BISPECIFIC ANTIBODY AND USE THEREOF
20230235090 · 2023-07-27
Assignee
Inventors
- Lei Shi (Shanghai, CN)
- Chen ZHONG (Shanghai, CN)
- Xiaodong Wu (Shanghai, CN)
- Yun He (Shanghai, CN)
- Fei Chen (Shanghai, CN)
- Xiaocheng LV (Shanghai, CN)
- Jinli XIE (Shanghai, CN)
- Yiping Rong (Shanghai, CN)
- Bing HUANG (Shanghai, CN)
- Fangfang DU (Shanghai, CN)
- Jianxun ZHAO (Shanghai, CN)
Cpc classification
C07K16/468
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
C07K2317/732
CHEMISTRY; METALLURGY
C07K2317/64
CHEMISTRY; METALLURGY
A61K47/6879
HUMAN NECESSITIES
A61K47/6803
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
C07K16/2878
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
Provided are a bispecific antibody and use thereof. The bispecific antibody comprises a B7-H4-targeting antigen-binding domain and a 4-1BB-targeting antigen-binding domain. The bispecific antibody has one or two or three sites for binding to 4-1BB, along with a novel fully human B7-H4 antibody. The bispecific antibody specifically binds to tumor cells by targeting B7-H4, reducing toxicity induced by 4-1BB activation. In addition, the bispecific antibody of the present invention comprises a human Fc fragment, and thus retains the binding of Fc to FcRn and has a longer half-life.
Claims
1-25. (canceled)
26. A bispecific antibody comprising a B7-H4-targeting antigen-binding domain and a 4-1BB-targeting antigen-binding domain, wherein the B7-H4-targeting antigen-binding domain comprises HCDR1 with a sequence as set forth in SEQ ID NO: 16, HCDR2 with a sequence as set forth in SEQ ID NO: 60, HCDR3 with a sequence as set forth in SEQ ID NO: 84, LCDR1 with a sequence as set forth in SEQ ID NO: 112, LCDR2 with a sequence as set forth in SEQ ID NO: 118 and LCDR3 with a sequence as set forth in SEQ ID NO: 133; HCDR1 with a sequence as set forth in SEQ ID NO: 16, HCDR2 with a sequence as set forth in SEQ ID NO: 46, HCDR3 with a sequence as set forth in SEQ ID NO: 84, LCDR1 with a sequence as set forth in SEQ ID NO: 112, LCDR2 with a sequence as set forth in SEQ ID NO: 118 and LCDR3 with a sequence as set forth in SEQ ID NO: 131; or HCDR1 with a sequence as set forth in SEQ ID NO: 23, HCDR2 with a sequence as set forth in SEQ ID NO: 59, HCDR3 with a sequence as set forth in SEQ ID NO: 98, LCDR1 with a sequence as set forth in SEQ ID NO: 113, LCDR2 with a sequence as set forth in SEQ ID NO: 118 and LCDR3 with a sequence as set forth in SEQ ID NO: 132.
27. The bispecific antibody according to claim 26, wherein the B7-H4-targeting antigen-binding domain comprises a VH with a sequence as set forth in SEQ ID NO: 160 and a VL with a sequence as set forth in SEQ ID NO: 168; a VH with a sequence as set forth in SEQ ID NO: 142 and a VL with a sequence as set forth in SEQ ID NO: 166; a VH with a sequence as set forth in SEQ ID NO: 159 and a VL with a sequence as set forth in SEQ ID NO: 167; or, a VH with a sequence as set forth in SEQ ID NO: 159 and a VL with a sequence as set forth in SEQ ID NO: 169.
28. The bispecific antibody according to claim 26, wherein the B7-H4-targeting antigen-binding domain comprises a heavy chain with a sequence as set forth in SEQ ID NO: 192 and a light chain with a sequence as set forth in SEQ ID NO: 200; a heavy chain with a sequence as set forth in SEQ ID NO: 174 and a light chain with a sequence as set forth in SEQ ID NO: 198; a heavy chain with a sequence as set forth in SEQ ID NO: 191 and a light chain with a sequence as set forth in SEQ ID NO: 199; or a heavy chain with a sequence as set forth in SEQ ID NO: 191 and a light chain with a sequence as set forth in SEQ ID NO: 201.
29. The bispecific antibody according to claim 26, wherein the B7-H4-targeting antigen-binding domain is in the form of single VH, tandem VH, ScFv, Fab or IgG.
30. The bispecific antibody according to claim 26, wherein the 4-1BB-targeting antigen-binding domain comprises: HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 61 and 95, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 17, 47 and 85, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 48 and 86, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 49 and 86, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 19, 49 and 94, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 49 and 87, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 19, 50 and 88, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 20, 51 and 89, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 52 and 90, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 49 and 90, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 21, 53 and 91, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 21, 54 and 92, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 19, 55 and 93, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 22, 56 and 86, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 57 and 95, respectively; HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 19, 58 and 96, respectively; or HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 14, 43 and 81, respectively;
31. The bispecific antibody according to claim 30, wherein the 4-1BB-targeting antigen-binding domain comprises one or more heavy chain variable regions with a sequence as set forth in SEQ ID NO: 161, 143-144, 153-154, 145-152, 155-157, 139 or 284.
32. The bispecific antibody according to claim 30, wherein the 4-1BB-targeting antigen-binding domain comprises a heavy chain with a sequence as set forth in SEQ ID NO: 193, 175-176, 185-186, 177-184, 187-189, 285 or 171.
33. The bispecific antibody according to claim 30, wherein the 4-1BB-targeting antigen-binding domain further comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 109, 118, and 128, respectively.
34. The bispecific antibody according to claim 30, wherein the 4-1BB-targeting antigen-binding domain is in the form of single VH or tandem VH, HCAb or ScFv.
35. The bispecific antibody according to claim 26, wherein the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 60 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 61 and 95, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 60 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 17, 47 and 85, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 60 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 48 and 86, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 60 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 49 and 86, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 133, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 60 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 19, 49 and 94, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 109, 118 and 128, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 14, 43 and 81, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 23, 59 and 98, respectively; and the 4-1BB-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 109, 118 and 128, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 14, 43 and 81, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 17, 47 and 85, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 48 and 86, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 49 and 87, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 19, 50 and 88, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 20, 51 and 89, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 52 and 90, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 21, 53 and 91, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 21, 54 and 92, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 19, 55 and 93, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 49 and 86, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 19, 49 and 94, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 22, 56 and 86, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 112, 118 and 131, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 16, 46 and 84, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 57 and 95, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 23, 59 and 98, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 48 and 86, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 23, 59 and 98, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 19, 58 and 96, respectively; the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 23, 59 and 98, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 57 and 95, respectively; or the B7-H4-targeting antigen-binding domain comprises LCDR1, LCDR2 and LCDR3 with sequences as set forth in SEQ ID NOs: 113, 118 and 132, respectively, and HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 23, 59 and 98, respectively; and the 4-1BB-targeting antigen-binding domain comprises HCDR1, HCDR2 and HCDR3 with sequences as set forth in SEQ ID NOs: 18, 49 and 90, respectively.
36. The bispecific antibody according to claim 35, wherein the B7-H4-targeting antigen domain is in the form of IgG and the 4-1BB-targeting antigen-binding domain is in the form of single VH; the B7-H4-targeting antigen domain is in the form of IgG and the 4-1BB-targeting antigen-binding domain is in the form of ScFv; the B7-H4-targeting antigen domain is in the form of IgG and the 4-1BB-targeting antigen-binding domain is in the form of a tandem of 2 or 3 VHs; the B7-H4-targeting antigen domain is in the form of Fab and the 4-1BB-targeting antigen-binding domain is in the form of HCAb or HCAb and VH (in the form of HCAb-VH); or the B7-H4-targeting antigen domain is in the form of Fab and the 4-1BB-targeting antigen-binding domain is in the form of single VH, or a tandem of 2 or 3 VHs.
37. The bispecific antibody according to claim 35, wherein the bispecific antibody is in a form where: (1) the bispecific antibody in this form comprises a polypeptide chain 1 shown as a formula of VLB7-H4-CL, and a polypeptide chain 2 shown as a formula of VHB7-H4-CH1-hinge-CH2-CH3-linker-(VH4-1BB)n; (2) the bispecific antibody in this form comprises a polypeptide chain 1 shown as a formula of VLB7-H4-CL, and a polypeptide chain 2 shown as a formula of VHB7-H4-CH1-hinge-CH2-CH3-linker-VH4-1BB-linker-VL4-1BB; (3) the bispecific antibody in this form comprises a polypeptide chain 1 shown as a formula of VHB7-H4-CH1, and a polypeptide chain 2 shown as a formula of VLB7-H4-CL-linker-VH4-1BB-linker-CH2-CH3; (4) the bispecific antibody in this form comprises a polypeptide chain 1 shown as a formula of VHB7-H4-CH1, and a polypeptide chain 2 shown as a formula of VLB7-H4-CL-linker-VH4-1BB-linker-CH2-CH3-VH4-1BB; or (5) the bispecific antibody in this form comprises three polypeptide chains: a polypeptide chain 1 shown as a formula of VLB7-H4-CL, a polypeptide chain 2 shown as a formula of VHB7-H4-CH1-hinge-CH2-CH3, and a polypeptide chain 3 shown as a formula of VH4-1BB-linker-CH2-CH3 or (VH4-1BB)n-linker-CH2-CH3.
38. The bispecific antibody according to claim 37, wherein the linker is selected from the group consisting of SEQ ID NOs: 241-261, 282 and 288-289.
39. The bispecific antibody according to claim 37, wherein the linker in form (1) comprises a sequence as set forth in SEQ ID NO: 245; the linker in form (2) comprises a sequence as set forth in any one of SEQ ID NOs: 243 and 245-247 and SEQ ID NOs: 288-289; the linker in form (3) comprises a sequence as set forth in SEQ ID NO: 250; the linker in form (4) comprises a sequence as set forth in SEQ ID NO: 282; and the linker in form (5) comprises a sequence as set forth in SEQ ID NO: 245.
40. The bispecific antibody according to claim 37, wherein the amino acid sequence of polypeptide chain 1 is as set forth in SEQ ID NO: 200, and the amino acid sequence of polypeptide chain 2 is as set forth in any one of SEQ ID NOs: 237, 235-236, 218-225, 263-268, 274-281, 286 and 287; the amino acid sequence of polypeptide chain 1 is as set forth in SEQ ID NO: 198, and the amino acid sequence of polypeptide chain 2 is as set forth in any one of SEQ ID NOs: 202-215; the amino acid sequence of polypeptide chain 1 is as set forth in SEQ ID NO: 201, and the amino acid sequence of polypeptide chain 2 is as set forth in SEQ ID NO: 217, 230, 238, 240, 226, 262 or 239; the amino acid sequence of polypeptide chain 1 is as set forth in SEQ ID NO: 201, the amino acid sequence of polypeptide chain 2 is as set forth in SEQ ID NO: 227, and the amino acid sequence of polypeptide chain 3 is as set forth in SEQ ID NO: 228, 229, 231 or 232; or the amino acid sequence of polypeptide chain 1 is as set forth in SEQ ID NO: 233, and the amino acid sequence of polypeptide chain 2 is as set forth in any one of SEQ ID NOs: 234 and 269-273.
41. An isolated nucleic acid encoding the bispecific antibody according to claim 26.
42. A pharmaceutical composition comprising the bispecific antibody according to claim 26.
43. A chimeric antigen receptor comprising the bispecific antibody according to claim 26.
44. An antibody-drug conjugate comprising a cytotoxic agent and the bispecific antibody according to claim 26.
45. The antibody-drug conjugate according to claim 44, wherein the cytotoxic agent is MMAF or MMAE.
46. A method for treating and/or preventing a 4-1BB and/or B7-H4-mediated disease or disorder comprising administering to a patient in need thereof a therapeutically effective amount of the bispecific antibody according to claim 26, wherein the disease or disorder is a tumor.
47. The method according to claim 46, wherein the disease or disorder is breast cancer, ovarian cancer, endometrial cancer, renal cancer, melanoma, lung cancer, gastric cancer, liver cancer, esophageal cancer, cervical cancer, head and neck tumor, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma or colorectal cancer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0157] The present invention is further illustrated by the following examples, which are not intended to limit the present invention. Experimental procedures without specified conditions in the following examples are performed in accordance with conventional procedures and conditions, or in accordance with instructions.
