGGGVVQPGRSLRLD (Nivolu VH-CH1) CKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYA DSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Nivolu VH 155 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQA PGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLF LQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS Nivolu VH - CDR1 156 GMH Nivolu VH - CDR2 157 VIWYDGSKRYYADSVKG Nivolu VH - CDR3 158 NDDY Short Chain #2 (Ipili 159 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGER VL-hCK) ATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATG IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ipili VL 160 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQK PGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSPWTFGQGTKVEIK Ipili VL - CDR1 161 RASQSVGSSYLA Ipili VL - CDR2 162 GAFSRAT Ipili VL - CDR3 163 YGSSPWT NBS3-C-Ig LongChain 164 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGER CTLA4 (IpiliVL-hCk- ATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATG (ipilimumab)/ NivoluVH-hCg4): IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT PD-1(nivolumab) FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECQVQ LVESGGGVVQPGRSLRLDCKASGIITSNSGMHWVRQAPGK GLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQM NSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFP LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK IpiliVL 165 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQK PGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSPWTFGQGTKVEIK IpiliVL - CDR1 166 RASQSVGSSYLA IpiliVL - CDR2 167 GAFSRAT IpiliVL - CDR3 168 QQYGSS NivoluVH 169 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQA PGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLF LQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS NivoluVH - CDR1 170 NSGMH NivoluVH - CDR2 171 VIWYDGSKRYYADSVKG NivoluVH - CDR3 172 NDDY Short Chain #1 (Ipili 173 MEFGLSWLFLVAILKGVQCQVQLVESGGGVVQPGRSLRLS VH-CH1) CAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGW LGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Ipili VH 174 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQA PGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS Ipili VH - CDR1 175 SYTMH Ipili VH - CDR2 176 FISYDGNNKYYADSVKG Ipili VH - CDR3 177 TGWLGPFDY Short Chain #2 178 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGER (Nivolu VL-hCK) ATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Nivolu VL 179 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQSSNWPRTFGQGTKVEIK Nivolu VL - CDR1 180 RASQSVSSYLA Nivolu VL - CDR2 181 DASNRAT Nivolu VL - CDR3 182 QQSSNWPRT NBS3R-C- Long Chain (Nivolu 183 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGER Ig VL-hCk-Ipili VH- ATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PD- hCg1mut) PARESGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTF 1(nivolumab)/ GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN CTLA4 FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST (ipilimumab) LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECQVQL VESGGGVVQPGRSLRLSCAASGFITSSYTMHWVRQAPGKG LEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK Nivolu VL 184 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQSSNWPRTFGQGTKVE1K Nivolu VL - CDR1 185 RASQSVSSYLA Nivolu VL - CDR2 186 DASNRAT Nivolu VL - CDR3 187 QQSSNWPRT Ipili VH 188 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQA PGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS Ipili VH - CDR1 189 SYTMH Ipili VH - CDR2 190 FISYDGNNKYYADSVKG Ipili VH - CDR3 191 TGWLGPFDY Short Chain #1 192 MEFGLSWLFLVAILKGVQCQVQLVESGGGVVQPGRSLRLD (Nivolu VH-IgG4- CKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYA CH1) DSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDD YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES Nivolu VH 193 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQA PGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLF LQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS Nivolu VH - CDR1 194 NSGMH Nivolu VH - CDR2 195 VIWYDGSKRYYADSVKG Nivolu VH - CDR3 196 NDDY Short Chain #2 (Ipili 197 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGER VL-hCK) ATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATG IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ipili VL 198 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQK PGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSPWTFGQGTKVEIK Ipili VL - CDR1 199 RASQSVGSSYLA Ipili VL - CDR2 200 GAFSRAT Ipili VL - CDR3 201 QQYGSSPWT
[0443] The long chain and short chain comprise a leader sequence, which can be either MDMRVPAQLLGLLLLWFPGSRC (SEQ ID NO: 487), or MEFGLSWLFLVAILKGVQC (SEQ ID NO: 488).
TABLE-US-00023 TABLE 22 SEC Profile/Expression level in 293E cells: FIT-Ig Monomer % in SEC Expression level(mg/L) NBS3 >95% 24.6 NBS3R 100% 6.3 NBS3-C 98.28 9.6 NBS3R-C 100% 4.0
[0444] Expression level was evaluated in small scale production without any optimization, except NBS3. All SEC samples are under one step Protein A purification. FIT-Ig in both IgG1 and IgG4 format can be produced as homogeneous protein from 293 cells.
Functional Studies
[0445] Functional binding data for these antibodies is provided below in Table 23. The data suggests that the affinity was not affected by changing IgG constant sequences, but can be improved by place certain Fab in upper domain.
TABLE-US-00024 TABLE 23 Functional binding data Captured Antibody Antigen ka (1/Ms) kd (1/s) KD (M) NBS3R-C PD1-his 3.412E+5 0.001407 4.123E−9 NBS3R 3.315E+5 0.001402 4.228E−9 NBS3-C 1.746E+5 0.002457 1.408E−8 NBS3 1.868E+5 0.002605 1.395E−8 NBS3R-C CTLA-4, his 7.79E+04 0.001573 2.02E−08 NBS3R 9.85E+04 0.001166 1.18E−08 NBS3-C 1.93E+05 0.001123 5.82E−09 NBS3 2.05E+05 0.001096 5.34E−09
[0446] ELISA binding study: ELISA binding of NBS3R was tested. Briefly, for binding to CTLA-4, human CTLA-4Ig (R&D Systems) was immobilized on 96-well plates, followed by routine wash and blocking procedures. Then FIT-Ig or related monoclonal antibodies at various concentrations were added to the plate, followed by incubation and multiple wash steps, and detected with anti-human Fab-HRP. For binding to PD-1, human PD-1 (with a his tag) (R&D Systems) was immobilized on 96-well plates, followed by routine wash and blocking procedures. Then NBS3R or Nivolumab at various concentrations was added to the plate, followed by incubation and multiple wash steps, and detected with anti-human Fc-HRP. It appears that NBS3R was able to bind both CTLA-4 (A) and PD-1 (B), respectively. The result is shown in
[0447] Multiple binding study: A multiple binding study of NBS3 was also carried out. The result is shown in Figure. 10.
[0448] Thermo stability study: Thermo stability test was also performed on these antibodies, the result of which is shown in Table 24. The melting temperature was measured by DSC.
TABLE-US-00025 TABLE 24 Thermo stability test Tm (° C.) Antibody Code T.sub.m1 (° C.) T.sub.m2 (° C.) T.sub.m3 (° C.) T.sub.m4 (° C.) NBS3R-c 57.38 70.24 74.42 82.38 NBS3R 69.78 75.54 82.75 NBS3-c 68.4 75.03 NBS3 69.38 75.01 82.52
[0449] Storage stability study: The storage stability of NBS3 was assessed by SEC-HPLC method, and result is shown in Table 25. Samples were treated by freeze/thaw cycle for one time, two times or three times, no aggregation or degradation was observed by SEC-HPLC profile. Samples was treated at 4° C., 25° C. or 40° C. for 1 day, 3 days or 7 days, no aggregation or degradation was observed by SEC-HPLC profile.
TABLE-US-00026 TABLE 25 Storage stability of NBS3 Rel. Area % Rel. Area % Rel. Area % Sample Name 1 2 3 NBS3_D0 0.77 99.23 n.a. NBS3_F/T1 1.23 98.77 n.a. NBS3_F/T2 1.32 98.68 n.a. NBS3_F/T3 1.39 98.61 n.a. NBS3_4C-D1 1.45 98.55 n.a. NBS3_25C-D1 1.45 98.55 n.a. NBS3_40C-D1 1.44 98.56 n.a. NBS3_4C-D3 1.49 98.51 n.a. NBS3_25C-D3 1.50 98.50 n.a. NBS3_40C-D3 1.62 97.85 0.53 NBS3_4C-D7 1.61 98.39 n.a. NBS3_25C-D7 1.67 98.33 n.a. NBS3_40C-D7 1.76 97.70 0.55
[0450] NBS3 was further tested in rat PK study, and the result is shown in
[0451] NBS3, NBS3-C, and NBS3R-C were tested in cell-based receptor blocking assay. Briefly, PD1-Fc was immobilized on 96-well plates, followed by routine wash and blocking procedures. Then diluted FIT-Ig and biotinylated PD-L1-Fc was added to each well, followed by incubation and multiple wash steps, and detected with Streptavidin-HRP. The result is shown in
[0452] NBS3, NBS3-C, and NBS3R-C were further tested in MLR (Mixed Lymphocyte Reaction) assays and PBMC SEB-stimulation assay for their functional activity.
[0453] In the MLR assays, mixed lymphocyte reaction was performed using monocyte-derived dendritic cells from one donor and allogeneic CD4 T cells from another donor. The whole blood samples were collected from healthy donors, and PBMC were isolated from whole blood using Ficoll-Pague gradient centrifugation. On day 1, PBMC from one donor was isolated and diluted with serum-free RPMI 1640 at 1×10.sup.6/ml. The diluted PBMC was seeded into 6-well tissue culture plate at 3 ml/well and incubated for 3 h. Supernatant was removed and unattached cells were washed off. The attached monocyte were polarized into dendritic cells with 250 U/ml IL-4 and 500 U/ml GM-CSF in RPMI1640 with 10% FBS. The medium was replaced with fresh IL-4 and GM-CSF at day 4. At day 7, immature DC was collected and treated with 1 μg/ml LPS in RPMI 1640 with 10% FBS for additional 24 h for maturation. At Day 8, CD4 T cells were isolated from another donor PBMC by negative selection and adjusted to final concentration at 2×10.sup.6 cells/ml. Mature DC were treated with mitomycin C at 37° C. for 1.5 hr. Then DC were washed with PBS and adjusted to final concentration at 1×10.sup.6 cells/ml. CD4 T cells (Responder cells) were added into 96 well plate at 100 μl/well and pre-treated with test antibody at diluted concentration for 30 minutes. Then mature DC (Stimulator cells) were added into the well at 100 μl/well. The final volume of each well is 200 μl. The MLR were incubated at 37 degree for 72 hr for IL-2 test and 120 hr for IFN-gamma test respectively using ELISA. The result is shown in
[0454] In the PBMC SEB-stimulation assays, PBMC were isolated from healthy donor blood by Ficoll-Pague gradient centrifugation. The isolated PBMC were seeded into 96-well tissue culture plate at 1×10.sup.5 cells/well. Then the PBMC were pre-treated with diluted test antibodies for 30 min. Staphylococcus enterotoxin B (SEB) was added into cell culture medium with final concentration at 100 ng/ml. The final assay volume was 200 μl/well. The cells were cultured for 96 hr and the culture supernatant was collected. IL-2 cytokine production in the supernatant was detected by ELISA. The result is shown in
Example 7: Construction, Expression, and Purification of Additional Anti-EGFR/PD-L1 Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0455] New FIT-Igs having specificity for EGFR and PD-L1 were constructed as in the foregoing Examples. These exemplary FIT-Igs and their corresponding sequences are provided below in Table 26. Table 27 provides the expression level in 293E cells and the SEC profile for each of the FIT-Ig.
TABLE-US-00027 TABLE 26 Amino acid sequences of additional exemplary FIT-Ig for EGFR and PD-L1 Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT012b - Ig Long Chain (Pani 202 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDR EGFR VL-hCk-1B12VH- VTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGV (panitumumab)/ hCg1) PSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAF PD-L1 (1B12) GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECQVQL VQSGAEVKKPGSSVKVSCKTSGDTFSSYAISWVRQAPGQG LEWMGGIIPIFGRAHYAQKFQGRVTITADESTSTAYMELS SLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK Pani VL 203 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKP GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP EDIATYFCQHFDHLPLAFGGGTKVEIK Pani VL - CDR1 204 QASQDISNYL Pani VL - CDR2 205 DASNLET Pani VL - CDR3 206 QHFDHLPLA 1B12VH 207 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSSYAISWVRQA PGQGLEWMGGIIPIFGRAHYAQKFQGRVTITADESTSTAY MELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVT VSS 1B12VH - CDR1 208 SYAIS 1B12VH - CDR2 209 GIIPIFGRAHYAQKFQG 1B12VH - CDR3 210 KFHFVSGSPFGMDV Short Chain #1 211 MEFGLSWLFLVAILKGVQCQVQLQESGPGLVKPSETLSLT (Pani VH-CH1h) CTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNY NPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDR VTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C Pani VH 212 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIR QSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQF SLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS Pani VH - CDR1 213 SGDYYWT Pani VH - CDR2 214 HIYYSGNTNYNPSLKS Pani VH - CDR3 215 DRVTGAFDI Short Chain #2 216 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGER (1B12 VL-hCk) ATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFG QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 1B12 VL 217 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPTFGQGTKVEIK 1B12 VL - CDR1 218 RASQSVSSYLA 1B12 VL - CDR2 219 DASNRAT 1B12 VL - CDR3 220 QQRSNWPT Long Chain (Pani 221 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDR VL-hCk-10A5VH- VTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGV hCg1) PSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECQVQL VQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQR LEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELS SLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK Pani VL 222 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKP GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP EDIATYFCQHFDHLPLAFGGGTKVEIK Pani VL - CDR1 223 QASQDISNYLN Pani VL - CDR2 224 DASNLET Pani VL - CDR3 225 QHFDHLPLA 10A5VH 226 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQA PGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAY MELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSS 10A5VH - CDR1 227 SYDVH 10A5VH - CDR2 228 WLHADTGITKFSQKFQG 10A5VH - CDR3 229 ERIQLWFDY Short Chain #1 230 MEFGLSWLFLVAILKGVQCQVQLQESGPGLVKPSETLSLT (Pani VH-CH1h) CTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNY NPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDR VTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C Pani VH 231 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIR QSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQF SLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS Pani VH - CDR1 232 SGDYYWT Pani VH - CDR2 233 HIYYSGNTNYNPSLKS Pani VH - CDR3 234 DRVTGAFDI Short Chain #2 235 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDR (10A5 VL-hCk) VTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTF GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10A5 VL 236 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKP EKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYNSYPYTFGQGTKLEIK 10A5 VL - CDR1 237 RASQGISSWLA 10A5 VL - CDR2 238 AASSLQS 10A5 VL - CDR3 239 QQYNSYPYT
TABLE-US-00028 TABLE 27 SEC Profile/Expression level in 293E cells: Monomer % Expression FIT-Ig in SEC level(mg/L) FIT012b - Ig 74.72% 5.3 FIT012d - Ig .sup. >87% 1.05
[0456] The SEC profile and expression data suggest that FIT012d exhibited better purity than FIT012b by changing PD-L1 antibody sequences.
[0457] Functional binding data for these two antibodies is provided below in Table 28 and Table 29. The data suggests that the affinity was not affected by changing IgG constant sequences, but can be improved by place certain Fab in upper domain.
TABLE-US-00029 TABLE 28 Functional binding data of FIT012b Ig Target Kon Koff KD IC50 Panitumumab 1(EGFR) 2.42E+05 4.48E−04 1.85E−09 FIT012b 3.08E+05 7.83E−04 2.54E−09 1B12 2(PD-L1) 2.35E+05 2.14E−03 9.08E−09 FIT012b 6.97E+05 2.71E−03 3.89E−09
TABLE-US-00030 TABLE 29 Functional binding data of FIT012d Ig Target Kon Koff KD IC50 Panitumumab 1(EGFR) 1.05E+05 4.91E−05 4.66E−10 FIT012d 1.03E+05 4.92E−05 4.76E−10 10A5 2(PD-L1) 8.91E+05 6.54E−04 7.35E−10 FIT012d 1.03E+06 6.00E−04 5.80E−10
[0458] A multiple binding study of FIT012b and FIT012d was also carried out. The result is shown in
Example 8: Construction, Expression, and Purification of Anti-cMet/EGFR Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0459] New FIT-Ig having specificity for cMet and EGFR was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 30. Table 31 provides the expression level in 293E cells and the SEC profile for each of the FIT-Ig.
TABLE-US-00031 TABLE 30 Amino acid sequences of additional exemplary FIT-Ig for cMet and EGFR Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT013a Long Chain (h1332 240 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDR cMet (h1332 VL-hCk-Pani VH- VTITCRASQGINTWLAWYQQKPGKAPKLLIYAASSLKSGV (13.3.2L- hCg1) PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTF A91T,H- GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN 42K,S97T)) FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECQVQL QESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPG KGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKL SSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK h1332 VL 241 DIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQKP GKAPKLLIYAASSLKSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQANSFPLTFGGGTKVEIK h1332 VL - CDR1 242 RASQGINTWLA h1332 VL - CDR2 243 AASSLKS h1332 VL - CDR3 244 QQANSFPLT Pani VH 245 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIR QSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQF SLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS Pani VH - CDR1 246 SGDYYWT Pani VH - CDR2 247 HIYYSGNTNYNPSLKS Pani VH - CDR3 248 DRVTGAFDI Short Chain #1 249 MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASVKVS (h1332 VH-CH1) CKASGYTFTSYGFSWVRQAPGQGLEWMGWISASNGNTYYA QKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVYA DYADYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC h1332 VH 250 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGFSWVRQA PGQGLEWMGWISASNGNTYYAQKLQGRVTMTTDTSTSTAY MELRSLRSDDTAVYYCARVYADYADYWGQGTLVTVSS h1332 VH - CDR1 251 SYGFS h1332 VH - CDR2 252 WISASNGNTYYAQKLQG h1332 VH - CDR3 253 VYADYADY Short Chain #2 254 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDR (Pani VL-hCk) VTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGV PSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Pani VL 255 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKP GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP EDIATYFCQHFDHLPLAFGGGTKVEIK Pani VL - CDR1 256 QASQDISNYLN Pani VL - CDR2 257 DASNLET Pani VL - CDR3 258 QHFDHLPLA
TABLE-US-00032 TABLE 31 SEC Profile/Expression level in 293E cells: FIT-Ig Monomer % in SEC Expression level(mg/L) FIT013a 98.48% 22
Stability Data
[0460] The storage stability of FIT013a was assessed by SEC-HPLC method, and result is shown in Table 32. Samples were treated by freeze/thaw cycle for one time, two times or three times, no aggregation or degradation was observed by SEC-HPLC profile. Samples was treated at 4° C., 25° C. or 40° C. for 1 day, 3 days or 7 days, no aggregation or degradation was observed by SEC-HPLC profile.
TABLE-US-00033 TABLE 32 Storage stability of FIT013a Rel. Area % Rel. Area % Rel. Area % Sample Name agg mono clip FIT013a_D0 2.95 97.05 n.a. FIT013a_F/T1 3.26 96.74 n.a. FIT013a_F/T2 3.38 96.62 n.a. FIT013a_F/T3 3.47 96.53 n.a. FIT013a_4C-D1 3.56 96.44 n.a. FIT013a_25C-D1 3.66 96.34 n.a. FIT013a_40C-D1 3.72 96.22 0.06 FIT013a_4C-D3 3.63 96.37 n.a. FIT013a_25C-D3 3.65 96.35 n.a. FIT013a_40C-D3 3.76 96.16 0.08 FIT013a_4C-D7 3.64 96.36 n.a. FIT013a_25C-D7 3.72 96.28 n.a. FIT013a_40C-D7 3.95 95.92 0.12
[0461] Functional Study: Protein based binding data for FIT013a is provided below in Table 33 and Table 34.
TABLE-US-00034 TABLE 33 Functional binding data of FIT013a (human cMet/human EGFR) Ig Target Kon Koff KD IC50 H1332 1 (human 4.14E+05 5.27E−04 1.27E−09 FIT013a cMet) 4.75E+05 5.02E−04 1.06E−09 Panitumumab 2 (human 8.43E+04 5.10E−05 6.05E−10 FIT013a EGFR) 1.09E+05 5.84E−05 5.34E−10
TABLE-US-00035 TABLE 34 Functional binding data of FIT013a (Cyno cMEt/Cyno EGFR) Ig Target Kon Koff KD IC50 H1332 1 (Cyno 2.21E+05 1.00E−03 4.53E−09 FIT013a cMet) 2.86E+05 9.74E−04 3.40E−09 Panitumumab 2 (Cyno 5.87E+05 2.12E−04 3.62E−10 FIT013a EGFR) 2.66E+05 1.54E−04 5.77E−10
[0462] A multiple binding study of FIT013a was also carried out. The result is shown in
[0463] Further, the binding activity of FIT013a in cancer cell lines was determined by flow cytometry using BD FACSVerse. Cells grown in culture were detached from flask with trypsin-free medium and collected. The collected cells were washed in PBS buffer containing 2% FBS. Cells were then aliquot and incubated with 1:5 serially diluted FIT013a on ice for 1 hr. The starting working concentration of FTI013a was 20 μg/ml. Cells were washed, resuspended and incubated with 1:100 diluted Alexa Fluor® 488 labeled mouse anti-human IgG1 (Invitrogen, Cat. No. A-10631) on ice protected from light for 1 hr. Cells were washed and signal was detected with a BD FACSVerse flow cytometer according to manufacture's protocols.
[0464] These experiments demonstrate that FIT013a can bind to many cancer cell lines, like NCI-H1993, HCC827, MKN-45, SGC-7901, EBC-1, A549, KP4, NCI-H292, NCI-H1975, etc. Geometric mean fluorescence intensity (MFI) and EC50 for each cell lines are listed in the Table 35.