[0158] In the present application, the term “antibody” generally refers to a protein comprising a moiety that binds to an antigen, and optionally a scaffold or framework moiety that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antibody to the antigen. An antibody may typically comprise an antibody light chain variable region (VL) or an antibody heavy chain variable region (VH), or both. For example, the “heavy-chain antibody” in the present application comprises no VL regions but comprises VH regions only. The VH or VL regions can be further divided into hypervariable regions termed complementarity determining regions (CDRs), which are scattered over more conserved regions termed framework regions (FRs). Each VH or VL can consist of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. Examples of the antibody include, but are not limited to, full-length antibodies, heavy-chain antibodies (HCAbs), antigen-binding fragments (Fab, Fab′, F(ab)2, Fv fragments, F(ab′).sub.2, scFv, di-scFv and/or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, fusion proteins, etc., provided that they exhibit the desired antigen-binding activity.
[0159] In the present application, the term “variable” generally refers to the fact that certain portions of the sequences of the variable domains of antibodies vary considerably, resulting in the binding and specificity of various particular antibodies to their particular antigens. However, variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light and heavy chain variable regions called CDRs or hypervariable regions (HVRs). FRs are more highly conserved portions of the variable domains. The variable domains of native heavy and light chains each comprise four FRs largely in a β-sheet configuration. The FRs are connected by three CDRs to form a loop connection, and in some cases to form part of a β-sheet structure. The CDRs in each chain are held in close proximity by the FRs and form, together with the CDRs from the other chain, antigen-binding sites of the antibody. The constant regions are not directly involved in the binding of the antibody to antigens, but they exhibit different effector functions, for example, being involved in antibody-dependent cytotoxicity of the antibody.
[0160] In this application, the term “fully human antibody” generally refers to an antibody that is expressed by a genetically engineered antibody gene-deleted animal into which the entire gene that encodes an antibody in human is transferred. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. The fully human antibody can greatly reduce the immune side effects caused in the human body by the heterologous antibody. Methods for obtaining fully human antibodies in the art can include phage display, transgenic mice, and the like.
[0161] As used herein, the term “nucleic acid” refers to a DNA molecule and an RNA molecule. It may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
[0162] In the present application, the term “specifically bind to” generally refers to that an antibody binds to an epitope via its antigen-binding domain, and that the binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind to” an antigen when the antibody more easily binds to an epitope via its antigen-binding domain than binds to a random, unrelated epitope.
[0163] In the present application, the term “Fab” generally refers to the portion of a conventional antibody (e.g., IgG) that binds to an antigen, including the heavy chain variable region VH, the light chain variable region VL, the heavy chain constant region domain CH1 and the light chain constant region CL of the antibody. In conventional antibodies, the C-terminus of VH is linked to the N-terminus of CH1 to form a heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form a light chain, and the C-terminus of CH1 is further linked to the hinge region and other constant region domains of the heavy chain to form a heavy chain. In some embodiments, “Fab” also refers to a variant structure of the Fab. For example, in certain embodiments, the C-terminus of VH is linked to the N-terminus of CL to form one polypeptide chain, and the C-terminus of VL is linked to the N-terminus of CH1 to form another polypeptide chain, in which case an Fab (cross VH/VL) structure is formed; in certain embodiments, CH1 of the Fab is not linked to the hinge region, but rather the C-terminus of CL is linked to the hinge region of the heavy chain, in which case an Fab (cross Fd/LC) structure is formed.
[0164] In the present application, the term “VH” generally refers to the heavy chain variable region VH domain of an antibody, i.e., the heavy chain variable region VH of a conventional antibody (H2L2 structure) from human or other animals, the heavy chain variable region VHH of a heavy-chain antibody (HCAb structure) from animals such as those of Camelidae species, or the heavy chain variable region VH of a fully human heavy-chain antibody (HCAb structure) produced using a Harbour HCAb transgenic mouse.
[0165] In the art, the CDRs of an antibody can be defined using a variety of methods, such as the Kabat scheme based on sequence variability (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health (U.S.), Bethesda, Md. (1991)), and the Chothia scheme based on the location of the structural loop regions (see A1-Lazikani et al., J Mol Biol 273: 927-948, 1997). In the present application, the Combined scheme comprising the Kabat scheme and the Chothia scheme can also be used to determine the amino acid residues in variable domain sequences and full-length antibody sequences (Table 1). In the present invention, each sequence is determined according to the Chothia scheme.
TABLE-US-00001 TABLE 1 Definition schemes available for the CDRs of the antibody of the present invention (see http://bioinf.org.uk/abs/) CDR Kabat scheme Chothia scheme Combined scheme LCDR1 L24--L34 L24--L34 L24--L34 LCDR2 L50--L56 L50--L56 L50--L56 LCDR3 L89--L97 L89--L97 L89--L97 HCDR1 H31--H35 H26--H32 H26--H35 HCDR2 H50--H65 H52--H56 H50--H65 HCDR3 H95--H102 H95--H102 H95--H102
[0166] Laa-Lbb can refer to an amino acid sequence from position aa (the Chothia scheme) to position bb (the Chothia scheme) beginning at the N-terminus of the light chain of the antibody; and Haa-Hbb can refer to an amino acid sequence from position aa (the Chothia scheme) to position bb (the Chothia scheme) beginning at the N-terminus of the heavy chain of the antibody. For example, L24-L34 can refer to the amino acid sequence from position 24 to position 34 according to the Chothia scheme beginning at the N-terminus of the light chain of the antibody; H26-H32 can refer to the amino acid sequence from position 26 to position 32 according to the Chothia scheme beginning at the N-terminus of the heavy chain of the antibody. The present invention uses the Chothia scheme to define the CDRs of an antibody.
[0167] Antibody Fc domain-mediated effector functions such as ADCC and CDC are also very important biological functions. Different IgG subtypes have different ADCC or CDC functions; for example, IgG1 and IgG3 have strong ADCC and CDC effects, while IgG2 and IgG4 have relatively weak effects. In addition, the inherent effector functions of Fc can be modulated by altering the binding ability of Fc to Fc receptors by amino acid mutation or modification. For example, the “LALA” double mutant (L234A/L235A) in IgG1 can significantly reduce the affinity for FcγRIIIA (CD16A), thereby reducing the ADCC effect. In addition, the P329G mutation can significantly reduce binding to a variety of Fcγ receptors (see Schlothauer T, Herter S, Koller C F, Grau-Richards S, Steinhart V, Spick C, Kubbies M, Klein C, Umaña P, Mössner E. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein EngDes Sel. 2016 October; 29(10):457-466. doi: 10.1093/protein/gzw040. Epub 2016 Aug. 29. PubMed PMID: 27578889). In the present application, in order to reduce the binding of B7-H4×4-1BB bispecific antibodies to Fcγ receptors, the “LALA” double mutant (L234A/L235A) or “LALAPG” triple mutant (L234A/L235A/P329G) is introduced into the Fc of these antibodies.
Example 1. Sequence Analysis, Expression and Purification, and Characterization and Analysis of Physicochemical Properties of Antibodies
[0168] 1.1 Sequence Analysis and Optimization of Antibodies
[0169] The sequences of the heavy chain variable domain of the antibody are derived from events such as gene rearrangements of germline gene V, D and J segments of heavy chain gene clusters and somatic hypermutations on chromosomes; the sequences of the light chain variable domain are derived from the events such as gene rearrangements of germline gene V, D and J segments of light chain gene clusters and somatic hypermutations. Gene rearrangement and somatic hypermutation are major factors in increasing antibody diversity. Antibodies derived from the same germline V gene segment may also produce different sequences, but with relatively high similarity overall. The germline gene segments that are likely to undergo gene rearrangement can be deduced from the antibody variable domain sequences using algorithms such as IMGT/DomainGapAlign (http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi) or NCBI/IgBLAST (https://www.ncbi.nlm nih.gov/igblast/).
[0170] Chemical modifications, sometimes introduced after amino acid chains of a protein or polypeptide is translated and synthesized in a cell, are called post-translational modifications (PTMs). For antibodies, some PTM sites are very conservative. For example, the conservative amino acid asparagine (Asn) at position 297 (EU numbering) of the constant domain of the human IgG1 antibody is often glycosylated to form a saccharide chain whose structure is critical for antibody structure and associated effector functions. However, PTMs may have a greater effect on antigen binding or result in changes in the physicochemical properties of the antibody, if they are present in the variable domains, particularly in the antigen binding regions (e.g., CDRs) of an antibody. For example, glycosylation, deamidation, isomerization, oxidation, and the like may increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development. Thus, it is very important for the development of therapeutic antibodies to avoid some potential PTMs. As experience has accumulated, it has been found that some PTMs are highly correlated with the composition of amino acid sequences, especially the “pattern” of the composition of adjacent amino acids, which makes it possible to predict potential PTMs from the primary amino acid sequences of a protein. For example, it can be predicted that there is an N-linked glycosylation site from the N-x-S/T sequence pattern (asparagine at the first position, any amino acid other than non-proline at the second position, and serine or threonine at the third position). The amino acid sequence patterns leading to PTMs may be derived from germline gene sequences, e.g., the human germline gene fragment IGHV3-33 naturally having a glycosylation pattern NST in the FR3 region; or they may also be derived from somatic hypermutations. For example, NGS or NLT may be a glycosylation site, NS may be a deamidation site, and DG may cause isomerization of aspartic acid.
[0171] The amino acid sequence patterns of PTMs may be disrupted by amino acid mutations, thereby reducing or eliminating the formation of specific PTMs. There are different methods for designing mutations depending on the antibody sequences and PTM sequence patterns. One method is to replace a “hot spot” amino acid (e.g., N or S in the NS pattern) with an amino acid with similar physicochemical properties (e.g., to mutate N into Q). If the PTM sequence pattern is derived from somatic hypermutations and is not present in the germline gene sequence, the other method can be to replace the sequence pattern with the corresponding germline gene sequence. In practice, a variety of methods for designing mutations may be used for the same PTM sequence pattern.
[0172] 1.2. Expression and Purification of Antibodies
[0173] This example describes a general method of antibody preparation in mammalian host cells (e.g., human embryonic kidney cell HEK293 or Chinese hamster ovary CHO cells and derived cells thereof) by using such techniques as transient transfection and expression, and affinity capture and separation. This method is applicable to an antibody of interest comprising an Fc region. The antibody of interest may consist of one or more protein polypeptide chains, and may be derived from one or more expression plasmids.
[0174] The amino acid sequences of the polypeptide chains of the antibody were converted into nucleotide sequences by codon optimization. The encoding nucleotide sequences were synthesized and cloned into expression vectors compatible with the host cell. The mammalian host cells were transfected simultaneously with plasmids encoding the polypeptide chains of the antibody in a particular ratio, and the recombinant antibody with correct folding and assembly of polypeptide chains could be obtained by the conventional recombinant protein expression and purification techniques. Specifically, FreeStyle™ 293-F cells (Thermo, #R79007) were expanded in FreeStyle™ F17 Expression Medium (Thermo, #A1383504). Before transient transfection, the cells were adjusted to a concentration of 6-8×10.sup.5 cells/mL, and cultured in a shaker at 37° C. with 8% CO.sub.2 for 24 h at a concentration of 1.2×10.sup.6 cells/mL. 30 mL of cultured cells were taken. Plasmids encoding the polypeptide chains of the antibody were mixed in a certain ratio, and a total of 30 μg of the plasmids (the ratio of the plasmids to cells was 1 μg:1 mL) were dissolved in 1.5 mL of Opti-MEM reduced serum medium (Thermo, #31985088). The resulting mixture was filtered through a 0.22 μm filter membrane for sterilization. Then, 1.5 mL of Opti-MEM was dissolved in 120 μL of 1 mg/mL PEI (Polysciences, #23966-2), and the mixture was left to stand for 5 min. PEI was slowly added to the plasmids, and the mixture was incubated at room temperature for 10 min. The mixed solution of plasmids and PEI was slowly added dropwise while shaking the culture flask, and the cells were cultured in a shaker at 37° C. with 8% CO.sub.2 for 5 days. Cell viability was measured after 5 days. The culture was collected and centrifuged at 3300 g for 10 min, and then the supernatant was collected and centrifuged at high speed to remove impurities. A gravity column (Bio-Rad, #7311550) containing MabSelect™ (GE Healthcare, #71-5020-91) was equilibrated with a PBS buffer (pH 7.4) and rinsed with 2-5 column volumes of PBS. The column was loaded with the supernatant sample, and rinsed with 5-10 column volumes of PBS buffer, followed by 0.1 M glycine at pH 3.5 to elute the target protein. The eluate was adjusted to neutrality with Tris-HCl at pH 8.0, and concentrated and buffer exchanged into PBS buffer or a buffer with other components with an ultrafiltration tube (Millipore, #UFC901024) to obtain a purified solution of the recombinant antibody. Finally, the purified antibody solution was determined for concentration using NanoDrop (Thermo, NanoDrop™ One), subpackaged and stored for later use.
[0175] 1.3 Analysis of Protein Purity and Polymers by SEC-HPLC
[0176] In this example, analytical size-exclusion chromatography (SEC) was used to analyze the protein sample for purity and polymer form. An analytical chromatography column TSKgel G3000SWx1 (Tosoh Bioscience, #08541, 5 μm, 7.8 mm×30 cm) was connected to a high-pressure liquid chromatograph HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with a PBS buffer at room temperature for at least 1 h. A proper amount of the protein sample (at least 10 μg) was filtered through a 0.22 μm filter membrane and then injected into the system, and an HPLC program was set: the sample was passed through the chromatography column with a PBS buffer at a flow rate of 1.0 mL/min for a maximum of 25 min. An analysis report was generated by the HPLC, with the retention time of the components with different molecular sizes in the sample reported.
[0177] 1.4 Determination of Thermostability of Protein Molecules by DSF
[0178] Differential scanning fluorimetry (DSF) is a commonly used high-throughput method for determining the thermostability of proteins. In this method, changes in the fluorescence intensity of the dye that binds to unfolded protein molecules were monitored using a real-time quantitative fluorescence PCR instrument to reflect the denaturation process of the protein and thus to reflect the thermostability of the protein. In this example, the thermal denaturation temperature (Tm) of a protein molecule was measured by DSF. 10 μg of protein was added to a 96-well PCR plate (Thermo, #AB-0700/W), followed by the addition of 2 μL of 100× diluted dye SYPRO™ (Invitrogen, #2008138), and then the mixture in each well was brought to a final volume of 40 μL by adding buffer. The PCR plate was sealed, placed in a real-time quantitative fluorescence PCR instrument (Bio-Rad CFX96 PCR System), and incubated at 25° C. for 5 min, then at a temperature gradually increased from 25° C. to 95° C. at a gradient of 0.2° C./0.2 min, and at a temperature decreased to 25° C. at the end of the test. The FRET scanning mode was used and data analysis was performed using Bio-Rad CFX Maestro software to calculate the Tm of the sample.
Example 2. Acquisition of Anti-B7-H4 Fully Human Antibodies
[0179] The Harbour H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying an immune repertoire of human immunoglobulins that produces antibodies with intact human antibody variable domains and rat constant domains.
[0180] Harbour H2L2 mice were subjected to multiple rounds of immunization with soluble recombinant human B7-H4-ECD-mFc fusion protein (Sino Biological, #10738-H05H), or with an immunogenic reagent prepared by mixing human B7-H4-overexpressing CHO-K1 cells transfected with mCD40L with an immunoadjuvant.
When the titer of the B7-H4-specific antibody in the serum of mice was detected to reach a certain level, spleen cells of the mice were harvested. Screening was performed using the hybridoma fusion technique, B cell clone technique and phage library technique. The monoclonal antibodies obtained by screening were further identified, and clones 80C8-2E9 and 1025_B-1H11 were selected according to parameters such as the binding ability to human B7-H4 and the binding ability to cynomolgus monkey B7-H4. The nucleotide sequences encoding the variable domains of the antibody molecules and the corresponding amino acid sequences were obtained through conventional sequencing means. The candidate antibody molecules were then subjected to sequence analysis and optimization to obtain several variant sequences. The VL and VH sequences of the antibody were fused to the corresponding human κ light chain constant region and IgG1 heavy chain constant region sequences and expressed to obtain recombinant fully human antibody molecules.
Anti-B7-H4 recombinant fully human IgG antibodies in this example are listed in Table 2.
TABLE-US-00002 TABLE 2 Sequence numbers of anti-B7-H4 H2L2 antibodies (CDRs are all defined according to the Chothia scheme) Antibody Light Heavy No. Notes chain chain VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 PR001476 80C8-2E9 clone 198 174 166 142 112 118 131 16 46 84 PR002037 1025_B-1H11 clone 199 191 167 159 113 118 132 23 59 98 PR002408 PR001476 variant 200 192 168 160 112 118 133 16 60 84 PR002410 PR002037 variant 201 191 169 159 113 118 132 23 59 98
[0181] In addition, a control antibody 1, also called 1D11.v1.9varC2, was designed by referring to patent WO2016040724A1 as a control in subsequent experiments.
TABLE-US-00003 TABLE 3 Sequence numbers of B7-H4 control antibody (SEQ ID NO:) Name of Antibody Light Heavy antibody No. chain chain VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 Positive PR000157 194 170 162 138 108 117 127 13 42 80 control 1
[0182] 2.1 FACS Assays for the Binding Ability of Anti-Human B7-H4 H2L2 Monoclonal Antibodies at the Cellular Level
[0183] This example is intended to investigate the in vitro binding activity of anti-human B7-H4 H2L2 monoclonal antibody to human/cynomolgus monkey/mouse B7-H4. Antibody binding experiments at the cellular level were conducted using a CHO-K1 cell strain overexpressing cynomolgus monkey B7-H4 (CHO-K1/cyno B7-H4, prepared in-house by Harbour Biomed), a CHO-K1 cell strain overexpressing mouse B7-H4 (CHO-K1/m B7-H4, prepared in-house by Harbour Biomed) and an SK-BR-3 cell line highly expressing human B7-H4 (ATCC® HTB-30). Briefly, the CHO-K1/cyno B7-H4 cells, CHO-K1/m B7-H4 cells or SK-BR-3 cells were digested and resuspended with PBS containing 2% BSA. The cell density was adjusted to 1×10.sup.6 cells/mL. The cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 μL/well, followed by the addition of test antibodies diluted in a 3-fold gradient at a concentration that was 2 times the final concentration, each at 100 μL/well. The cells were incubated at 4° C. for 2 h away from light. Thereafter, the cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% BSA, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Then 100 μL of a fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-06, 1:500 diluted) was added to each well. The plate was incubated away from light at 4° C. for 1 h. The cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% BSA, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Finally, the cells in each well were resuspended in 200 μL of pre-cooled PBS containing 2% BSA, and the fluorescence signal values were read using an ACEA Novocyte3000 flow cytometer.
[0184]
[0185]
[0186]
Example 3. Acquisition of 4-1BB Fully Human Antibodies
[0187] 3.1 Acquisition of Anti-4-1BB Fully Human HCAb Antibodies
[0188] The Harbour HCAb mouse (Harbour Antibodies BV, WO2010/109165A2) is a transgenic mouse carrying an immune repertoire of human immunoglobulins, capable of producing heavy chain-only antibodies that are only half the size of conventional IgG antibodies. The antibodies produced have only human antibody heavy chain variable domains and mouse Fc constant domains.
[0189] Harbour HCAb mice were subjected to multiple rounds of immunization with a soluble recombinant human 4-1BB-Fc fusion protein (ChemPartner) or human 4-1BB-overexpressing NIH-3T3 cells (ChemPartner). When the titer of the 4-1BB-specific antibody in the serum of mice was detected to reach a certain level, spleen cells of the mice were taken, from which B cells were isolated, and the CD138-positive plasma cells and human 4-1BB antigen-positive B cell populations were sorted using a BD FACS Ariall cell sorter. The human VH gene was amplified from plasma cells using conventional molecular biology techniques, and the amplified human VH gene fragments were constructed into mammalian cell expression plasmid pCAG vectors encoding the sequence of the heavy chain Fc region of the human IgG1 antibody. Mammal host cells (e.g., human embryonic kidney cell HEK293) were transfected with the plasmids and allowed to express antibodies to obtain a supernatant with fully human HCAb antibodies. Positive HCAb antibodies were identified by testing the supernatant with HCAb antibodies for binding to CHO-K1 cell CHO-K1/hu4-1BB highly expressing human 4-1BB by FACS. These HCAb antibodies were further identified, and several candidate HCAb antibody molecules were preferentially selected according to parameters such as the binding ability to human 4-1BB, the binding ability to cynomolgus monkey 4-1BB, and the T cell activation ability. The candidate HCAb antibody molecules were then subjected to sequence analysis and optimization to obtain several variant sequences. The VH sequence of the HCAb antibody and the Fc sequence of the heavy chain of human IgG1 were fused and expressed to obtain fully human recombinant HCAb antibody molecules. PR004469 is a PTM variant of PR001838. PR007381 is a germline variant of PR004469. The specific mutation sites are shown in Table 5.
TABLE-US-00004 TABLE 4 Sequence listing for anti-4-lBB recombinant fully human HCAb antibodies Antibody No. Heavy chain VH HCDR1 HCDR2 HCDR3 PR001758 175 143 17 47 85 PR001760 176 144 18 48 86 PR001763 177 145 18 49 87 PR001764 178 146 19 50 88 PR001767 179 147 20 51 89 PR001768 180 148 18 52 90 PR001771 181 149 18 49 90 PR001774 182 150 21 53 91 PR001780 183 151 21 54 92 PR001781 184 152 19 55 93 PR001830 185 153 18 49 86 PR001833 186 154 19 49 94 PR001836 187 155 22 56 86 PR001838 188 156 18 57 95 PR001840 189 157 19 58 96 PR001842 190 158 19 49 97 PR004469 193 161 18 61 95 PR007381 285 284 18 61 95
TABLE-US-00005 TABLE 5 Mutation site designs for 4-1BB HCAb sequences Initial Variable region Recombinant antibody Variant mutations antibody subtype PR001838 PR004469 G53A Human IgG1 PR001838 PR007381 F37V, P40A, E42G, Human IgG1 T43K, K46E, G53A
[0190] 3.2 Acquisition of Anti-4-1BB Fully Human H2L2 Antibodies
[0191] The Harbour H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying an immune repertoire of human immunoglobulins that produces antibodies with intact human antibody variable domains and rat constant domains. Harbour H2L2 mice were subjected to multiple rounds of immunization with a soluble recombinant human 4-1BB-Fc fusion protein. When the titer of the 4-1BB-specific antibody in the serum of mice was detected to reach a certain level, spleen cells of the mice were taken and fused with a myeloma cell line to obtain hybridoma cells. After multiple rounds of screening and cloning of the hybridoma cells, hybridoma cell strains expressing anti-4-1BB monoclonal antibody molecules were separated. The nucleotide sequences encoding the variable domains of the antibody molecules and the corresponding amino acid sequences were obtained through conventional sequencing means for hybridomas. The candidate antibody molecules were then subjected to sequence analysis and optimization to obtain several variant sequences. The VL and VH sequences of the antibody were fused to the corresponding human κ light chain constant region and IgG1 heavy chain constant region sequences and expressed to obtain recombinant fully human antibody molecules.
TABLE-US-00006 TABLE 6 Sequence listing for anti-4-1BB recombinant fully human IgG antibodies Antibody No. Light chain Heavy chain VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 PR000448 195 171 163 139 109 118 128 14 43 81
[0192] In addition, control antibodies were designed as controls in subsequent experiments.
TABLE-US-00007 TABLE 7 Sequence listing for control antibodies Name of Antibody Light Heavy antibody No. chain chain VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 Urelumab analog PR000628 197 173 165 141 111 120 130 14 45 83 Utomilumab PR000483 196 172 164 140 110 119 129 15 44 82 analog
[0193] 3.3 FACS Assays for the Binding Ability of Anti-Human 4-1BB Monoclonal Antibodies at the Cellular Level
[0194] This example is intended to investigate the in vitro binding activity of anti-human 4-1BB HCAb and H2L2 monoclonal antibodies to human and cynomolgus monkey 4-1BB. Antibody binding experiments at the cellular level were conducted using a CHO-K1 cell strain overexpressing human 4-1BB (CHO-K1-hu 4-1BB, Genescript) and a CHO-K1 cell strain overexpressing cynomolgus monkey 4-1BB (CHO-K1-cyno 4-1BB, Genescript). Briefly, the CHO-K1-hu 4-1BB and CHO-K1-cyno 4-1BB cells were digested and resuspended in DMEM complete medium, and the cell density was adjusted to 1×10.sup.6 cells/mL. The cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 μL/well, followed by the addition of test antibodies diluted in a 3-fold gradient at a concentration that was 2 times the final concentration, each at 100 μL/well. The cells were incubated at 4° C. for 1 h away from light. Thereafter, the cells in each well were rinsed twice with 100 μL of pre-cooled PBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Then 100 μL of a fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #: 109-545-06, 1:500 diluted) was added to each well. The plate was incubated away from light at 4° C. for 30 min. The cells in each well were rinsed twice with 100 μL of pre-cooled PBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Finally, the cells in each well were resuspended in 200 μL of pre-cooled PBS, and the fluorescence signal values were read using a BD FACS CANTOII.