TABLE-US-00036 TABLE 35 Cell based binding data for FIT013a Cell lines MFI EC50 (nM) NCI-H1993 447 37.14 HCC827 2000 41.33 MKN-45 2050 8.89 SGC-7901 590 2.53 EBC-1 6000 1.5 A549 1000 0.62 KP4 345 0.31 NCI-H292 920 0.58 HCI-H1975 507 —
[0465] In addition, FACS assays were conducted to measure FIT013a's dual binding to membrane c-Met and EGFR to show its multiple binding activity.
[0466] Several cell lines were used n this assay. MKN-45 (human gastric adenocarcinoma cells) expressed high level of c-Met and low level of EGFR. SGC-7901 (human gastric cancer) expressed high level of EGFR and low level of c-Met. NCI-H1975 (human non-small cell lung cancer) expressed equal level of c-Met and EGFR.
[0467] Cells grown in culture were detached from flask with trypsin-free medium and collected. The collected cells were washed in PBS buffer containing 2% FBS. Cells were aliquot and incubated with serially diluted FIT013a or FIT013a-Fab on ice for 1 hr. Cells were then washed and incubated with 1 μg/ml biotinylated human c-MET or biotinylated EGFR on ice for 1 hr. Cells were resuspended and incubated with 4 μg/ml Alexa Fluor® 488 labeled Streptavidin (Invitrogen, Cat. No S32354) on ice protected from light for 1 hr. Cells were washed and signal was detected with a BD FACSVerse flow cytometer according to manufacture's protocols.
[0468] When cell membrane expression level c-Met is much higher than EGFR (e.g., on MKN-45 cell), c-Met binding cite of FIT013a and FIT013a-Fab can be occupied by membrane c-Met, the free EGFR binding cite of FIT013a and FIT013a-Fab can be detected by biotinylated EGFR. When cell membrane expression level EGFR is much higher than c-Met (e.g., on SGC-7901), EGFR binding cite of FIT013a and FIT013a-Fab can be occupied by membrane EGFR, the free c-Met binding cite of FIT013a and FIT013a-Fab can be detected by biotinylated c-Met. When cell membrane expression level c-Met is equal to EGFR (e.g., on NCI-H1975 cell), c-Met and EGFR binding cites of FIT013a and FIT013a-Fab are occupied simultaneously, no free EGFR or c-Met binding cite of FIT013a and FIT013a-Fab can be detected. As demonstrated in
[0469] Signaling assay: Next, FIT013a was used to inhibit HGF induced AKT phosphorylation in cells. NCI-H292 cells were plated at 2×10.sup.5 per well in 96-well plate and serum starved overnight. Serially diluted FIT013a or other related Abs were added to plate and incubate for 30 mins and then 40 ng/ml HGF was added to the assay plate for 5 mins. The cells were lysed and AKT phosphorylation was detected by ERK phospho-T202/Y204 kit (Cisbio, Cat: 64AKSPEG). The experiment demonstrates that FIT013a shows superior activity than mAbs combo in neutralizing HGF induced AKT phosphorylation in NCI-H292 cell. FIT013a inhibited 80% of AKT phosphorylation while EGFR and c-Met antibody combo inhibit 60% of AKT phosphorylation, see
[0470] Agonist assay: Agonist effect of c-Met, EGFR antibody in FIT013a was tested by AKT phosphorylation. NCI-H441 cells were plated at 2×10.sup.5 per well in 96-well plate and serum starved overnight. Serially diluted FIT013a or other related Abs were added to plate and incubate for 30 mins. The cells were lysed and AKT phosphorylation was detected by ERK phospho-T202/Y204 kit (Cisbio, Cat: 64AKSPEG). As shown in
[0471] Receptor depletion study: FIT013a was tested to see if it can deplete both c-Met and EGFR on cell membrane. The c-Met antibody, EGFR antibody and FIT013a were incubated with H441 cell for more than 16 hrs at 37° C. and then EGFR and c-Met remaining on the cell surface was detected by FACS. The result indicates that FIT013a can deplete near 70% of cell membrane c-Met and EGFR, higher than the c-Met antibody or the EGFR antibody, see Table 36.
TABLE-US-00037 TABLE 36 Receptor depletion by FIT013a EGFR c-Met H441 cell Depletion (%) Depletion (%) h1332 0.0 54.0 Panitumumab 56.6 0.0 FIT013a 65.9 64.5
[0472] Rat PK study: The purpose of this study was to evaluate the pharmacokinetic of FIT013a (c-met/EGFR) following single intravenous (IV) or subcutaneous (SC) administrations in SD rats. For IV and SC dosing, the test article FIT013a (c-met/EGFR) was dissolved (in 10 mM sodium citrate, 50 mM NaCl, pH 6.0) at 2.3 mg/mL (lot: 160408001). A total of 8 male SD rats, approximately 195-208 g of body weight, purchased from SLAC Laboratory Animal Co. LTD. with Qualification No.: SCXK (SH) 2008001659659, were used in this study. The dosing and sampling is designed as Table 37.
TABLE-US-00038 TABLE 37 Dosing and Sampling Design Route Dose Solution Dose Treatment No. of of Level Conc. Volume Group Treatment animals admin. (mg/kg) (mg/mL) (mL/kg) Time points 1 FIT013a 4 IV 5 2 2.5 Sampling at 0, (c- 10 min, 1, 4, 8, 24 hr, met/EGFR) 2, 4, 7, 10, 14, 21, 28 d, serial bleeding via tail vein for serum only. ~240 uL blood per time point. 2 FIT013a 4 SC 5 2 2.5 Sampling at 0, (c- 30 min, 1, 4, 8, 12, met/EGFR) 24 hr, 2, 4, 7, 10, 14, 21, 28 d, serial bleeding via tail vein for serum only. ~240 uL blood per time point.
[0473] The IV dose was administered via foot dorsal vein injection and SC dose was administered via subcutaneous injection. At the designated time-points, the animals were restrained manually, and approximately 240 μL blood/time point was collected via tail vein puncture or cardiac puncture into tubes. The blood samples were placed at room temperature for 0.5 hr. Then blood samples were centrifuged (10000 g, 5 min under 4° C.) to obtain the serum samples. The serum samples were immediately stored at −80° C. until analysis. The samples were detected by ELISA using c-Met and EGFR protein respectively. The concentration-time data of FIT013a (c-met/EGFR) in rat serum for IV and SC studies are listed in Table 38 to Table 41, and are illustrated in
TABLE-US-00039 TABLE 38 Serum concentration-time data and pharmacokinetic parameters of FIT013a (c-met/EGFR) after an IV dose at 5 mg/kg in male SD rats (c-met plate) Sampling Concentration Dose Dose time (μg/mL) Mean (mg/kg) route (Day) Rat #1 Rat #2 Rat #3 Rat #4 (μg/mL) SD CV (%) 5 IV 0 BQL BQL BQL BQL BQL NA NA c-met plate 0.00694 138 118 82.0 108 112 23.3 20.9 0.0417 114 105 74.3 99.3 98.2 17.0 17.4 0.167 103 97.8 71.7 83.8 89.0 14.0 15.8 0.333 79.1 87.9 62.8 70.9 75.2 10.8 14.4 1 60.8 62.8 56.3 53.6 58.4 4.17 7.14 2 51.0 54.2 45.4 42.6 48.3 5.27 10.9 4 44.0 45.5 37.7 39.0 41.5 3.81 9.17 7 31.9 33.1 29.6 28.6 30.8 2.06 6.68 10 24.7 22.7 24.4 21.8 23.4 1.40 5.97 14 20.0 19.7 18.5 18.0 19.1 0.946 4.96 21 12.3 11.9 12.1 11.5 12.0 0.319 2.67 28 7.68 8.12 9.53 7.43 8.19 0.938 11.5 PK parameters Unit Rat #1 Rat #2 Rat #3 Rat #4 Mean SD CV (%) CL mL/day/kg 6.24 6.14 6.21 6.86 6.36 0.335 5.27 Vss mL/kg 82.5 82.7 105 97.6 91.8 11.0 12.0 V1 mL/kg 38.2 44.4 65.3 47.6 48.9 11.6 23.8 Alpha t.sub.1/2 day 0.235 0.491 0.879 0.309 0.478 0.288 60.2 Beta t.sub.1/2 day 9.46 9.80 12.3 10.21 10.4 1.26 12.1 AUC Day × μg/mL 802 815 805 729 788 39.5 5.02 MRT day 13.2 13.5 16.8 14.2 14.4 1.65 11.4
TABLE-US-00040 TABLE 39 Serum concentration-time data and pharmacokinetic parameters of FIT013a (c-met/EGFR) after a SC dose of 5 mg/kg in male SD rats (c-met plate) Sampling Concentration Dose Dose time (μg/mL) Mean (mg/kg) route (Day) Rat #5 Rat #6 Rat #7 Rat #8 (μg/mL) SD CV (%) 5 SC 0 BQL BQL BQL BQL BQL NA NA c-Met plate 0.00694 0.0780 BQL 0.0500 BQL 0.0640 NA NA 0.0417 0.167 0.150 0.172 0.228 0.179 0.0338 18.9 0.167 0.865 2.38 1.70 3.28 2.06 1.03 49.9 0.333 2.44 4.89 3.66 6.17 4.29 1.60 37.4 0.5 5.69 8.74 7.37 10.8 8.16 2.18 26.7 1 21.3 29.5 23.6 28.3 25.7 3.85 15.0 2 39.4 47.9 40.9 43.7 43.0 3.73 8.69 4 47.4 36.8 42.3 47.2 43.4 5.01 11.5 7 36.8 38.5 34.4 37.7 36.9 1.76 4.78 10 28.8 25.7 27.0 29.9 27.9 1.85 6.64 14 19.8 *0.753 18.5 18.6 19.0 0.748 3.94 21 14.2 BQL 12.8 16.2 14.4 1.67 11.6 28 11.6 BQL 9.52 10.5 10.5 1.03 9.74 PK parameters Unit Rat #5 Rat #6 Rat #7 Rat #8 Mean SD CV (%) T.sub.max day 4.00 2.00 4.00 4.00 3.50 1.00 28.6 C.sub.max μg/ml 47.4 47.9 42.3 47.2 46.2 2.63 5.70 Terminal t.sub.1/2 day 11.6 11.6 11.4 12.0 11.7 0.262 2.24 AUC.sub.last Day × μg/ml 656 344 611 680 573 155 27.1 AUC.sub.INF Day × μg/ml 850 775 767 863 814 49.8 6.12 CL/F mL/day/kg 5.88 6.45 6.52 5.79 6.16 0.376 6.11 F % 108 98.4 97.4 110 103 6.32 6.12 *The serum concentration of these time point were excluded from mean value and PK parameters calculation due to the posibility of anti-drug antibody.