[0195]
[0196]
[0197] As shown in
[0198] As shown in
[0199] 3.4 Antigen-Binding Proteins can Activate the 4-1BB Pathway In Vitro
[0200] CHO-K1 cells overexpressing human CD32b (CHO-K1/CD32b, Genscript, #M00600) were treated with 10 μg/mL mitomycin (Beijing Ruitaibio, #10107409001) at 37° C. for 30 min and then washed 4 times with F-12K culture medium containing 10% FBS. The treated cells were placed in a 96-well plate at 1.5×10.sup.4 cells/well and incubated in an incubator at 37° C. overnight. The next day, human CD3 positive T cells were isolated from human PBMCs using an MACS kit (Miltenyi Biotec, #130-096-535). The number of the cells was determined firstly, and then corresponding amounts of MACS buffer and Pan-T cell biotin antibody were added according to the number of the cells. The mixture was well mixed and left to stand at 4° C. for 5 min. Then, a corresponding amount of magnetic microbeads was added, and the mixture was left to stand at 4° C. for 10 min What passed through the LS column were CD3 positive T cells. The previous day's culture medium was washed off the 96-well plate, and purified T cells were added at 1×10.sup.5 cells/well. Then a 4-1BB antibody or a control antibody was added at a corresponding concentration, and OKT3 (eBiosciences, #16-0037-85) was added at a final concentration of 0.3 μg/mL. The wells were cultured in an incubator at 37° C. for 72 h. After 72 hours, the supernatant was collected and assayed for IFN-γ content using an ELISA kit (Invitrogen, #88-7316-88). A coating antibody was added to a 96-well flat-bottom plate, and the plate was incubated at 4° C. overnight. The next day, ELISA buffer was added, and the plate was incubated at room temperature for 1 h. The collected supernatant was added, and the plated was incubated at room temperature for 2 h. The plate was washed twice, and test antibodies were added. The plate was incubated at room temperature for 1 h. The plate was washed twice, and HRP-streptavidin was added. The plate was incubated at room temperature for 1 h. Then TMB substrate was added, and ELISA stop buffer (BBI, #E661006-0200) was added later. The absorbance values at 450 nm and 570 nm (0D450-0D570) were read using a microplate reader (PerkinElemer, #Enspire), and the IFN-γ concentration was calculated.
[0201] The results are shown in
Example 4: Preparation of B7-H4×4-1BB Bispecific Antibodies
[0202] The anti-B7-H4 antibodies selected in Example 2 and the anti-4-1BB antibodies selected in Example 3 were combined and used for the preparation of bispecific antibodies, which could bind to two targets simultaneously, with one terminus being capable of recognizing B7-H4 specifically expressed on tumor cell surfaces and the other terminus being capable of binding to a 4-1BB molecule on T cells. After binding to the surface of a tumor cell, the B7-H4×4-1BB bispecific antibody molecule can recruit and activate T cells in the vicinity of the tumor cell, thereby killing the tumor cell.
[0203] The B7-H4×4-1BB bispecific antibodies prepared in this example include a variety of molecular structures.
[0204]
[0205] 4.1 Construction of Bispecific Antibodies of IgG-scFv Tetravalent Symmetric Structure
[0206] Bispecific antibodies of an IgG-scFv tetravalent symmetric structure were constructed using anti-B7-H4 H2L2 antibodies and anti-4-1BB H2L2 antibodies. A binding protein of an IgG-scFv tetravalent symmetric structure (as shown in
[0207] 4.2 Construction of Bispecific Antibodies of IgG-VH Tetravalent Symmetric Structure
[0208] Bispecific antibodies of an IgG-VH tetravalent symmetric structure were constructed using anti-B7-H4 H2L2 antibodies and anti-4-1BB HCAb antibodies. A binding protein of an IgG-VH tetravalent symmetric structure (as shown in
[0209] 4.3 Construction of Bispecific Antibodies of IgG-VH-VH Hexavalent Symmetric Structure
[0210] Bispecific antibodies of an IgG-VH-VH hexavalent symmetric structure were constructed using anti-B7-H4 H2L2 antibodies and anti-4-1BB HCAb antibodies. A binding protein of an IgG-VH-VH hexavalent symmetric structure (as shown in
[0211] 4.4 Construction of Bispecific Antibody Molecules of Fab-HCAb Structure
[0212] Bispecific antibodies of an Fab-HCAb symmetric structure were constructed using anti-B7-H4 H2L2 antibodies and anti-4-1BB HCAb antibodies. Two different structures were included: Fab(CL)-VH-Fc (as shown in
[0213] 4.5 Construction of Fab-Fc-VH(n) Asymmetric Structure
[0214] Bispecific antibodies of an Fab-Fc-VH(n) asymmetric structure were constructed using anti-B7-H4 H2L2 antibodies and anti-4-1BB HCAb antibodies. A binding protein of an Fab-Fc-VH(n) asymmetric structure (as shown in
[0215] To minimize the formation of byproducts with mismatched heavy chains (e.g., mismatching of two heavy chains of the anti-4-1BB antibody or two heavy chains of the anti-B7-H4 antibody), a mutant heterodimeric Fc region carrying a “knob-hole” mutation and a modified disulfide bond was used, as described in WO2009080251 and WO2009080252.
[0216] 4.6 Construction of Fab-VH-Fc-VH Symmetric Structures
[0217] Bispecific antibodies of an Fab-VH-Fc-VH symmetric structure were constructed using anti-B7-H4 H2L2 antibodies and anti-4-1BB HCAb antibodies. A binding protein of an Fab-VH-Fc-VH symmetric structure (as shown in
TABLE-US-00008 TABLE 8 Sequence listing for linker peptides Name of linker Length of Sequence of Sequence No. peptide linker peptide linker peptide SEQ ID NO: GS_4 4 GSGS 241 GS_5 5 GGGGS 242 GS_6 6 GGSGGS 243 GS_7 7 GGGGSGS 244 GS_15 15 GGGGSGGGGSGGGGS 245 GS_20 20 GGGGSGGGGSGGGGSGGGGS 246 GS_25 25 GGGGSGGGGSGGGGSGGGGSGG 247 GGS Human IgG1 15 EPKSCDKTHTCPPCP 248 hinge Human IgG1 15 EPKSSDKTHTCPPCP 249 hinge (C220S) H1_15 15 EPKSSDKTHTPPPPP 250 G5-LH 15 GGGGGDKTHTCPPCP 251 H1_15-RT 17 EPKSSDKTHTPPPPPRT 252 L-GS_15-RT 18 LGGGGSGGGGSGGGGSRT 253 L-H1_15-RT 18 LEPKSSDKTHTPPPPPRT 254 KL-H1_15-RT 19 KLEPKSSDKTHTPPPPPRT 255 KL-H1_15-AS 19 KLEPKSSDKTHTPPPPPAS 256 RT-GS_5-KL 9 RTGGGGSKL 257 RT-GS_15-KL 19 RTGGGGSGGGGSGGGGSKL 258 RT-GS_25-KL 29 RTGGGGSGGGGSGGGGSGGGGS 259 GGGGSKL EPKSSD 6 EPKSSD 260 AS-GS_15 17 ASGGGGSGGGGSGGGGS 261 H2_13 13 ERKCCVECPPCP 282 GS_2 2 GS (G2S)2 6 GGSGGS 288 (G4S)2 10 GGGGSGGGGS 289
[0218] The amino acid sequences of the polypeptide chains of the bispecific antibody molecules obtained in the present invention are shown in the sequence listing below.
TABLE-US-00009 TABLE 9 Sequence listing for the bispecific antibody molecules obtained in the present invention Polypeptide Polypeptide Polypeptide Antibody No. chain 1 chain 2 chain 3 PR002789 198 202 PR002972 201 217 PR002790 198 203 PR002791 198 204 PR002792 198 205 PR002793 198 206 PR002794 198 207 PR002795 198 208 PR002798 198 209 PR002800 198 210 PR002801 198 211 PR002802 198 212 PR002804 198 213 PR002805 198 214 PR002806 198 215 PR002808 198 216 PR003334 200 218 PR003335 200 219 PR003336 200 220 PR003337 200 221 PR003338 200 222 PR004162 201 230 PR004357 201 238 PR004359 201 240 PR004280 200 235 PR004281 200 236 PR004282 200 237 PR003487 200 223 PR003488 200 224 PR003489 200 225 PR004158 201 226 PR004358 201 239 PR004160 201 227 228 PR004161 201 227 229 PR004181 201 227 231 PR004182 201 227 232 PR004279 233 234 PR004995 201 262 PR005183 200 263 PR005184 200 264 PR005185 200 265 PR005186 200 266 PR005187 200 267 PR005188 200 268 PR005189 233 269 PR005190 233 270 PR005827 200 274 PR005828 200 275 PR005829 200 276 PR005830 200 277 PR005649 233 271 PR005650 233 272 PR005651 233 273 PR005838 200 278 PR005839 200 279 PR005866 200 280 PR007165 200 281 PR007379 200 286 PR007380 200 287
[0219] The CDR sequence numbers of the antigen domains of the B7-H4×4-1BB bispecific antibodies are shown in the table below. In Table 10, the number 1# refers to a B7-H4-binding antigen domain, and the number 2# refers to a 4-1BB-binding antigen domain.
TABLE-US-00010 TABLE 10 CDR sequence numbers of antigen-binding domains of bispecific antibodies (SEQ ID NO:) Structure Antigen-binding No. Antibody No. domain No. LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 A PR002789 #1 112 118 131 16 46 84 #2 109 118 128 14 43 81 A PR002972 #1 113 118 132 23 59 98 #2 109 118 128 14 43 81 B PR002790 #1 112 118 131 16 46 84 #2 17 47 85 B PR002791 #1 112 118 131 16 46 84 #2 18 48 86 B PR002792 #1 112 118 131 16 46 84 #2 18 49 87 B PR002793 #1 112 118 131 16 46 84 #2 19 50 88 B PR002794 #1 112 118 131 16 46 84 #2 20 51 89 B PR002795 #1 112 118 131 16 46 84 #2 18 52 90 B PR002798 #1 112 118 131 16 46 84 #2 21 53 91 B PR002800 #1 112 118 131 16 46 84 #2 21 54 92 B PR002801 #1 112 118 131 16 46 84 #2 19 55 93 B PR002802 #1 112 118 131 16 46 84 #2 18 49 86 B PR002804 #1 112 118 131 16 46 84 #2 19 49 94 B PR002805 #1 112 118 131 16 46 84 #2 22 56 86 B PR002806 #1 112 118 131 16 46 84 #2 18 57 95 B PR002808 #1 112 118 131 16 46 84 #2 19 49 97 B PR003334 #1 112 118 133 16 60 84 #2 17 47 85 B PR003335 #1 112 118 133 16 60 84 #2 18 48 86 B PR003336 #1 112 118 133 16 60 84 #2 18 49 86 B PR003337 #1 112 118 133 16 60 84 #2 19 49 94 B PR003338, #1 112 118 133 16 60 84 PR004280, #2 18 61 95 PR004281, PR004282, PR005183, PR005184, PR005185, PR005186, PR005187, PR005188, PR005827, PR005828, PR005829, PR005830, PR005838, PR005839, PR005866 B PR004162 #1 113 118 132 23 59 98 #2 18 48 86 B PR004357 #1 113 118 132 23 59 98 #2 19 58 96 B PR004359 #1 113 118 132 23 59 98 #2 18 57 95 B PR004995 #1 113 118 132 23 59 98 #2 18 61 95 C PR003487, #1 112 118 133 16 60 84 PR003488, #2 18 48 86 PR003489 C PR004158 #1 113 118 132 23 59 98 #2 18 48 86 C PR004358 #1 113 118 132 23 59 98 #2 19 58 96 D PR004279, #1 112 118 133 16 60 84 PR005189, #2 18 48 86 PR005649 D PR005190 #1 112 118 133 16 60 84 #2 18 61 95 E PR007165 #1 112 118 133 16 60 84 #2 18 48 86 G PR004160 #1 113 118 132 23 59 98 #2 18 48 86 H PR004161 #1 113 118 132 23 59 98 #2 18 48 86 H PR004181 #1 113 118 132 23 59 98 #2 18 49 90 H PR004182 #1 113 118 132 23 59 98 #2 19 58 96 I PR005650 #1 112 118 133 16 60 84 #2 18 48 86 I PR005651 #1 112 118 133 16 60 84 #2 18 61 95
[0220] Tables 11-(A)-(F) show the structures of the bispecific antibody molecules of the present invention.
TABLE-US-00011 TABLE 11-(A) B7-H4 × 4-1BB bispecific antibody molecules of an IgG-scFv symmetric structure First linker Second linker peptide peptide Length Length Bispecific B7-H4 4-1BB (between (between of first of second antibody antibody antibody Structure of Fc and VH_B and Fc type linker linker molecules (IgG) (scFv) scFv end scFv) VL_B) (mutation) peptide peptide PR002789 PR001476 PR000448 VH-linker H1_15-RT GS_15 Human IgG1 17 15 peptide-VL (L234A, L235A, P329G) PR002972 PR002410 PR000448 VH-linker H1_15-RT GS_15 Human IgG1 17 15 peptide-VL (L234A, L235A, P329G)
TABLE-US-00012 TABLE 11-(B) B7-H4 × 4-1BB bispecific antibody molecules of an IgG-VH tetravalent symmetric structure Linker peptide Bispecific B7-H4 4-1BB (between Length antibody antibody antibody VH_B position VH_B Fc type of linker molecule (IgG) (VH_B) relative to IgG and IgG) (mutation) peptide PR002790 PR001476 PR001758 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002791 PR001476 PR001760 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002792 PR001476 PR001763 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002793 PR001476 PR001764 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002794 PR001476 PR001767 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002795 PR001476 PR001768 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002798 PR001476 PR001774 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002800 PR001476 PR001780 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002801 PR001476 PR001781 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002802 PR001476 PR001830 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002804 PR001476 PR001833 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002805 PR001476 PR001836 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002806 PR001476 PR001838 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR002808 PR001476 PR001842 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR003334 PR002408 PR001758 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A) PR003335 PR002408 PR001760 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A) PR003336 PR002408 PR001830 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A) PR003337 PR002408 PR001833 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A) PR003338 PR002408 PR004469 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A) PR004162 PR002410 PR001760 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A) PR004357 PR002410 PR001840 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A, P329G) PR004359 PR002410 PR001838 C-terminus of H1_15-RT Human IgG1 17 heavy chain (L234A, L235A) PR004280 PR002408 PR004469 C-terminus of EPKSSD Human IgG1 6 heavy chain (L234A, L235A) PR004281 PR002408 PR004469 C-terminus of None Human IgG1 0 heavy chain (L234A, L235A) PR004282 PR002408 PR004469 C-terminus of GS_6 Human IgG1 6 heavy chain (L234A, L235A) PR004995 PR002410 PR004469 C-terminus of None Human IgG1 0 heavy chain (L234A, L235A) PR005183 PR002408 PR004469 C-terminus of None Human IgG1 0 heavy chain (L234A, L235A, P329G) PR005184 PR002408 PR004469 C-terminus of None Human IgG1 0 heavy chain (del233-235) PR005185 PR002408 PR004469 C-terminus of None Human hIgG4 0 heavy chain (S228P, FALA) PR005186 PR002408 PR004469 C-terminus of None Human IgG1 0 heavy chain (L234A, G237A) PR005187 PR002408 PR004469 C-terminus of None Human IgG1 0 heavy chain (LALA, YTE) PR005188 PR002408 PR004469 C-terminus of None Human IgG1 0 heavy chain (LALA, DHS) PR005827 PR002408 PR004469 C-terminus of (G4S)2 Human IgG1 10 heavy chain (L234A, L235A) PR005828 PR002408 PR004469 C-terminus of GS_15 Human IgG1 15 heavy chain (L234A, L235A) PR005829 PR002408 PR004469 C-terminus of GS_20 Human IgG1 20 heavy chain (L234A, L235A) PR005830 PR002408 PR004469 C-terminus of GS_25 Human IgG1 25 heavy chain (L234A, L235A) PR005838 PR002408 PR004469 C-terminus of GS_6 Human IgG1 6 heavy chain (L234A, L235A, G237A) PR005839 PR002408 PR004469 C-terminus of GS_6 Human IgG1 6 heavy chain (L234A, G237A) PR005866 PR002408 PR004469 C-terminus of (G4S)2 Human IgG1 10 heavy chain (N297A) PR007379 PR002408 PR004469 VH C-terminus of (G2S)2 Human IgG1 6 (H:gm) heavy chain (L234A, L235A) PR007380 PR002408 PR004469_VH_gm C-terminus of (G2S)2 Human IgG1 6 (H:gm) heavy chain (L234A, L235A)
TABLE-US-00013 TABLE 11-(C) B7-H4 × 4-1BB bispecific antibody molecules of an IgG-VH-VH hexavalent symmetric structure First linker Second linker VH_B peptide peptide Length Length Bispecific B7-H4 4-1BB position (between (between of first of second antibody antibody antibody relative VH_B and VH_B and Fc type linker linker molecules (IgG) (VH_B) to IgG IgG) VH_B) (mutation) peptide peptide PR003487 PR002408 PR001760 C-terminus of H1_15-RT None Human IgG1 17 0 heavy chain (L234A, L235A) PR003488 PR002408 PR001760 C-terminus of H1_15-RT GS_5 Human IgG1 17 5 heavy chain (L234A, L235A) PR003489 PR002408 PR001760 C-terminus of H1_15-RT GS_15 Human IgG1 17 15 heavy chain (L234A, L235A) PR004158 PR002410 PR001760 C-terminus of H1_15-RT GS_5 Human IgG1 17 5 heavy chain (L234A, L235A) PR004358 PR002410 PR001840 C-terminus of H1_15-RT GS_5 Human IgG1 17 5 heavy chain (L234A, L235A, P329G)
TABLE-US-00014 TABLE 11-(D) B7-H4 × 4-1BB bispecific antibody molecules of an Fab-HCAb tetravalent symmetric structure First linker Second linker peptide peptide Length Length Bispecific B7-H4 4-1BB (between (between of first of second antibody antibody antibody Fab and VH_B Fc type linker linker molecules (IgG) (VH_B) VH_B) and CH2) (mutation) peptide peptide PR004279 PR002408 PR001760 H1_15 Human IgG1 Human IgG1 15 15 hinge (C220S) (L234A, L235A) PR005189 PR002408 PR001760 None Human IgG1 Human IgG1 0 15 hinge (C220S) (L234A, L235A) PR005190 PR002408 PR004469 H1_15 Human IgG1 Human IgG1 15 15 hinge (C220S) (L234A, L235A) PR005649 PR002408 PR001760 None Human IgG1 Human IgG1 0 15 hinge (C220S) (L234A, L235A) PR007165 PR002408 PR001760 None Human IgG1 Human IgG1 0 15 hinge (C220S) (L234A, L235A)
TABLE-US-00015 TABLE 11-(E) B7-H4 × 4-1BB bispecific antibody molecules of an Fab-Fc-VH(n) asymmetric structure Length Length Bispecific B7-H4 4-1BB Number of First Second Fc type of Fc type of of first of second antibody antibody antibody repetition linker linker Fab end VH_B end linker linker molecules (IgG) (VH_B) n for VH_B peptide peptide (mutation) (mutation) peptide peptide PR004160 PR002410 PR001760 2 GS_15 None Human IgG1 Human IgG1 15 0 (knob, LALA) (hole, LALA) PR004161 PR002410 PR001760 3 GS_15 AS-GS_15 Human IgG1 Human IgG1 15 17 (knob, LALA) (hole, LALA) PR004181 PR002410 PR001771 3 GS_15 GS_15 Human IgG1 Human IgG1 15 15 (knob, LALA) (hole, LALA) PR004182 PR002410 PR001840 3 GS_15 GS_15 Human IgG1 Human IgG1 15 15 (knob, LALA) (hole, LALA)
TABLE-US-00016 TABLE 11-(F) B7-H4 × 4-1BB bispecific antibody molecules of an Fab-VH-Fc-VH structure Length Length Bispecific B7-H4 4-1BB First Second of first of second antibody antibody antibody linker linker Fc type linker linker molecules (IgG) (VH_B) peptide peptide (mutation) peptide peptide PR005650 PR002408 PR001760 None None Human IgG1 0 0 (L234A, L235A) PR005651 PR002408 PR004469 None None Human IgG1 0 0 (L234A, L235A)
[0221] Tables 12-(A)-(F) show the protein expression and physicochemical properties of the bispecific antibody molecules of the present invention.
TABLE-US-00017 TABLE 12-(A) Expression and physicochemical properties of bispecific antibody molecule proteins of an IgG-scFv symmetric structure Plasmid transfection Bispecific Expression ratio (short Yield (mg/L) antibody system and chain:long after first HPLC-SEC DSF Tm1 molecules volume chain) purification purity (%) (° C.) PR002789 HEK293F (30 ml) 3:2 23 59 51.4 PR002972 HEK293F (30 ml) 3:2 59 50.6
TABLE-US-00018 TABLE 12-(B) Expression and physicochemical properties of bispecific antibody molecule proteins of an IgG-VH symmetric structure Plasmid transfection Bispecific ratio (short Yield (mg/L) antibody Expression system and chain:long after first HPLC-SEC molecules volume chain) purification purity (%) PR002790 Expi293F (10 ml) 3:2 74 95 PR002791 Expi293F (10 ml) 3:2 60 95 PR002792 Expi293F (10 ml) 3:2 52 92 PR002793 Expi293F (10 ml) 3:2 49 92 PR002794 Expi293F (10 ml) 3:2 47 94 PR002795 Expi293F (10 ml) 3:2 69 93 PR002798 Expi293F (10 ml) 3:2 67 94 PR002800 Expi293F (10 ml) 3:2 73 85 PR002801 Expi293F (10 ml) 3:2 57 89 PR002802 Expi293F (10 ml) 3:2 33 76 PR002804 Expi293F (10 ml) 3:2 26 83 PR002805 Expi293F (10 ml) 3:2 3 83 PR002806 Expi293F (10 ml) 3:2 20 88 PR002808 Expi293F (10 ml) 3:2 16 71 PR003334 Expi-CHOs (200 ml) 3:2 38.2 98 PR003335 Expi-CHOs (200 ml) 3:2 68.9 99 PR003336 HEK293-F (30 ml) 3:2 66.47 91 PR003337 HEK293-F (30 ml) 3:2 91.41 93 PR003338 HEK293-F (30 ml) 3:2 74.6 98 PR004162 HEK293-F (30 ml) 3:2 57 44.81 PR004357 HEK293-F (30 ml) 3:2 66 47.53 PR004359 HEK293-F (30 ml) 3:2 81 96.82 PR004280 HEK293-F (30 ml) 3:2 39.5 98.63 PR004281 HEK293-F (30 ml) 3:2 28.5 98.73 PR004282 HEK293-F (30 ml) 3:2 34.25 99.09 PR004995 293-6E (40 ml) 3:2 42 95.69 PR005183 Expi-CHOs (1000 ml) 3:2 89.6 99.916 PR005184 293-6E (40 ml) 3:2 7.15 90.77 PR005185 293-6E (40 ml) 3:2 7.425 89.1 PR005186 293-6E (40 ml) 3:2 10.5 83.89 PR005187 293-6E (40 ml) 3:2 8 97.86 PR005188 293-6E (40 ml) 3:2 10.15 94.19 PR005827 HEK293-F (100 ml) 3:2 35.2 99.526 PR005828 HEK293-F (100 ml) 3:2 38.4 99.311 PR005829 HEK293-F (100 ml) 3:2 31.8 99.457 PR005830 HEK293-F (100 ml) 3:2 27.3 99.680 PR005838 Expi-CHOs (2000 ml) 3:2 11.5 98.833 PR005839 Expi-CHOs (2000 ml) 3:2 33.2 98.104 PR005866 Expi-CHOs (2000 ml) 3:2 5.3 98.241 PR007379 HEK293-F (40 ml) 3:2 33.5 98.625 PR007380 HEK293-F (40 ml) 3:2 38.0 198.453
TABLE-US-00019 TABLE 12-(C) Expression and physicochemical properties of bispecific antibody molecule proteins of an IgG-VH-VH hexavalent symmetric structure Plasmid Yield HPLC- Bispecific transfection ratio (mg/L) SEC antibody Expression system (short chain:long after first purity molecules and volume chain) purification (%) PR003487 HEK293-F (30 ml) 3:2 19 84 PR003488 HEK293-F (30 ml) 3:2 17.39 85 PR003489 HEK293-F (30 ml) 3:2 16.03 76 PR004158 HEK293-F (30 ml) 3:2 31.92 63 PR004358 HEK293-F (30 ml) 3:2 18.9 98.72
TABLE-US-00020 TABLE 12-(D) Expression and physicochemical properties of bispecific antibody molecule proteins of an Fab-HCAb tetra valent symmetric structure Plasmid Yield HPLC- Bispecific transfection ratio (mg/L) SEC antibody Expression system (short chain:long after first purity molecules and volume chain) purification (%) PR004279 HEK293-F (30 ml) 3:2 11.375 79.18 PR005189 HEK293-F (100 ml) 3:2 4 97.59 PR005649 HEK293-F (100 ml) 3:2 30.4 91.928 PR007165 HEK293-F (100 ml) 3:2 39 94.70
TABLE-US-00021 TABLE 12-(E) Expression and physicochemical properties of bispecific antibody molecule proteins of an Fab-Fc-VH(n) asymmetric structure Plasmid transfection Bispecific Expression ratio (short Yield (mg/L) antibody system and chain dong after first HPLC-SEC DSF Tm1 molecules volume chain) purification purity (%) (° C.) PR004160 HEK293-F (30 ml) 1:1:1 29.36 89.41 64.4 PR004161 HEK293-F (30 ml) 1:1:1 31.62 85.47 63.0 PR004181 HEK293-F (30 ml) 1:1:1 87.25 87.19 64.2 PR004182 HEK293-F (30 ml) 1:1:1 24.75 79.14 51
TABLE-US-00022 TABLE 12-(F) Expression and physicochemical properties of bispecific antibody molecule proteins of an Fab-VH-Fc-VH symmetric structure Plasmid transfection Bispecific ratio (short Yield (mg/L) antibody Expression system chain:long after first HPLC-SEC molecules and volume chain) purification purity (%) PR005650 HEK293-F (100 ml) 3:2 21.5 97.476 PR005651 HEK293-F (100 ml) 3:2 18.2 98.129
Example 5. FACS Assays for In Vitro Binding of B7-H4×4-1BB Bispecific Antibodies on CHO-K1 Cell Strain Overexpressing Human and Cynomolgus Monkey 4-1BB
[0222] This example is intended to investigate the in vitro binding activity of the 4-1BB arms of B7-H4×4-1BB bispecific antibodies to human and cynomolgus monkey 4-1BB. Antibody binding experiments at the cellular level were conducted using a CHO-K1 cell strain overexpressing human 4-1BB (CHO-K1/hu 4-1BB, Genscript, #M00538) and a CHO-K1 cell strain overexpressing cynomolgus monkey 4-1BB (CHO-K1/cyno 4-1BB, Genscript, #M00569). Briefly, the CHO-K1-hu 4-1BB and CHO-K1/cyno 4-1BB cells were digested, resuspended in F12K complete medium, and washed once with PBS. The cell density was adjusted to 1×10.sup.6 cells/mL with PBS. The cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 μL/well, followed by the addition of test antibodies diluted in a 3-fold gradient at a concentration that was 2 times the final concentration, each at 100 μL/well. The cells were incubated at 4° C. for 1 h away from light. Thereafter, the cells in each well were rinsed twice with 100 μL of pre-cooled PBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Then 100 μL of a fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, #:109-545-06, 1:500 diluted) or (Alexa Fluor® 647, Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch, #: 109-605-098, 1:1000 diluted) was added to each well. The plate was incubated away from light at 4° C. for 30 min. The cells in each well were rinsed twice with 100 μL of pre-cooled PBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Finally, the cells in each well were resuspended in 200 μL of pre-cooled PBS, and the fluorescence signal values were read using a BD FACS CANTOII or an ACEA NovoCyte flow cytometer.