TABLE-US-00041 TABLE 40 Serum concentration-time data and pharmacokinetic parameters of FIT013a (c-met/EGFR) after an IV dose at 5 mg/kg in male SD rats (EGFR plate) Sampling Concentration Dose Dose time (μg/mL) Mean (mg/kg) route (Day) Rat #1 Rat #2 Rat #3 Rat #4 (μg/mL) SD CV (%) 5 IV 0 BQL BQL BQL BQL BQL NA NA EGFR plate 0.00694 130 115 80.0 105 107 21.0 19.6 0.0417 111 103 75.8 97.3 96.9 15.2 15.7 0.167 98.0 95.5 72.3 83.4 87.3 11.9 13.6 0.333 76.2 83.6 62.1 70.8 73.2 9.06 12.4 1 57.0 59.3 56.6 53.1 56.5 2.59 4.58 2 47.6 50.9 45.6 44.6 47.2 2.79 5.92 4 45.6 45.3 39.9 39.8 42.6 3.27 7.67 7 32.7 33.8 31.1 29.5 31.8 1.88 5.90 10 25.1 23.6 25.2 22.3 24.0 1.37 5.69 14 19.2 19.7 19.1 18.4 19.1 0.524 2.75 21 12.8 12.2 12.8 12.1 12.5 0.365 2.92 28 8.38 8.62 10.0 7.71 8.68 0.973 11.2 PK parameters Unit Rat #1 Rat #2 Rat #3 Rat #4 Mean SD CV (%) CL mL/day/kg 6.14 6.09 5.98 6.66 6.22 0.302 4.86 Vss mL/kg 86.3 85.2 102 96.0 92.3 7.94 8.61 V1 mL/kg 39.7 44.8 65.3 48.7 49.6 11.1 22.3 Alpha t.sub.1/2 day 0.222 0.368 0.795 0.306 0.423 0.255 60.3 Beta t.sub.1/2 day 10.0 10.0 12.3 10.3 10.7 1.09 10.2 AUC Day × μg/mL 815 821 836 751 806 37.6 4.67 MRT day 14.1 14.0 17.0 14.4 14.9 1.44 9.70
TABLE-US-00042 TABLE 41 Serum concentration-time data and pharmacokinetic parameters of FIT013a (c-met/EGFR) after a SC dose at 5 mg/kg in male SD rats (EGFRplate) Sampling Concentration Dose Dose time (μg/mL) Mean (mg/kg) route (Day) Rat #5 Rat #6 Rat #7 Rat #8 (μg/mL) SD CV(%) 5 SC 0 BQL BQL BQL BQL BQL NA NA EGFR plate 0.00694 0.0760 BQL 0.0490 BQL 0.0625 NA NA 0.0417 0.164 0.144 0.167 0.231 0.177 0.0377 21.4 0.167 0.836 2.32 1.66 3.30 2.03 1.04 51.4 0.333 2.45 4.45 3.74 6.37 4.25 1.64 38.5 0.5 5.85 8.61 7.69 11.6 8.45 2.42 28.6 1 21.6 27.7 24.5 29.8 25.9 3.61 14.0 2 41.5 45.3 40.0 43.8 42.6 2.39 5.60 4 46.2 38.9 41.9 46.2 43.3 3.56 8.22 7 35.9 37.3 34.7 39.6 36.9 2.13 5.77 10 28.5 25.0 26.8 30.6 27.7 2.40 8.65 14 19.3 *0.768 19.4 19.0 19.2 0.183 0.950 21 14.7 BQL 13.9 16.5 15.0 1.33 8.84 28 11.7 BQL 10.5 11.4 11.2 0.601 5.37 PK parameters Unit Rat #5 Rat #6 Rat #7 Rat #8 Mean SD CV (%) T.sub.max day 4.00 2.00 4.00 4.00 3.50 1.00 28.6 C.sub.max μg/ml 46.2 45.3 41.9 46.2 44.9 2.03 4.51 Terminal t.sub.1/2 day 13.1 9.38 12.4 12.3 11.8 1.66 14.0 AUC.sub.last Day × μg/ml 653 339 625 694 578 162 28.0 AUC.sub.INF Day × μg/ml 875 677 813 896 815 98.8 12.1 CL/F mL/day/kg 5.72 7.39 6.15 5.58 6.21 0.823 13.3 F % 109 84.0 101 111 101 12.3 12.1 *The serum concentration of these time point were excluded from mean value and PK parameters calculation due to the posibility of anti-drug antibody.
NCI-H1975-HGF Xenograft Model Tumor Distribution Study
[0474] In this study, serum and tumor concentration of FIT013a was measured 24 hrs after single IP dose in NCI-H1975-HGF tumor bearing nude BALB/c Mice.
[0475] NCI-H1975-HGF cells were subcutaneously inoculated to nude BALB/c mice. When the average tumor volume reached to 200-250 mm.sup.3, the mice were randomly allocated to four groups, FIT013a, H1332, Panitumumab and vehicle group. FIT013a group was single IP dose, 16 mg/kg; H1332 or Panitumumab was single IP dose, 10 mg/kg. Vehicle group was dosed with formulation buffer 10 mM sodium citrate, 50 mM NaCl, pH 6.0. 24 hrs after dosing, tumor and serum were collect. Tumors were homogenized and the supernatant was collected for the following ELISA study. FIT013a, Panitumumab, and H1332 were quantified by using generic hIgG ELISA method for both serum and tumor.
[0476] The experiment demonstrates that FIT013a showed comparable distribution activity in serum and tumor with monoclonal antibodies, see
NCI-H1975-HGF Xenograft Model Efficacy Study
[0477] In this study, the efficacy of FIT013a was measured in NCI-H1975-HGF xenograft model.
[0478] NCI-H1975-HGF cells were subcutaneously inoculated to nude BALB/c mice. When the average tumor volume reached to 100-130 mm.sup.3, and the largest tumor volume was less than 140 mm.sup.3. The mice were randomly allocated to four groups, FIT013a, H1332, Panitumumab and vehicle group. The antibodies were dosed two times/week i.p. for three weeks. The dosing for FIT013a was 16 mg/kg, for H1332 or Panitumumab was 10 mg/kg. Vehicle group was dosed with formulation buffer, 10 mM sodium citrate, 50 mM NaCl, pH 6.0. The tumor volume and mouse body weight was measured twice/week. Percentage tumor growth inhibition (% TGI) was defined as the difference between the control-treated group mean tumor volume (MTV) and the test antibody-treated group MTV.
[0479] The experiment demonstrates that FIT013a showed FIT013a showed better efficacy than EGFR or c-Met monoclonal Ab, see
Example 9: Study of Anti-Factor IXa/Factor X Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0480] FIT-Ig having specificity for Factor IXa and Factor X was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 42. Table 43 provides the expression level in 293E cells and the SEC profile for each of the FIT-Ig.
TABLE-US-00043 TABLE 42 Amino acid sequences of additional exemplary FIT-Ig for cMEt and EGFR Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT014a Long Chain (FIX 259 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDR Factor IX VL-hCk-FX-VH- VTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGV (Factor IX hCg4) PDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTF Ab)/Factor X GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN (Factor X Ab) FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECQVQL VQSGSELKKPGASVKVSCKASGYTFTDNNMDWVRQAPGQG LEWMGDINTRSGGSIYNEEFQDRVIMTVDKSTDTAYMELS SLRSEDTATYHCARRKSYGYYLDEWGEGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSP FIX VL 260 DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKP GQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQP EDIATYYCQQYSDPPLTFGGGTKVEIK FIX VL - CDR1 261 KASRNIERQLA FIX VL - CDR2 262 QASRKES FIX VL - CDR3 263 QQYSDPPLT FX-VH 264 QVQLVQSGSELKKPGASVKVSCKASGYTFTDNNMDWVRQA PGQGLEWMGDINTRSGGSIYNEEFQDRVIMTVDKSTDTAY MELSSLRSEDTATYHCARRKSYGYYLDEWGEGTLVTVSS FX-VH - CDR1 265 DNNMD FX-VH - CDR2 266 DINTRSGGSIYNEEFQD FX-VH - CDR3 267 RKSYGYYLDE Short Chain #1 (F- 268 MEFGLSWLFLVAILKGVQCQVQLVESGGGLVQPGGSLRLS IX VH-CH1h) CAASGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYR REVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTG REYGGGWYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSC F-IX VH 269 QVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDIQWVRQA PGKGLEWVSSISPSGQSTYYRREVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARRTGREYGGGWYFDYWGQGTLVT VSS F-IX VH - CDR1 270 SYYDIQ F-IX VH - CDR2 271 SISPSGQSTYYRREVKG F-IX VH - CDR3 272 RTGREYGGGWYFDY Short Chain #2 (F-X 273 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDR VL-hCk) VTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGV PDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC F-X VL 274 DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKP GQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQP EDIATYYCQQYSDPPLTFGGGTKVEIK F-X VL - CDR1 275 KASRNIERQLA F-X VL - CDR2 276 QASRKES F-X VL - CDR3 277 QQYSDPPLT
TABLE-US-00044 TABLE 43 SEC Profile/Expression level in 293E cells: Expression FIT-Ig Monomer % in SEC level (mg/L) FIT014a 98.8% 10.5
Functional Study
[0481] Affinity measurement by surface plasmon resonance: The kinetics of FIT-Ig binding to hFactor IX and hFactor X (Enzyme Research Laboratory) was determined by surface plasmon resonance with a Biacore X100 instrument (Biacore AB, Uppsala, Sweden) using HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25° C. Briefly, goat anti-human IgG Fc fragment specific polyclonal antibody (Pierce Biotechnology Inc, Rockford, Ill.) was directly immobilized across a CM5 research grade biosensor chip using a standard amine coupling kit according to manufacturer's instructions. Purified FIT-Ig samples were diluted in HEPES-buffered saline for capture across goat anti-human IgG Fc specific reaction surfaces and injected over reaction matrices at a flow rate of 5 μl/min. The association and dissociation rate constants, kon (M-1s-1) and koff (s-1) were determined under a continuous flow rate of 30 μL/min. Rate constants were derived by making kinetic binding measurements at 500 nM antigen concentrations. The equilibrium dissociation constant (M) of the reaction between FIT-Ig and the target proteins was then calculated from the kinetic rate constants by the following formula: KD=koff/kon. Aliquots of antigen samples were also simultaneously injected over a blank reference and reaction CM surface to record and subtract any nonspecific binding background to eliminate the majority of the refractive index change and injection noise. Surfaces were regenerated with two subsequent 25 ml injections of 10 mM Glycine (pH 1.5) at a flow rate of 10 μL/min. The anti-Fc antibody immobilized surfaces were completely regenerated and retained their full capture capacity over twelve cycles. Protein based binding data for FIT014a is provided below in Table 44.