[0223]
[0224]
[0225] As shown in
[0226] As shown in
Example 6. FACS Assays for In Vitro Binding of B7-H4×4-1BB Bispecific Antibodies on SK-BR-3 Cell Line Highly Expressing B7-H4
[0227] This example is intended to investigate the binding activity of the B7-H4 arms of B7-H4×4-1BB bispecific antibodies to human B7-H4. Experiments of binding to human B7-H4 at the cellular level were conducted using an SK-BR-3 cell line highly expressing B7-H4. Briefly, SK-BR-3 cell suspensions were collected and the cell densities were adjusted to 2×10.sup.6 cells/mL. The cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 50 μL/well, followed by the addition of test antibodies diluted in a 3-fold gradient at a concentration that was 2 times the final concentration, each at 50 μL/well. The cells were incubated at 4° C. for 2 h away from light. Thereafter, the cells in each well were rinsed twice with 100 μL of pre-cooled PBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Then 100 μL of a fluorescent secondary antibody (Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, #: 109-605-098, 1:1000 diluted) was added to each well. The plate was incubated away from light at 4° C. for 1 h. The cells in each well were rinsed twice with 100 μL of pre-cooled PBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Finally, the cells in each well were resuspended in 200 μL of pre-cooled FACS, and the fluorescence signal values were read using ACEA Novocyte3000.
[0228]
[0229] As shown in
Example 7. T Cell Activation Experiments for B7-H4×4-1BB Bispecific Antibodies in the Presence of SK-BR-3 Cell Line Highly Expressing B7-H4, and Release of Cytokines
[0230] To study the ability of B7-H4×4-1BB bispecific antibodies to mediate activation of target T cells in vitro, activation experiments were conducted in vitro using human pan T cells as effector cells and an SK-BR-3 cell line highly expressing B7-H4 as crosslinking-mediating cells, and the release of cytokines was determined. Specifically, a 96-well flat-bottom plate (Corning, #3599) was coated with OKT3; the density of pan T cells was adjusted to 2×10.sup.6 cells/mL, and the density of SK-BR-3 cells was adjusted to 2×10.sup.5 cells/mL; each of the two cell suspensions was seeded into the 96-well flat-bottom plate (Corning, #3599) at 50 μL/well, and then 50 μL of test antibodies serially diluted 3-fold with concentrations that were 3 times the final concentrations were added to each well, with the highest final concentration of the antibodies being 100 nM, 30 nM, 20 nM or 6 nM; 6, 3 or 2 concentrations were set for each antibody, the final effector-to-target ratio was 10:1, and two replicates were set. Meanwhile, an isotype IgG control group was set in the plate. The 96-well plate was incubated in a carbon dioxide incubator at 37° C. for 2 or 3 days. After incubation, the supernatant was collected and added to a 96-well plate (Corning, #3599), and centrifuged at 500 g at 4° C. for 5 min. The supernatant was incubated for 2 days and then analyzed for the release of cytokine IL-2; or incubated for 3 days and then analyzed for the release of IFN-gamma. The instructions of the IL-2 (IL-2 Human Uncoated ELISA Kit, Thermo, #88-7025-88) kit and IFN gamma (IFN gamma Human Uncoated ELISA Kit, Thermo, #88-7316-88) were referred to for the ELISA method.
[0231]
[0232]
[0233] As shown in
[0234] The linker also affected the bispecific antibodies' activation of T cells when varying in length; for example, PR004281 and PR004282 performed better than PR003338 and urelumab (
[0235] The B7H4 arm affected the bispecific antibodies' activation of T cells when comprising different antigenic epitopes; for example, PR004281 and PR004282 performed better than PR004995 and urelumab (
[0236] The bispecific antibodies' activation of T cells was affected when different Fc mutation sites were comprised; for example, PR005185, PR005186 and PR004282 were superior to urelumab (
Example 8. Activation of T Cells by B7-H4×4-1BB Bispecific Antibodies Being Independent of FcγR Cells
[0237] To investigate whether the activity of the B7-H4×4-1BB bispecific antibodies was dependent on the expression of FcγR, activation experiments were conducted in vitro using human pan T cells as effector cells and a CHO-K1/CD32b cell line highly expressing FcγRIIb or a CHO-K1/CD64 cell line highly expressing FcγRI as crosslinking-mediating cells, and the release of cytokines was determined. Specifically, a 96-well flat-bottom plate (Corning, #3599) was coated with OKT3; the density of pan T cells was adjusted to 2×10.sup.6 cells/mL, and the density of FcγR cells was adjusted to 2×10.sup.5 cells/mL; each of the two cell suspensions was seeded into the 96-well flat-bottom plate (Corning, #3599) at 50 μL/well, and then 100 μL of test antibodies serially diluted 2-fold with a concentration that was twice the final concentration were added to each well; the final effector-to-target ratio was 10:1, and two replicates were set. Meanwhile, an isotype IgG control group was set in the plate. The 96-well plate was incubated in a carbon dioxide incubator at 37° C. for 2 days. After incubation, 100 μL of supernatant was collected and added to a 96-well plate (Corning, #3894), and centrifuged at 500 g at 4° C. for 5 min. The supernatant was collected and analyzed for the release of cytokine IL-2. The instructions of the IL-2 (IL-2 Human Uncoated ELISA Kit, Thermo, #88-7025-88) kit were referred to for the ELISA method.
[0238]
Example 9. Activation of T Cells by B7-H4×4-1BB Bispecific Antibodies Being Dependent on Expression of B7-H4
[0239] To investigate whether the activity of the B7-H4×4-1BB bispecific antibodies was dependent on the expression of B7-H4, activation experiments were conducted in vitro using human pan T cells as effector cells and cells of cell lines SK-BR-3 and MDA-MB-468 highly expressing B7-H4 and COV644, JIMT-1 and MDA-MB-231 cells not expressing B7-H4 as crosslinking-mediating cells, and the release of cytokines was determined. Specifically, a 96-well flat-bottom plate (Corning, #3599) was coated with OKT3; the density of pan T cells was adjusted to 2×10.sup.6 cells/mL, and the density of tumor cells was adjusted to 2×10.sup.5 cells/mL; each of the two cell suspensions was seeded into the 96-well flat-bottom plate (Corning, #3599) at 50 μL/well, and then 50 μL of test antibodies with a concentration that was 3 times the final concentration were added to each well; the final effector-to-target ratio was 10:1, and two replicates were set. Meanwhile, an isotype IgG control group was set in the plate. The 96-well plate was incubated in a carbon dioxide incubator at 37° C. for 2 days. After incubation, 100 μL of supernatant was collected and added to a 96-well plate (Corning, #3894), and centrifuged at 500 g at 4° C. for 5 min. The supernatant was collected and analyzed for the release of cytokine IL-2. The instructions of the IL-2 (IL-2 Human Uncoated ELISA Kit, Thermo, #88-7025-88) kit were referred to for the ELISA method.
[0240]
[0241]
Example 10. Study on Serum Stability of B7-H4×4-1BB Bispecific Antibodies
[0242] To investigate the stability of B7-H4×4-1BB bispecific antibodies in human serum with high concentration, PR0003334 and PR0003335 were serially diluted 1:3 with 90% human serum from an initial concentration of 100 nM to obtain 8 concentration points. Six samples were prepared, incubated at 37° C. for 0 days, 1 day, 2 days, 4 days, 7 days and 14 days, respectively, then snap frozen in liquid nitrogen, and stored at −70° C. After the B7-H4×4-1BB bispecific antibodies were stored in high-concentration serum at 37° C. for different periods of time, their in vitro binding to the SK-BR-3 cell line and CHO-K1-hu4-1BB cells highly expressing B7-H4 was determined by FACS, as in Example 3.3 and Example 6.
[0243] PR0004282 was serially diluted with 95% human serum and divided into 6 tubes, which were incubated at 37° C. for 0 days, 1 day, 2 days, 4 days, 7 days and 14 days, respectively, then snap frozen in liquid nitrogen, and stored at −80° C. After PR004282 was stored in high-concentration human serum at 37° C. for different periods of time, its in vitro binding to B7H4+ cells and 4-1BB+ cells was determined by FACS as follows.
[0244] CHO-K1/h4-1BB, CHO-K1/cyno4-1BB, CHO-K1/hB7H4 and CHO-K1/cynoB7H4 cells were digested, resuspended in DMEM complete medium, and washed with PBS. Then the cell density was adjusted to 1×10.sup.6 cells/mL. The cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 μL/well, followed by the addition of the antibody PR004282 serially diluted 3-fold with a concentration that was twice the final concentration and treated with test human serum, each at 100 μL/well (each dose of the antibody treated for a different period of time contained 4.5% human serum). The cells were incubated at 4° C. for 2 h away from light. Then, the cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% FBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Then 100 μL of a fluorescent secondary antibody (Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, #109-545-06, 1:1000 diluted) was added to each well. The plate was incubated away from light at 4° C. for 1 h. The cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% FBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Finally, the cells in each well were resuspended in 200 μL of pre-cooled PBS containing 2% FBS, and the fluorescence signal values were read using an ACEA NovoCyte flow cytometer.