TABLE-US-00045 TABLE 44 Functional binding data of FIT014 Ig Target Kon Koff KD Factor IX mAb Factor IX 2.74E+04 3.55E−04 1.30E−08 FIT-Ig 014a 3.35E+04 3.32E−04 9.91E−09 Factor X mAb Factor X 3.15E+04 1.42E−03 4.51E−08 FIT-Ig 014a 7.75E+04 7.76E−04 1.00E−08
[0482] Factor VIIIa-like activity assay: FVIIIa-like activity of the FIT-Ig was evaluated by an enzyme assay according to manufacturer's instructions of BIOPHEN FVIII:C kit (Hyphen-Biomed, 221402-RUO). The result indicates that FIT014a has comparable FVIIIa like activity with Emicizumab and purified FVIIIa, while monoclonal antibody of Factor IX and Factor X, as well as a combination of them, has no activity, see
[0483] Multiple-antigen binding study: this study was done using OctetRed to determine if FIT014a is able to bind hFactor IX and hFactor X simultaneously. Briefly, FIT014a was immobilize on AR2G sensor at concentration of 10 μg/ml, followed by binding of hFactor IX and then hFactor X (Enzyme Research Laboratory) in assay buffer (PBS pH 7.4, 0.1% BSA, 0.02% Tween), with concentration at 500 nM. At the end of the experiment, the surface was regenerated with 10 mM glycine at pH1.5 five times. This experiment shows that FIT014a is able to bind hFactor X when it had already bound to hFactor IX, indicating that FIT014a is able to bind both hFactor IX and hFactor X simultaneously, see
[0484] Stability study: FIT014a protein samples in citrate buffer (pH=6.0) were individually incubated at constant 4° C., 25° C. and 40° C. for 1 day, 3 days or 7 days. Similarly, FIT014a protein samples were freeze-thawed once, twice or three times. The fractions of intact full monomeric protein of all samples was detected by SEC-HPLC, with 10 μg of each protein sample injected into Ultimate 3000 HPLC equipping Superdex200 5/150 GL at flow rate 0.3 mL/min for 15 min, and data was recorded and analyzed using Chromeleon software supplied by the manufacturer. Table 45 shows that FIT014a remained full intact monomeric molecule under these thermo-challenged conditions.
TABLE-US-00046 TABLE 45 Storage stability of FIT014 Rel. Area % Rel. Area % Rel. Area % Sample Name 1 2 3 FIT014_D0 6.28 93.32 0.40 FIT014_F/T1 6.86 92.25 0.89 FIT014-F/T2 6.72 92.36 0.93 FIT014_F/T3 6.80 92.03 1.16 FIT014_4C-D1 6.19 93.51 0.30 FIT014_25C-D1 6.81 92.24 0.95 FIT014_40C-D1 6.60 92.36 1.04 FIT014_4C-D3 6.63 92.42 0.95 FIT014_25C-D3 6.92 92.02 1.07 FIT014_40C-D3 6.60 92.15 1.25 FIT014_4C-D7 6.68 92.45 0.87 FIT014_25C-D7 6.58 92.39 1.03 FIT014_40C-D7 6.94 91.74 1.32
[0485] Rat PK study: FIT014a was subjected to PK study in rat, and the result is shown in
TABLE-US-00047 TABLE 46 Rat PK data for FIT014a hIgG plate Factor X plate PK IV, SC, IV, SC, parameters 5 mg/kg 5 mg/kg 5 mg/kg 5 mg/kg CL, 11.2 NA 14.5 NA mL/day/kg Alpha t.sub.1/2, 0.329 NA 0.233 NA Day Beta t.sub.1/2, 8.79 6.90 7.03 5.76 day V1, 54.0 NA 52.6 NA mL/kg T.sub.max, day 0.00347 3.00 0.00347 2.50 C.sub.max, μg/mL 104 30.7 105 16.3 F (%) NA 73.8 NA 48.3
Example 10: Study of Anti-HER3/IGF-1R Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0486] FIT-Ig having specificity for HER3 and IGF-1R was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 47. Table 48 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.
TABLE-US-00048 TABLE 47 Amino acid sequences of additional exemplary FIT-Ig for HER3 and IGF-1R Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT016a Long Chain (paritu VL- 278 MDMRVPAQLLGLLLLWFPGSRCDIEMTQSPDSLAVSL HER3 hCk-FigituVH-hCg1) GERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKL (Patritumab)/IGF LIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV 1R AVYYCQQYYSTPRTFGQGTKVEIKRTVAAPSVFIFPP (Figitumumab) SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGECEVQLLESGGGLVQPGGS LRLSCTASGFTFSSYAMNWVRQAPGKGLEWVSAISGS GGTTFYADSVKGRFTISRDNSRTTLYLQMNSLRAEDT AVYYCAKDLGWSDSYYYYYGMDVWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK paritu VL 279 DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRN YLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSG TDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVE IK paritu VL - CDR1 280 RSSQSVLYSSSNRNYLA paritu VL - CDR2 281 WASTRES paritu VL - CDR3 282 QQYYSTPRT FigituVH 283 EVQLLESGGGLVQPGGSLRLSCTASGFTFSSYAMNWV RQAPGKGLEWVSAISGSGGTTFYADSVKGRFTISRDN SRTTLYLQMNSLRAEDTAVYYCAKDLGWSDSYYYYYG MDVWGQGTTVTVSS FigituVH - CDR1 284 SYAMN FigituVH - CDR2 285 AISGSGGTTFYADSVKG FigituVH - CDR3 286 DLGWSDSYYYYYGMDV Short Chain #1 287 MEFGLSWLFLVAILKGVQCQVQLQQWGAGLLKPSETL (PatritumabVH - CH1) SLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSG STNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAV YYCARDKWTWYFDLWGRGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSC PatritumabVH 288 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWI RQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETS KNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGT LVTVSS PatritumabVH - CDR1 289 GYYWS PatritumabVH - CDR2 290 EINHSGSTNYNPSLKS PatritumabVH - CDR3 291 DKWTWYFDL Short Chain #2 (Figitu 292 MDMRVPAQLLGLLLLWFPGSRCDIQMTQFPSSLSASV VL-hCk) GDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAAS RLHRGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCL QHNSYPCSFGQGTKLEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Figitu VL 293 DIQMTQFPSSLSASVGDRVTITCRASQGIRNDLGWYQ QKPGKAPKRLIYAASRLHRGVPSRFSGSGSGTEFTLT ISSLQPEDFATYYCLQHNSYPCSFGQGTKLEIK Figitu VL - CDR1 294 RASQGIRNDLG Figitu VL - CDR2 295 AASRLHR Figitu VL - CDR3 296 LQHNSYPCS
TABLE-US-00049 TABLE 48 SEC Profile/Expression level in 293E cells: Expression FIT-Ig Monomer % in SEC level(mg/L) FIT016a 99.54% 16
Functional Studies
[0487] Functional binding study: Functional binding data for FIT016a is provided below in Table 49.
TABLE-US-00050 TABLE 49 Functional binding data Ig Target Kon Koff KD IC50 Patritumab 1 (HER3) 3.17E+05 2.85E−04 9.00E−10 FIT016a 3.19E+05 3.19E−04 1.00E−09 Figitumumab 2 (IGF1R) 1.30E+05 9.48E−05 7.29E−10 FIT016a 3.38E+04 9.19E−05 2.72E−09
[0488] Multiple-antigen binding study: this study was done using OctetRed to determine if FIT016a is able to bind Her3 and IGF1R simultaneously. This experiment shows that FIT016a is able to bind Her3 when it had already bound to IGF1R, indicating that FIT016a is able to bind both Her3 and IGF-1R simultaneously, see
Example 11: Study of Anti-DLL4/VEGF Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0489] FIT-Ig having specificity for DLL4 and IGF1R was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 50. Table 51 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.
TABLE-US-00051 TABLE 50 Amino acid sequences of additional exemplary FIT-Ig for HER3 and IGF-IR Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT017a Long Chain (Demci 297 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSPDSLAVSLGE DLL4(demcizumab)/ VL-hCk-Bevci VH- RATISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAAS VEGF(bevcizumab) hCg1) NQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQS KEVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGECEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYG MNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSL DTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFD VWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Demci VL 298 DIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKW FQQKPGQPPKLLIYAASNQGSGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQSKEVPWTFGGGTKVEIK Demci VL - CDR1 299 RASESVDNYGISFMK Demci VL - CDR2 300 AASNQGS Demci VL - CDR3 301 QQSKEVPWT Bevci VH 302 EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQ APGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKST AYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGT LVTVSS Bevci VH - CDR1 303 NYGMN Bevci VH - CDR2 304 WINTYTGEPTYAADFKR Bevci VH - CDR3 305 YPHYYGSSHWYFDV Short Chain #1 306 MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASVKI (Demci VH-CH1h) SCKASGYSFTAYYIHWVKQAPGQGLEWIGYISSYNGATN YNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCAR DYDYDVGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSC Demci VH 307 QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQ APGQGLEWIGYISSYNGATNYNQKFKGRVTFTTDTSTST AYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTV SS Demci VH - CDR1 308 AYYIH Demci VH - CDR2 309 YISSYNGATNYNQKFKG Demci VH - CDR3 310 DYDYDVGMDY Short Chain #2 311 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGD (Bevci VL-hCk)) RVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVP WTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC Bevci VL 312 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQK PGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQYSTVPWTFGQGTKVEIK Bevci VL - CDR1 313 SASQDISNYLN Bevci VL - CDR2 314 FTSSLHS Bevci VL - CDR3 315 QQYSTVPWT
TABLE-US-00052 TABLE 51 SEC Profile/Expression level in 293E cells: Expression FIT-Ig Monomer % in SEC level (mg/L) FIT017a >82% 2.2
Functional Studies
[0490] Functional binding study: Functional binding data for FIT017a is provided below in Table 52.
TABLE-US-00053 TABLE 52 Functional binding data Ig Target Kon Koff KD IC50 demcizumab 1 (DLL4) 1.74E+05 1.28E−04 7.36E−09 FIT017a 1.88E+05 1.28E−04 6.81E−09 bevcizumab 2 (VEGF) 3.46E+05 2.43E−06 7.04E−12 FIT017a 3.50E+05 1.28E−05 3.65E−11
[0491] Multiple-antigen binding study: this study was done using OctetRed to determine if FIT017a is able to bind DLL4 and VEGF simultaneously. This experiment shows that FIT017a is able to bind DLL4 when it had already bound to VEGF, indicating that FIT017a is able to bind both DLL4 and VEGF simultaneously, see
Example 12: Study of Anti-CD20/CD3 Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0492] FIT-Ig having specificity for CD20 and CD3 was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 53. Table 54 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.