[0245]
[0246]
Example 11. FACS Assays for Binding Ability of B7-H4×4-1BB Bispecific Antibody PR004282 at 4-1BB+ Cell Level
[0247] This example is intended to investigate the in vitro binding activity of the B7-H4×4-1BB bispecific antibody PR004282 to human/cynomolgus monkey/mouse 4-1BB. Antibody binding experiments at the cellular level were conducted using a CHO-K1 cell strain overexpressing human 4-1BB (CHO-K1/h4-1BB, Gensript, #M00538), a CHO-K1 cell strain expressing cynomolgus monkey 4-1BB (CHO-K1/cyno4-1BB, Genscript, #M00569) and a CHO-K1 cell strain overexpressing mouse 4-1BB (CHO-K1/m4-1BB, Genscript, #M00568). Briefly, the CHO-K1/h4-1BB cells, CHO-K1/cyno 4-1BB cells and CHO-K1/m 4-1BB cells were digested and resuspended with PBS containing 2% FBS. The cell density was adjusted to 1×10.sup.6 cells/mL. The cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 μL/well, followed by the addition of test antibodies diluted in a 3-fold gradient at a concentration that was 2 times the final concentration, each at 100 μL/well. The cells were incubated at 4° C. for 2 h away from light. Thereafter, the cells in each well were rinsed twice with 100 μL of a pre-cooled FACS buffer, PBS containing 2% FBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Then 100 μL of a fluorescent secondary antibody (Alexa Fluor® 647, Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch, #109-605-098, 1:1000 diluted) was added to each well. The plate was incubated away from light at 4° C. for 1 h. The cells in each well were rinsed twice with 100 μL of pre-cooled FACS buffer, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Finally, the cells in each well were resuspended in 200 μL of pre-cooled FACS buffer, and the fluorescence signal values were read using an ACEA Novocyte3000 flow cytometer.
[0248]
[0249]
[0250]
Example 12. FACS Assays for Binding Ability of B7-H4×4-1BB Bispecific Antibody PR004282 at B7H4+ Cell Level
[0251] This example is intended to investigate the in vitro binding activity of the B7-H4×4-1BB bispecific antibody PR004282 to human/cynomolgus monkey/mouse B7-H4. Antibody binding experiments at the cellular level were conducted using a CHO-K1 cell strain overexpressing human B7-H4 (CHO-K1/hB7-H4, prepared in-house by Harbour Biomed), a CHO-K1 cell strain expressing cynomolgus monkey B7-H4 (CHO-K1/cynoB7-H4, prepared in-house by Harbour Biomed) and a CHO-K1 cell strain overexpressing mouse B7-H4 (CHO-K1/mB7-H4, prepared in-house by Harbour Biomed). Briefly, the CHO-K1/hB7H4 cells, CHO-K1/cyno B7-H4 cells and CHO-K1/mB7-H4 cells were digested and resuspended with PBS containing 2% FBS. The cell density was adjusted to 1×10.sup.6 cells/mL. The cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 μL/well, followed by the addition of test antibodies diluted in a 3-fold gradient at a concentration that was 2 times the final concentration, each at 100 μL/well. The cells were incubated at 4° C. for 2 h away from light. Thereafter, the cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% FBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Then 100 μL of a fluorescent secondary antibody (Alexa Fluor® 647, Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch, #109-605-098, 1:1000 diluted) was added to each well. The plate was incubated away from light at 4° C. for 1 h. The cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% FBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Finally, the cells in each well were resuspended in 200 μL of pre-cooled PBS containing 2% BSA, and the fluorescence signal values were read using an ACEA Novocyte3000 flow cytometer.
[0252]
[0253]
[0254]
Example 13. FACS Assays for Ability of B7-H4×4-1BB Bispecific Antibody PR004282 to Bind to Both CHO-K1/h4-1BB and SK-BR-3
[0255] This example is intended to investigate the ability of the B7-H4×4-1BB bispecific antibody PR004282 to bind to both CHO-K1/h4-1BB and SK-BR-3. Briefly, the CHO-K1/h4-1BB cells and SK-BR-3 cells were digested, and the cell density was adjusted to 1×10.sup.6 cells/mL with PBS. The SK-BR-3 cells and CHO-K1/h4-1BB cells were stained with 0.5 μM Far-red (lifetechnologies, #C34572) and 0.5 μM CFSE (lifetechnologies, #C34544), respectively, at room temperature for 5 min. The stained cells were centrifuged and washed once with >20 mL of medium containing 1% FBS. The washed cells were resuspended in FACS buffer, and the cell density was adjusted to 2×10.sup.6/mL. To each well of a 96-well V-bottom plate (Corning, #3894) were added 50 μL of SK-BR-3 cells (1×10.sup.5 cells per well), 25 μL of CHO-K1/h4-1BB cells (1×10.sup.5 cells per well) and 25 μL of a test antibody serially diluted 4-fold with FACS buffer. The mixtures were well mixed and incubated at 4° C. for 1 h. The percentage of the double positive FITC+CHO-K1/h4-1BB cells and Alexa647+SK-BR-3 was determined using an ACEA Novocyte3000 flow cytometer. Data were analyzed using FlowJo software (Tree Star, Ashland, Oreg.). The percentage of double stained cells was used to determine the bispecific antibody-mediated co-binding rate=(Q2/(Q1+Q2).
[0256]
Example 14. FACS Assays for Binding Ability of B7-H4×4-1BB Bispecific Antibody PR004282 at Primary Cell Level
[0257] This example is intended to investigate the in vitro binding activity of the B7-H4×4-1BB bispecific antibody PR004282 to activated human/cynomolgus monkey primary T cells. Monkey CD3+ T cells were isolated from cryopreserved monkey PBMCs (PharmaLegacy, #SC1702051) using a non-human primate CD3 isolation kit (Miltenyi, #130-092-012), resuspended to 2×10.sup.6/mL in medium RPMI1640+10% FBS+1% sodium pyruvate (Thermo, #11360-070)+1% non-essential amino acid solution (Thermo, #11140-050). 1 mL of the cell suspension was added to a 6-well plate, and 1 mL of medium containing 20 ng/mL PMA (Sigma, #P1585-1MG) and 5 nM Ionomycin (Sigma, #407952-5MG) was added. The plate was incubated in an incubator at 37° C. with CO.sub.2 for 16 h. Human CD3+ T cells were isolated from cryopreserved human PBMCs (Saily, XFB-HP100B) using a human CD3 isolation kit (Miltenyi, #130-096-535), and other procedures were the same as those for the monkey CD3+ T cells.
[0258] The activated monkey T cells was adjusted to a density of 1×10.sup.6 cells/mL with PBS containing 2% FBS and seeded at 100 μL CD3+ T cells/well in a 96-well V-bottom plate (Corning, #3894), followed by the addition of test antibodies PR004282/hIgG1/urelumab/hIgG4 serially diluted 3-fold with a concentration that was twice the final concentration at 100 μL/well. The cells were incubated away from light at 4° C. for 2 h. Thereafter, the cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% FBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Then 100 μL of a fluorescent secondary antibody (Alexa Fluor® 647, Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch, #109-605-098, 1:1000 diluted) was added to each well. The plate was incubated away from light at 4° C. for 1 h. The cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% FBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Finally, the cells in each well were resuspended in 100 μL of pre-cooled PBS containing 2% FBS, and the fluorescence signal values were read using an ACEA Novocyte3000 flow cytometer.
[0259] The activated human T cells were incubated with Zombie NIR™ dye (Biolegend, #77184) away from light at room temperature for 15-30 min and washed once with FACS buffer. The cell density was adjusted to 1×10.sup.6 cells/mL. The cells were seeded at 100 μL CD3+ T cells/well in a 96-well V-bottom plate (Corning, #3894), followed by the addition of test antibodies PR004282/hIgG1 serially diluted 3-fold with a concentration that was twice the final concentration at 100 μL/well. The cells were incubated away from light at 4° C. for 2 h. Thereafter, the cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% FBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Then 100 μL of a fluorescent secondary antibody (Alexa Fluor® 488, Goat Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch, #109-545-098, 1:1000 diluted) was added to each well. The plate was incubated away from light at 4° C. for 1 h. The cells in each well were rinsed twice with 100 μL of pre-cooled PBS containing 2% FBS, and centrifuged at 500 g at 4° C. for 5 min, and then the supernatant was discarded. Finally, the cells in each well were resuspended in 100 μL of pre-cooled PBS containing 2% FBS, and the fluorescence signal values were read using an ACEA Novocyte3000 flow cytometer.
[0260] As shown in
Example 15. ELISA Assays for Cross-Reactivity of B7-H4×4-1BB Bispecific Antibody PR004282 with Other Members of B7 Family or TNFR Family
[0261] 15.1 ELISA Assays for Cross-Reactivity of B7-H4×4-1BB Bispecific Antibody PR004282 with Other Members of B7 Family
[0262] The proteins of the B7 family (Table 13) were each diluted to 1 μg/mL with PBS, added to a 96-well plate (Corning, #9018) at 100 μL per well, and incubated overnight at 4° C. After the liquid was discarded, the plate was washed 3 times with PBST buffer (pH 7.4, containing 0.05% tween-20), and 250 μL of 2% BSA blocking buffer was added. The plate was incubated at 37° C. for 1 h. The blocking buffer was discarded, and the plate was washed 3 times with PBST buffer. The test antigen-binding protein was diluted to 3 concentrations: 100 nM, 10 nM and 1 nM, and added at 100 μL per well. The plate was incubated at 37° C. for 1 h. After the plate was washed 3 times with PBST buffer, a 5000-fold diluted goat anti-human HRP secondary antibody (Invitrogen, #A18805) was added. The plate was incubated away from light at 37° C. for 1 h. After the plate was washed 3 times with PBST buffer, TMB (Biopanda, #TMB-S-003) was added at 100 μL/well. The plate was left away from light at room temperature for about 30 min. The reactions were terminated by adding 50 μL of stop buffer (BBI life sciences, #E661006-0200) to each well, and the absorbance values at 450 nm (0D450) was measured using a microplate reader (PerkinElemer, #Enspire).
[0263]
TABLE-US-00023 TABLE 13 Suppliers of other members of the B7 family Other members of B7 family Supplier Catalog number Alias Human B7-1/CD80 protein, Fc Tag (HPLC-verified) Acrobiosystem B71-H5259 CD80 Human B7-2/CD86 protein, Fc Tag Acrobiosystem CD6-H5257 CD86 Human B7-DC/PD-L2/CD273 recombinant protein (Fc Tag) Sino Biological H10292-H02H PD-L2 Human PD-L1/B7-H1 protein, His Tag (HPLC verified) Acrobiosystem PD1-H5229 PD-L1 Human CD275/ICOS ligand recombinant protein (Fc Tag) Sino Biological 11559-H02H ICOS-L Human B7-H3/CD276 recombinant protein (ECD, Fc Tag) Sino Biological 11188-H02H Human B7H4 Fc chimeric recombinant protein R&D 8870-B7-050 B7S1 Human VISTA/B7H5/PD-1H Fc chimeric recombinant protein R&D 7126-B7-050 VISTA Human B7-H6/NCR3LG1 chimeric recombinant protein (Fc Tag) Sino Biological 16140-H02H Human B7H7/HHLA2 chimeric recombinant protein (Fc Tag) R&D 8084-B7-050 HHLA2
[0264] 15.2 ELISA Assays for Cross-Reactivity of B7-H4×4-1BB Bispecific Antibody PR004282 with Other Members of TNFR Family
[0265] The proteins of the TNFR family (Table 14) were each diluted to 1 μg/mL with PBS, added to a 96-well plate (Corning, #9018) at 100 μL per well, and incubated overnight at 4° C. After the liquid was discarded, the plate was washed 3 times with PBST buffer (pH 7.4, containing 0.05% tween-20), and 250 μL of 2% BSA blocking buffer was added. The plate was incubated at 37° C. for 1 h. The blocking buffer was discarded, and the plate was washed 3 times with PBST buffer. The test antigen-binding protein was diluted to 3 concentrations: 100 nM, 10 nM and 1 nM, and added at 100 μL per well. The plate was incubated at 37° C. for 1 h. An isotype antibody was used as a control. After the plate was washed 3 times with PBST buffer, a 5000-fold diluted goat anti-human HRP secondary antibody (Invitrogen, #A18805) was added. The plate was incubated away from light at 37° C. for 1 h. After the plate was washed 3 times with PBST buffer, TMB (Biopanda, #TMB-S-003) was added at 100 μL/well. The plate was left away from light at room temperature for about 30 min. The reactions were terminated by adding 50 μL of stop buffer (BBI life sciences, #E661006-0200) to each well, and the absorbance values at 450 nm (0D450) was measured using a microplate reader (PerkinElemer, #Envision).