TABLE-US-00054 TABLE 53 Amino acid sequences of additional exemplary FIT-Ig for CD20 and CD3 Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT018a Long Chain 316 MDMRVFAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPG CD20(Ofatumumab)/ (OfatuVL-hCk- ERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNR CD3 mAb CD3mAb VH- ATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS (described in hCg1mut) NWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTA US2009/0252683, SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS incorporated by KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT reference) KSFNRGECEVQLLESGGGLVQPGGSLKLSCAASGFTFN TYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGN SYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK Ofatu VL 317 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFAVYYCQQRSNWPITFGQGTRLEIK OfatuVL - CDR1 318 RASQSVSSYLA OfatuVL - CDR2 319 DASNRAT OfatuVL - CDR3 320 QQRSNWPIT CD3mAb VH 321 EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDD SKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAY WGQGTLVTVSS CD3mAb VH - CDR1 322 TYAMN CD3mAb VH - CDR2 323 RIRSKYNNYATYYADSVKD CD3mAb VH - CDR3 324 HGNFGNSYVSWFAY Short Chain #1 (Ofatu 325 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGRSLR VH-CH1) LSCAASGETFNDYAMHWVRQAPGKGLEWVSTISWNSGS IGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYY CAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSC Ofatu VH 326 EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVR QAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAK KSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQ GTTVTVSS Ofatu VH - CDR1 327 DYAMH Ofatu VH - CDR2 328 TISWNSGSIGYADSVKG Ofatu VH - CDR3 329 DIQYGNYYYGMDV Short Chain #2 330 MTWTPLLFLTLLLHCTGSLSELVVTQEPSLTVSPGGTV (CD3mAb VL-hCL) TLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKR APGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWY SNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANK ATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQ SNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS CD3mAb VL 331 ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWV QQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL CD3mAb VL - CDR1 332 RSSTGAVTTSNYAN CD3mAb VL - CDR2 333 GTNKRAP CD3mAb VL - CDR3 334 ALWYSNLWV
TABLE-US-00055 TABLE 54 SEC Profile/Expression level in 293E cells: Expression FIT-Ig Monomer % in SEC level (mg/L) FIT017a 97.03% 7.8
Functional Studies
[0493] Functional binding study: Functional binding data for FIT018a is provided below in Table 55.
TABLE-US-00056 TABLE 55 Functional binding data Ig Target Kon Koff KD IC50 Ofatumumab 1 (CD20) FIT018a CD3 mAb 2 (CD3e) 6.69E+05 8.86E−05 1.32E−10 FIT018a 7.79E+05 1.22E−04 1.57E−10
[0494] Cell based binding study: The binding activity of FIT018a to human or cynomolgus B cell and T cell were determined by flow cytometry using BD FACSVerse. Raji cell was used for detecting human B cell binding. Jurakt cell was used for detecting human T cell binding. Primary cynomolgus T cell was used for detecting cynomolgus T cell binding. HEK 293 cell transit transfected with cynoCD20 was used for detecting cynomolgus B cell binding. Cells were washed in PBS buffer containing 2% FBS. Cells were then aliquot and incubated with 1:5 serially diluted FIT018a on ice for 1 hr. The starting working concentration of FIT018a was 20 μg/ml. Cells were washed, resuspended and incubated with 1:100 diluted Alexa Fluor® 488 labeled mouse anti-human IgG1 (Invitrogen, Cat. No. A-10631) on ice protected from light for 1 hr. Cells were washed and signal was detected with a BD FACSVerse flow cytometer according to manufacture's protocols. These experiments demonstrate that FIT018a can bind to human B cell and T cell (Raji is human B cell line and Jurkat is human T cell line), see
[0495] B-cell depletion assay: The in vitro activity of FIT018a was measured by B3-cell depletion assay. Human PBMCs were isolated by Ficoll Paque Plus (GE HEALTHCARE, cat: GE17144002) according to manufacture's instruction. Target cell Raji was harvested and seeded to assay plate at 5×10.sup.4 per well. Antibodies were serially diluted and added to assay plate. 2.5×10.sup.5 per well PBMCs were added to assay plate and incubate for 2 days or 3 days. After incubation, B cell was detected by anti-CD19 antibody using FACS machine. The experiments demonstrate that FIT018a can induce B cell apoptosis, see
Example 13: Study of Anti-HER3/EGFR Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0496] FIT-Ig having specificity for HER3 and EGFR was constructed as in the foregoing Examples. This exemplary FIT-Ig and corresponding sequences are provided below in Table 56. Table 57 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.
TABLE-US-00057 TABLE 56 Amino acid sequences of additional exemplary FIT-Ig for HER3 and EGFR Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT019a - Ig Long Chain (patritu 335 MDMRVPAQLLGLLLLWFPGSRCDIEMTQSPDSLAVSLGE HER3(patritumab/ VL-hCk-PaniVH- RATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYW EGFR(Panitumumab) hCg1) ASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQ QYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGECQVQLQESGPGLVKPSETLSLTCTVSGGSVSS GDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK patritu VL 336 DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYL AWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFT LTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIK patritu VL - CDR1 337 RSSQSVLYSSSNRNYLA patritu VL - CDR2 338 WASTRES patritu VL - CDR3 339 QYYSTPRT PaniVH 340 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWI RQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKT QFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTV SS Pani VH - CDR1 341 SGDYYWT Pani VH - CDR2 342 HIYYSGNTNYNPSLKS Pani VH - CDR3 343 DRVTGAFDI Short Chain #1 344 MEFGLSWLFLVAILKGVQCQVQLQQWGAGLLKPSETLSL (Patritumab VH- TCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY CH1) NPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARD KWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSC Patritumab VH 345 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQ PPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQF SLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS Patritumab VH - 346 GYYWS CDR1 Patritumab VH - 347 EINHSGSTNYNPSLKS CDR2 Patritumab VH - 348 DKWTWYFDL CDR3 Short Chain #2 349 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGD (Pani VL-hCk) RVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSPRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHL PLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC Pani VL 350 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQK PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSL QPEDIATYFCQHFDHLPLAFGGGTKVEIK Pani VL - CDR1 351 QASQDISNYLN Pani VL - CDR2 352 DASNLET Pani VL - CDR3 353 QHFDHLPLA FIT019b - Ig Long Chain (Panitu 354 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGD EGFR VL-hCk-Patritu- RVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET (Panitumumab)/ hCg1) GVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLP HER3 LAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC (patritumab) LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG ECQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWI RQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKN QFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK Panitu VL 355 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQK PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSL QPEDIATYFCQHFDHLPLAFGGGTKVEIK Panitu VL - CDR1 356 QASQDISNYLN Panitu VL - CDR2 357 DASNLET Panitu VL - CDR3 358 QHFDHLPLA PatrituVH 359 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQ PPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQF SLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS Patritu VH - CDR1 360 GYYWS Patritu VH - CDR2 361 EINHSGSTNYNPSLKS Patritu VH - CDR3 362 DKWTWYFDL Short Chain #1 363 MEFGLSWLFLVAILKGVQCQVQLQESGPGLVKPSETLSL (Panitu VH-CH1) TCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNT NYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCV RDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSC Panitu VH 364 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWI RQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKT QFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTV SS Panitu VH - CDR1 365 DYYWT Panitu VH - CDR2 366 HIYYSGNTNYNPSLKS Panitu VH - CDR3 367 DRVTGAFDI Short Chain #2 368 MDMRVPAQLLGLLLLWFPGSRCDIEMTQSPDSLAVSLGE (Patritu VL-hCk) RATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYW ASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQ QYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC Patritu VL 369 DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYL AWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFT LTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIK Patritu VL - CDR1 370 RSSQSVLYSSSNRNYLA Patritu VL - CDR2 371 WASTRES Patritu VL - CDR3 372 QQYYSTPRT
TABLE-US-00058 TABLE 57 SEC Profile/Expression level in 293E cells: Expression level FIT-Ig Monomer % in SEC (mg/L) FIT019a >86% 15.6 FIT019b 96.7% 10.2
Functional Studies
[0497] Functional binding study: Functional binding data for FIT019a and FIT019b is provided below in Table 58 and Table 59, respectively.
TABLE-US-00059 TABLE 58 Functional binding data of FIT019a Ig Target Kon Koff KD IC50 Patritumab 1(Her3) 1.59E+05 4.80E−06 3.02E−11 FIT019a 1.57E+05 5.05E−06 3.21E−11 Panitumumab 2(EGFR) 4.45E+05 4.92E−04 1.10E−09 FIT019a 2.86E+05 3.91E−04 1.37E−09
TABLE-US-00060 TABLE 59 Functional binding data of FIT019b Ig Target Kon Koff KD IC50 Panitumumab 1(EGFR) 8.43E+04 5.10E−05 6.05E−10 FIT019b 2.84E+05 9.60E−05 3.38E−10 Patritumab 2(Her3) 3.17E+05 2.85E−04 9.00E−10 FIT019b 1.20E+05 2.37E−04 1.98E−09
[0498] Multiple binding study: A multiple binding study of FIT019a and FIT019b was carried out. The result is shown in
Example 14: Study of Anti-PD-L1/PD-1 Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0499] FIT-Ig having specificity for PD-L1 and PD-1 was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 60. Table 61 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.
TABLE-US-00061 TABLE 60 Amino acid sequences of additional exemplary FIT-Ig for PD-L1 and PD-1 Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT020b Long Chain (1B12 373 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE PD-L1 VL-hCk-Nivolu RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT (1B12)/PD-1 VH-hCg1Mut) GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP (nivolumab) TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE CQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVR QAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKN TLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK I1B12 VL 374 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWPTFGQGTKVEIK 1B12 VL - CDR1 375 RASQSVSSYLA 1B12 VL - CDR2 376 DASNRAT 1B12 VL - CDR3 377 QQRSNWPT NivoluVH 378 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQ APGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNT LFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS NivoluVH - CDR1 379 NSGMH NivoluVH - CDR2 380 VIWYDGSKRYYADSVKG NivoluVH - CDR3 381 NDDY Short Chain #1 382 MEFGLSWLFLVAILKGVQCQVQLVQSGAEVEKPGSSVKV (1B12 VH-CH1) SCKTSGDIFSSYAISWVRQAPGQGLEWMGGIIPIFGRAH YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCAR KFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSC 1B12 VH 383 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSSYAISWVRQ APGQGLEWMGGIIPIFGRAHYAQKFQGRVTITADESTST AYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGT TVTVSS 1B12 VH - CDR1 384 SYAIS 1B12 VH - CDR2 385 GIIPIFGRAHYAQKFQG 1B12 VH - CDR3 386 KFHFVSGSPFGMDV Short Chain #2 387 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE (Nivolu VL-hCK) RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP RTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC Nivolu VL 388 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQSSNWPRTFGQGTKVEIK Nivolu VL - CDR1 389 RASQSVSSYLA Nivolu VL - CDR2 390 DASNRAT Nivolu VL - CDR3 391 QQSSNWPRT
TABLE-US-00062 TABLE 61 SEC Profile/Expression level in 293E cells: Expression level FIT-Ig Monomer % in SEC (mg/L) FIT020b 100% 6.2
[0500] Affinity measurement by surface plasmon resonance: The kinetics of FIT-Ig binding to rhPD-L1 and rhPD-1 was determined by surface plasmon resonance with a Biacore X100 instrument (Biacore AB, Uppsala, Sweden) using HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25° C. Briefly, for rhPD-L1 and rhPD-1 was directly immobilized at 40 RU across a CM5 research grade biosensor chip using a standard amine coupling kit according to manufacturer's instructions. Rate constants were derived by making kinetic binding measurements at seven different antigen concentrations ranging from 1 to 40 nM. The equilibrium dissociation constant (M) of the reaction between FIT-Ig and the target proteins was then calculated from the kinetic rate constants by the following formula: KD=koff/kon. Aliquots of FIT-Ig/Ab samples were also simultaneously injected over a blank reference and reaction CM surface to record and subtract any nonspecific binding background to eliminate the majority of the refractive index change and injection noise. Surfaces were regenerated with two subsequent 25 ml injections of 10 mM Glycine (pH 1.5) at a flow rate of 10 μL/min. Functional binding data for FIT020b is provided below in Table 62.
TABLE-US-00063 TABLE 62 Functional binding data Ig Target Kon Koff KD IC50 1B12 1(PD-L1) 6.77E+05 2.72E−04 4.02E−10 FIT020b 2.92E+05 1.85E−04 6.34E−10 Nivolumab 2(PD1) 5.00E+05 1.76E−04 3.52E−10 FIT020b 8.89E+04 3.02E−04 3.39E−09
[0501] Functional activity test by MLR assay: Mixed lymphocyte reaction was performed using monocyte-derived dendritic cells from one donor and allogeneic CD4 T cells from another donor. The whole blood samples were collected from healthy donors, and PBMC were isolated from whole blood using Ficoll-Pague gradient centrifugation. On day 1, PBMC from one donor was isolated and diluted with serum-free RPMI 1640 at 1×10.sup.6/ml. The diluted PBMC was seeded into 6-well tissue culture plate at 3 ml/well and incubated for 3 h. Supernatant was removed and unattached cells were washed off. The attached monocyte were polarized into dendritic cells with 250 U/ml IL-4 and 500 U/ml GM-CSF in RPMI1640 with 10% FBS. The medium was replaced with fresh IL-4 and GM-CSF at day 4. At day 7, immature DC was collected and treated with 1 μg/ml LPS in RPMI 1640 with 10% FBS for additional 24 h for maturation. At Day 8, CD4 T cells were isolated from another donor PBMC by negative selection and adjusted to final concentration at 2×10.sup.6 cells/ml. Mature DC were treated with mitomycin C at 37° C. for 1.5 hr. Then DC were washed with PBS and adjusted to final concentration at 1×10.sup.6 cells/ml. CD4 T cells (Responder cells) were added into 96 well plate at 100 μl/well and pre-treated with test antibody at diluted concentration for 30 minutes. Then mature DC (Stimulator cells) were added into the well at 100 μl/well. The final volume of each well is 200 μl. The MLR were incubated at 37 degree for 72 hr for IL-2 test. The result is shown in
[0502] Multiple binding study: A multiple binding study of FIT020b was carried out. The result is shown in
Example 15: Study of Anti-CD20/CD-22 Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0503] FIT-Ig having specificity for CD20 and CD22 was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 63. Table 64 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.
TABLE-US-00064 TABLE 63 Amino acid sequences of additional exemplary FIT-Ig for CD20 and CD22 Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT021b Long Chain (Ofatu 392 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE CD20 VL-hCk-Epratu VH- RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT (Ofatumumab)/ hCg1) GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP CD22 ITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVC (Epratuzumab) LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG ECQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWV RQAPGQGLEWIGYINPRNDYTEYNQNFKDKATITADEST NTAYMELSSLRSEDTAFYFCARRDITTFYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK OfatuVL 393 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWPITFGQGTRLEIK Ofatu VL - CDR1 394 RASQSVSSYLA Ofatu VL - CDR2 395 DASNRAT Ofatu VL - CDR3 396 QQRSNWPIT Epratu VH 397 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQ APGQGLEWIGYINPRNDYTEYNQNFKDKATITADESTNT AYMELSSLRSEDTAFYFCARRDITTFYWGQGTTVTVSS Epratu VH - CDR1 398 SYWLH Epratu VH - CDR2 399 YINPRNDYTEYNQNFKD Epratu VH - CDR3 400 RDITTFY Short Chain #1 401 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGRSLRL (Ofatu VH-CH1) SCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIG YADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAK DIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSC Ofatu VH 402 EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQ APGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKS LYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTT VTVSS Ofatu VH - CDR1 403 DYAMH Ofatu VH - CDR2 404 TISWNSGSIGYADSVKG Ofatu VH - CDR3 405 DIQYGNYYYGMDV Short Chain #2 406 MDMRVPAQLLGLLLLWFPGSRCDIQLTQSPSSLSASVGD (Epratu VL-hCk) RVTMSCKSSQSVLYSANHKNYLAWYQQKPGKAPKLLIYW ASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCH QYLSSWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC Epratu VL 407 DIQLTQSPSSLSASVGDRVTMSCKSSQSVLYSANHKNYL AWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFT FTISSLQPEDIATYYCHQYLSSWTFGGGTKLEIK Epratu VL - CDR1 408 KSSQSVLYSANHKNYLA Epratu VL - CDR2 409 WASTRES Epratu VL - CDR3 410 HQYLSSWT
TABLE-US-00065 TABLE 64 SEC Profile/Expression level in 293E cells: Expression level FIT-Ig Monomer % in SEC (mg/L) FIT021b 100% 4.9
Functional Studies
[0504] Cell based binding study: The binding activity of FIT021b to B lymphoma cell lines Raji or Daudi were determined by flow cytometry using BD FACSVerse. Cells were washed in PBS buffer containing 2% FBS. Cells were then aliquot and incubated with 1:5 serially diluted FIT021b on ice for 1 hr. The starting working concentration of FIT021b was 20 μg/ml. Cells were washed, resuspended and incubated with 1:100 diluted Alexa Fluor® 488 labeled mouse anti-human IgG1 (Invitrogen, Cat. No. A-10631) on ice protected from light for 1 hr. Cells were washed and signal was detected with a BD FACSVerse flow cytometer according to manufacture's protocols. These experiments demonstrate that FIT021b can bind to B lymphoma cell lines Raji or Daudi, see
Example 16: Study of Anti-HER3/PD-1 Fabs-In-Tandem Immunoglobulin (FIT-h2)
[0505] FIT-Ig having specificity for HER3 and PD1 was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 65. Table 66 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.
TABLE-US-00066 TABLE 65 Amino acid sequences of additional exemplary FIT-Ig for HER3 and PD-1 Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT022a Long Chain (patritu 411 MDMRVPAQLLGLLLLWFPGSRCDIEMTQSPDSLAVSLGE HER3 VL-hCk-Nivolu RATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYW (patritumab)/ VH-hCg1mut) ASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQ PD-1 QYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG (nivolumab) TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGECQVQLVESGGGVVQPGRSLRLDCKASGITFSN SGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTI SRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK patrituVL 412 DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYL AWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFT LTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIK patritu VL - CDR1 413 RSSQSVLYSSSNRNYLA patritu VL - CDR2 414 WASTRES patritu VL - CDR3 415 QQYYSTPRT Nivolu VH 416 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQ APGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNT LFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS Nivolu VH - CDR1 417 NSGMH Nivolu VH - CDR2 418 VIWYDGSKRYYADSVKG Nivolu VH - CDR3 419 NDDY Short Chain #1 420 MEFGLSWLFLVAILKGVQCQVQLQQWGAGLLKPSETLSL (Patritumab-CH1) TCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARD KWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSC Patritumab VH 421 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQ PPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQF SLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS Patritumab VH - 422 GYYWS CDR1 Patritumab VH - 423 EINHSGSTNYNPSLKS CDR2 Patritumab VH - 424 DKWTWYFDL CDR3 Short Chain#2 425 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE (NivoVL-hCK) RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP RTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC Nivo VL 426 EIVLTQSPATLSLSPGERATLSCRASQSVSSYL AWYQQKPGQAPRLLIYDASNRATGIPARFSGSG SGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFG QGTKVEIK Nivo VL - CDR1 427 RASQSVSSYLA Nivo VL - CDR2 428 DASNRAT Nivo VL - CDR3 429 QQSSNWPRT
TABLE-US-00067 TABLE 66 SEC Profile/Expression level in 293E cells: Expression level FIT-Ig Monomer % in SEC (mg/L) FIT022a 100% 1.6
Functional Studies
[0506] Affinity measurement by surface plasmon resonance: The kinetics of FIT022a-Ig binding to Her-3-his and hPD-1-Fc was determined by surface plasmon resonance. The result is shown in Table 67.
TABLE-US-00068 TABLE 67 Functional binding data for FIT022a Ig Target Kon Koff KD IC50 Patritumab Her3-his 2.43E+05 3.12E−04 1.28E−09 FIT022a 2.68E+05 3.23E−04 1.21E−09 Nivolumab hPD1-Fc 5.00E+05 1.76E−04 3.52E−10 FIT022a 1.29E+05 3.01E−04 2.34E−09
[0507] Multiple binding study: A multiple binding study of FIT022a was carried out. The result is shown in
[0508] MLR functional assay: Mixed lymphocyte reaction was performed using monocyte-derived dendritic cells from one donor and allogeneic CD4 T cells from another donor. The whole blood samples were collected from healthy donors, and PBMC were isolated from whole blood using Ficoll-Pague gradient centrifugation. On day 1, PBMC from one donor was isolated and diluted with serum-free RPMI 1640 at 1×10.sup.6/ml. The diluted PBMC was seeded into 6-well tissue culture plate at 3 ml/well and incubated for 3 h. Supernatant was removed and unattached cells were washed off. The attached monocyte were polarized into dendritic cells with 250 U/ml IL-4 and 500 U/ml GM-CSF in RPMI1640 with 10% FBS. The medium was replaced with fresh IL-4 and GM-CSF at day 4. At day 7, immature DC was collected and treated with 1 μg/ml LPS in RPMI 1640 with 10% FBS for additional 24 h for maturation. At Day 8, CD4 T cells were isolated from another donor PBMC by negative selection and adjusted to final concentration at 2×10e6 cells/ml. Mature DC were treated with mitomycin C at 37° C. for 1.5 hr. Then DC were washed with PBS and adjusted to final concentration at 1×10.sup.6 cells/ml. CD4 T cells (Responder cells) were added into 96 well plate at 100 μl/well and pre-treated with test antibody at diluted concentration for 30 minutes. Then mature DC (Stimulator cells) were added into the well at 100 μl/well. The final volume of each well is 200 μl. The MLR were incubated at 37 degree for 72 hr for IL-2 test and 120 hr for IFN-gamma test respectively using ELISA. The result is shown in
Example 17: Study of Anti-cMet/PD-L1 Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0509] FIT-Ig having specificity for cMet and PD-L1 was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 68. Table 69 provides the expression level in 293E cells and the SEC profile for the FIT-Ig. Table 68. Amino acid sequences of additional exemplary FIT-Ig for cMet and PD-L1
TABLE-US-00069 TABLE 68 Amino acid sequences of additional exemplary FIT-Ig for cMet and PD-L1 Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT023a Long Chain(h1332 430 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGD cMet (h1332)/ VL-hCk-1B12 VH- RVTITCRASQGINTWLAWYQQKPGKAPKLLIYAASSLKS PD-L1 (1B12) hCg1) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFP LTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG ECQVQLVQSGAEVKKPGSSVKVSCKTSGDTFSSYAISWV RQAPGQGLEWMGGIIPIFGRAHYAQKFQGRVTITADEST STAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK h1332 VL 431 DIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQK PGKAPKLLIYAASSLKSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQANSFPLTFGGGTKVEIK h1332 VL - CDR1 432 RASQGINTWLA h1332 VL - CDR2 433 AASSLKS h1332 VL - CDR3 434 QQANSFPLT 1B12 VH 435 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSSYAISWVRQ APGQGLEWMGGIIPIFGRAHYAQKFQGRVTITADESTST AYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGT TVTVSS 1B12 VH-CDR1 436 SYAIS 1B12 VH-CDR2 437 GIIPIFGRAHY 1B12 VH-CDR3 438 KFHFVSGSPFGMDV Short Chain #1 439 MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASVKV (h1332 VH-CH1) SCKASGYTFTSYGFSWVRQAPGQGLEWMGWISASNGNTY YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR VYADYADYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSC h1332 VH 440 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGFSWVRQ APGQGLEWMGWISASNGNTYYAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARVYADYADYWGQGTLVTVSS h1332 VH - CDR1 441 SYGFS h1332 VH - CDR2 442 WISASNGNTYYAQKLQG h1332 VH - CDR3 443 VYADYADY Short Chain #2 444 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE (1B12 VL-hCK) RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C 1B12 VL 445 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWPTFGQGTKVEIK 1B12 VL-CDR1 446 RASQSVSSYLA 1B12 VL-CDR2 447 DASNRAT 1B12 VL-CDR3 448 QQRSNWPT
TABLE-US-00070 TABLE 69 SEC Profile/Expression level in 293E cells: Expression level FIT-Ig Monomer % in SEC (mg/L) FIT023a 97.49% 5.94
Functional Studies
[0510] Affinity measurement by surface plasmon resonance: The kinetics of FIT023a-Ig binding to cMet and PD-L1 was determined by surface plasmon resonance. The result is shown in Table 70.
TABLE-US-00071 TABLE 70 Functional binding data for FIT023a Ig Target Kon Koff KD H1332 1(cMet) 2.47E+05 5.39E−04 2.19E−09 FIT023a 3.42E+05 5.10E−04 1.49E−09 1B12 2(PD-L1) 3.21E+06 2.28E−03 7.08E−10 FIT023a 2.84E+06 2.08E−03 7.31E−10
[0511] Multiple binding study: A multiple binding study of FIT023a was carried out. The result is shown in
Example 18: Study of Anti-BTLA/PD-1 Fabs-In-Tandem Immunoglobulin (FIT-Ig)
[0512] FIT-Ig having specificity for BTLA and PD-L1 were constructed as in the foregoing Examples. This exemplary FIT-Ig and their corresponding sequences are provided below in Table 71. Table 72 provides the expression level in 293E cells and the SEC profile for the FIT-Ig. Both FIT024a and FIT024b have good purity after one-step Protein A purification.
TABLE-US-00072 TABLE 71 Amino acid sequences of additional exemplary FIT-Ig for BTLA and PD-1 Name Target (mAb) mAb1 (upper SEQ domain)/mAb2 ID (lower domain) Protein region NO Sequences FIT024a-Ig Long Chain (6A5 449 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGE BTLA (6A5)/ VL-hCk-Nivolu RATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRA PD-1 VH-hCg1mut) TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSS (Nivolumab) PPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGECQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMH WVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDN SKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 6A5 VL 450 EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQ KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR LEPEDFAVYYCQQYGSSPPITFGQGTRLEIK 6A5 VL - CDR1 451 RASQSVSSTYLA 6A5 VL - CDR2 452 GASSRAT 6A5 VL - CDR3 453 QQYGSSPPIT Nivolu VH 454 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQ APGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNT LFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS Nivolu VH - CDR1 455 NSGMH Nivolu VH - CDR2 456 VIWYDGSKRYYADSVKG Nivolu VH - CDR3 457 NDDY Short Chain #1 (6A5 458 MEFGLSWLFLVAILKGVQCQITLKESGPTLVKPTQTLTL VH-CH1) TCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIYWDDDK RYSPSLKSRLTITKDTSKNQVVLTMANMDPVDTATYYCA HIRITEVRGVIISYYGMDVWGQGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSC 6A5 VH 459 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWI RQPPGKALEWLALIYWDDDKRYSPSLKSRLTITKDTSKN QVVLTMANMDPVDTATYYCAHIRITEVRGVIISYYGMDV WGQGTTVTVSS 6A5 VH - CDR1 460 TSGVGVG 6A5 VH - CDR2 461 LIYWDDDKRYSPSLKS 6A5 VH - CDR3 462 IRITEVRGVIISYYGMDV Short Chain #2 463 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE (Nivolu VL-hCK) RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP RTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC Nivolu VL 464 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQSSNWPRTFGQGTKVEIK Nivolu VL-CDR1 465 RASQSVSSYLA Nivolu VL-CDR2 466 DASNRAT Nivolu VL-CDR3 467 QQSSNWPRT FIT024b-Ig Long Chain (Nivolu 468 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE PD-1 VL-hCk-6A5 VH- RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT (Nivolumab)/ hCg1mut) GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP BTLA (6A5) RTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG ECQITLKESGPTLVKPTQTLTLTCTFSGESLSTSGVGVG WIRQPPGKALEWLALIYWDDDKRYSPSLKSRLTITKDTS KNQVVLTMANMDPVDTATYYCAHIRITEVRGVIISYYGM DVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Nivolu VL 469 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQSSNWPRTFGQGTKVEIK Nivolu VL - CDR1 470 RASQSVSSYLA Nivolu VL - CDR2 471 DASNRAT Nivolu VL - CDR3 472 QQSSNWPRT 6A5 VH 473 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWI RQPPGKALEWLALIYWDDDKRYSPSLKSRLTITKDTSKN QVVLTMANMDPVDTATYYCAHIRITEVRGVIISYYGMDV WGQGTTVTVSS 6A5 VH - CDR1 474 TSGVGVG 6A5 VH - CDR2 475 LIYWDDDKRYSPSLKS 6A5 VH - CDR3 476 IRITEVRGVIISYYGMDV Short Chain #1 477 MEFGLSWLFLVAILKGVQCQVQLVESGGGVVQPGRSLRL (Nivolu VH-CH1) DCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRY YADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAT NDDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C Nivolu VH 478 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQ APGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNT LFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS Nivolu VH - CDR1 479 NSGMH Nivolu VH - CDR2 480 VIWYDGSKRYYADSVKG Nivolu VH - CDR3 481 NDDY Short Chain #2 (6A5 482 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGE VL-hCK) RATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRA TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSS PPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 6A5 VL 483 EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQ KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR LEPEDFAVYYCQQYGSSPPITFGQGTRLEIK 6A5 VL - CDR1 484 RASQSVSSTYLA 6A5 VL - CDR2 485 GASSRAT 6A5 VL - CDR3 486 QQYGSSPPIT
TABLE-US-00073 TABLE 72 SEC Profile/Expression level in 293E cells: Expression level FIT-Ig Monomer % in SEC (mg/L) FIT024a 99.68% 9.3 FIT024b 98.61% 13.0
Functional Studies
[0513] Affinity measurement by surface plasmon resonance: The kinetics of FIT024a-Ig and FIT024b-Ig binding to BTLA4 and PD-1 was determined by surface plasmon resonance. The result is shown in Table 73.
TABLE-US-00074 TABLE 73 Functional binding data for FIT024a and FIT024b Ig Target Kon Koff KD 6A5 BTLA-his 6.68E+04 7.34E−04 1.10E−08 FIT024a 6.16E+04 8.05E−04 1.31E−08 FIT024b 4.45E+04 8.14E−04 1.83E−08 Nivolumab hPD1-his 3.76E+05 1.39E−03 3.70E−09 FIT024a 1.76E+05 2.58E−03 1.46E−08 FIT024b 3.53E+05 1.48E−03 4.20E−09
[0514] Multiple binding study: A multiple binding study of FIT024a and FIT024b was carried out. The result is shown in
[0515] MLR functional assay: Mixed lymphocyte reaction was performed using monocyte-derived dendritic cells from one donor and allogeneic CD4 T cells from another donor. The whole blood samples were collected from healthy donors, and PBMC were isolated from whole blood using Ficoll-Pague gradient centrifugation. On day 1, PBMC from one donor was isolated and diluted with serum-free RPMI 1640 at 1×10.sup.6/ml. The diluted PBMC was seeded into 6-well tissue culture plate at 3 ml/well and incubated for 3 h. Supernatant was removed and unattached cells were washed off. The attached monocyte were polarized into dendritic cells with 250 U/ml IL-4 and 500 U/ml GM-CSF in RPMI1640 with 10% FBS. The medium was replaced with fresh IL-4 and GM-CSF at day 4. At day 7, immature DC was collected and treated with 1 μg/ml LPS in RPMI 1640 with 10% FBS for additional 24 h for maturation. At Day 8, CD4 T cells were isolated from another donor PBMC by negative selection and adjusted to final concentration at 2×10e6 cells/ml. Mature DC were treated with mitomycin C at 37° C. for 1.5 hr. Then DC were washed with PBS and adjusted to final concentration at 1×10.sup.6 cells/ml. CD4 T cells (Responder cells) were added into 96 well plate at 100 μl/well and pre-treated with test antibody at diluted concentration for 30 minutes. Then mature DC (Stimulator cells) were added into the well at 100 μl/well. The final volume of each well is 200 μl. The MLR were incubated at 37 degree for 72 hr for IL-2 test. The result is shown in
[0516] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference in its entirety.
[0517] Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.
[0518] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
[0519] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.