TABLE-US-00024 TABLE 14 Suppliers of other members of the TNFR family Other members of TNFR family Supplier Catalog No. CD30 Acrobiosystems CD0-H5229 CD27 Acrobiosystems CD7-H522b HVEM Acrobiosystems HVM-H52E9 GITR Acrobiosystems GIR-H5228 OX40 Acrobiosystems OX0-H5224 4-1BB Acrobiosystems 41B-H5227 CD40 Sino Biological 10774-H08H
[0266]
Example 16. Determination of ADCC Effect of B7-H4×4-1BB Bispecific Antibody PR004282
[0267] 16.1 This Example Used an ADCC Reporter Gene Cell Line to Determine the ADCC Effect of PR004282
[0268] The ADCC effect activity of PR004282 for CHO-K1/h4-1BB or SK-BR-3 was determined using Jurkat FcγRIIIa-V158/NFAT-Luc cells (prepared in-house by Harbour Biomed). The CHO-K1/h4-1BB or SK-BR-3 cells were centrifuged at 300 g for 5 min and then resuspended in RPMI1640+4% FBS serum medium. The cell density was adjusted to 6×10.sup.5 cells/mL, and 50 μL of the cell suspension was added to each well of a 96-well plate. The plate was incubated overnight at 37° C. The Jurkat FcγRIIIa-V158/NFAT-Luc cells were centrifuged at 400 g for 4 min and then resuspended in RPMI1640+4% FBS serum medium. The cell density was adjusted to 3×10.sup.6 cells/mL, and 50 μL of the cell suspension was added to each well of a 96-well plate. Antibodies were 5-fold diluted with RPMI1640+4% FBS medium to a maximum final concentration of 100 nM, and a total of 4 concentrations were prepared for each antibody. Two replicates were set. 50 μL of the antibody dilutions was added to each well of the 96-well plate. Meanwhile, an isotype IgG control group and a blank medium control group were set in the plate. The cells were incubated with the antibodies at 37° C. for 5 h. The 96-well plate was left to stand at room temperature for 30 min, and 60 μL of One-Glo chromogenic solution (Promega, #E6110) was added to each well at room temperature. Then, the sample was incubated away from light at room temperature for 10 min. Luminscence readings were obtained using PE Enspire. Fold=Luminscence reading of antibody well/Luminscence reading of blank medium control group. Plotting was performed using Prism 8 software. PR004469 and PR003369 (B7H4 monoclonal antibodies of an affinity mature variant, having ADCC-enhanced effects, comprising a heavy chain with a sequence as set forth in SEQ ID NO: 290 and a light chain with a sequence as set forth in SEQ ID NO: 291) were used as positive controls, and human Iso IgG1 (CrownBio, #C0001-4) was used as a negative control.
[0269]
[0270] In this experiment, the human NK cell was used as an effector cell and an SK-BR-3 cell line highly expressing B7H4 as a target cell. The killing efficiency was reflected by the conductivity of the target cell measured using an RTCA instrument from ACEA. A 96-well E-plate was first equilibrated with 50 μL of complete medium. SK-BR-3 cells were digested, resuspended in RPM1640 complete medium containing 10% fetal bovine serum and diluted to 4×10.sup.5/mL. The cell suspension was plated on the 96-well E-plate at 50 μL/well, i.e. 2×10.sup.4/well, and incubated overnight at 37° C. The next day NK cells were sorted using an EasySep™ Human CD56 positive selection kit (Stem cell, #17855). 50 μL of fresh culture medium containing 1×10.sup.5 PBMCs was added to each well, followed by the addition of 50 μL of 4× antibodies serially diluted 5-fold with a maximum final concentration of 100 nM. A total of 4 concentrations were set in duplicate for each antibody. PR003369 was used as a positive control and human Iso IgG1 (CrownBio, C0001-4) antibody as a negative control. The conductivity of the target cells was measured in real time. The value at hour 24 was used to calculate the target cell killing efficiency=(1−sample/iso control)×100%.
[0271] The results in
Example 17. Determination of Affinity of Antibodies for Human, Mouse and Cynomolgus Monkey Fcγ Receptor Proteins by BLI
[0272] An Octet Red 96e molecular interaction analyzer was used in the assay. The buffer used in the experiment was a 1× diluted kinetics buffer (ForteBio, #18-1105).
[0273] The rotational speed of the instrument was set to 1000 rpm. FAB2G sensors (Fortebio, #18-5125) arranged in a row were equilibrated in a test buffer for 10 min, and then allowed to capture antibodies at a capture height of 1 nm. After being equilibrated in buffer for 120 s, the FAB2G sensors were allowed to associate with Fcγ receptor proteins that were serially diluted 2-fold. The association time was set to 60 s, and the dissociation time to 30 s. The protein concentrations are shown in Table 15. Finally, the FAB2G sensor was immersed in a 10 mM glycine-hydrochloric acid solution at pH 1.5 for regeneration to elute the proteins bound to the sensor. The affinity assays of antibodies for FcRn were performed under the conditions of both pH 6.0 buffer and pH 7.4 buffer. For the avi-tagged cynomolgus monkey CD32b and CD64, an SA sensor (Fortebio, 18-5019) was used to capture the receptor proteins. Antibodies were analytes.
TABLE-US-00025 TABLE 15 Fc receptor proteins and analyte concentrations Receptor protein Supplier Catalog No. Concentration Human CD64 Novo CM60 800-25 Human CD32a (H) Protein CS35 8000-500 Human CD32b C444 8000-250 Human CD16a (V158) C441 8000-250 Human CD16a (F158) CS11 8000-250 Human CD 16b CB62 8000-250 Human FcRn CI01 800-50 Mouse CD32 (C-6his) C767 8000-250 Mouse CD16 (C-6his) CS29 4000-250 Mouse CD64 (his) Aero CD4-M5227 1000-125 Cynomolgus monkey FcRn Biosystems FCM-C52W9 800-25 Cynomolgus monkey CD32a CDA-C52H7 8000-250 Cynomolgus monkey CD32b CDB-C82E4 3000-187.5 Cynomolgus monkey CD 16 FC6-C52H9 2000-125 Cynomolgus monkey CD64 FCA-C82E8 400-12.5
[0274] When data analysis was performed using Octet Data Analysis software (Fortebio, version 11.0), 0 nM was used as a reference hole, and reference subtraction was performed; the “1:1 Global fitting” method was selected to fit the data, and the kinetics parameters of the binding of proteins to antibodies were calculated, with kon(1/Ms) values, kdis(1/s) values and KD(M) values obtained. For the interactions of fast association and fast dissociation, the “steady state” method was selected to fit the data to obtain KD(M) values.
[0275] As can be seen from Tables 16-18, PR004282 showed higher KD values than the control antibody urelumab, indicating that it binds weakly to the human, mouse and monkey Fcγ receptor protein.
TABLE-US-00026 TABLE 16 Affinity of PR004282 for human Fcγ receptors KD(M) Human Fcγ receptor Name of CD32a FcRn antibody CD64 CD16a(v) CD16a(F) CD 16b (H) CD32b (pH 6.0) PR002408 2.68E−11 1.23E−07 1.62E−06 3.80E−06 4.40E−07 3.30E−06 2.64E−07 PR004282 1.20E−07 1.52E−06 No binding >8E−06 No binding 2.35E−07 Urelumab 6.52E−10 2.39E−06 2.80E−06 3.20E−06 1.94E−07
TABLE-US-00027 TABLE 17 Affinity of PR004282 for monkey Fcγ receptors KD (M) Monkey Fcγ receptor Antibody CD64 CD32b CD32a CD16 FcRn(pH 7.4) FcRn(pH 6.0) PR002408 3.67E−10 1.51E−06 2.10E−06 1.29E−07 No binding 1.59E−07 PR004282 9.77E−08 5.10E−06 No binding 7.47E−07 1.43E−07 Urelumab 1.74E−10 5.97E−07 4.82E−06 8.92E−07 1.39E−07
TABLE-US-00028 TABLE 18 Affinity of PR004282 for MOUSE Fey receptors KD (M) Mouse Fcγ receptor Name of antibody CD16 CD32 CD64 PR002408 1.28E−07 6.10E−07 3.60E−08 PR004282 1.41E−06 >8E−06 No binding urelumab 7.81E−07 2.10E−06 1.63E−07
Example 18. Determination of Non-Specific Release of Cytokines Caused by B7-H4×4-1BB Bispecific Antibody
[0276] This example is intended to evaluate whether B7-H4×4-1BB bispecific antibodies cause non-specific release of cytokines. Monocytes were isolated using a human CD14 isolation kit (Meltenyi, #130-050-201). The monocytes or PBMCs of the same donor were resuspended in RPM1640 complete medium containing 10% fetal bovine serum and diluted to 2E6/mL. The cell suspension was plated on a flat-bottom 96-well plate (Costar, #3599) at 100 μL/well, i.e., 2E5/well, and then 100 μL of antibodies serially diluted 10-fold with concentrations that were twice the final concentrations were added. The highest concentration of the antibodies was 300 nM. A total of 3 concentrations were set in duplicate. The plate was incubated at 37° C. overnight. Urelumab was used as a positive control and utomilumab and human Iso IgG1 (CrownBio, C0001-4) as negative controls. After 24 h of incubation, the supernatant was collected and analyzed for the release of TNF-α (Thermo, 88-7066-88) and IL-6 (Thermo, 88-7346-77). After 72 h of incubation, the supernatant was collected and analyzed for the release of IFN-gamma (Thermo, 88-7316-77).
[0277]
Example 19. Pharmacokinetic Study of B7-H4×4-1BB Bispecific Antibodies in Normal Mice
[0278] This example determined the pharmacokinetic properties of fusion proteins as follows. Six female C57BL/6 mice weighing 18-22 g were selected and given a bispecific antibody drug by intravenous injection at a dose of 5 mg/kg PR003334 or 5 mg/kg PR003335 or 5 mg/kg PR004282. The whole blood of 3 mice in one group was collected before the administration and 15 min, 24 h (1 day), 4 days and 10 days after the administration, and the whole blood of 3 mice in the other group was collected before the administration and 5 h, 2 days, 7 days and 14 days after the administration. The whole blood was left to stand for 30 min for coagulation and centrifuged at 2,000 rpm for 5 min at 4° C., and the isolated serum sample was cryopreserved at −80° C. until it was taken for analysis. In this example, the drug concentration in the serum of mice was quantitatively determined by two ELISA methods. The ELISA method I, namely the overall detection method, was performed by capturing the fusion protein containing human Fc in the serum of mice using a goat anti-human Fc polyclonal antibody coating a 96-well plate, and then adding an HRP-labeled goat anti-human Fc secondary antibody; and the ELISA method II, namely the functional domain detection method, was performed by capturing the bispecific antibody containing human 4-1BB HCAb in the serum of mice using a human 4-1BB protein a 96-well plate was coated with, and then adding an HRP-labeled goat anti-human Fc secondary antibody. The plasma concentration data were analyzed using Phoenix WinNonlin software (version 8.2) by non-compartmental analysis (NCA) to evaluate the pharmacokinetics.
[0279]
TABLE-US-00029 TABLE 19 Pharmacokinetics of PR003334, PR003335 and PR004282 PR003334 PR003335 PR004282 PR003334 Functional PR003335 Functional PR004282 Functional PK Overall domain Overall domain Overall domain parameters detection detection detection detection detection detection T.sub.1/2 (hr) 193 218 336 295 274 197 V.sub.d (mL/kg) 76 89 81 81 133 119 AUC.sub.all 12,680 ± 11,471 ± 14,813 ± 14,286 ± 8,363 ± 8,125 ± (μg/mL hr) 428 497 683 560 102 150 AUC (%)* 100 90 100 96 100 97 Cl (mL/hr/kg) 0.28 0.28 0.17 0.19 0.36 0.43 C.sub.0 (μg/mL) 111 114 123 124 71 71
Example 20. Pharmacokinetic Study of B7H4/4-1BB Bispecific Antibody PR004282 in Cynomolgus Monkeys
[0280] This example studied the pharmacokinetics of the B7-H4/4-1BB bispecific antibody in cynomolgus monkeys. Specifically, PR004282 was diluted with 0.9% normal saline and intravenously injected into male cynomolgus monkeys at a dose of 1 mg/kg, and blood samples were collected before the administration and 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 168, 336, 504, 672, 840 and 1008 hours after the administration. The blood samples were left to stand at room temperature for 30 min and then centrifuged to obtain serum, and the B7-H4/4-1BB bispecific antibody content in the serum is determined by using the established specific ELISA method (the overall detection method).
[0281] As shown in
TABLE-US-00030 TABLE 20 Pharmacokinetics of PR004282 (in single doses) in normal monkeys Average PK parameters Monkey 1 Monkey 2 value C.sub.max μg/mL 32.1 48 40 Terminal t.sub.1/2 hr 45.3 82.3 63.8 AUC.sub.0-last hr * μg/mL 1472 2300 1886 AUC.sub.0-INF hr * μg/mL 1574 2932 2253 CL mL/hr/kg 0.635 0.341 0.488 V.sub.ss mL/kg 35.1 35.5 35.3
Example 21. Evaluation of Anti-tumor Effect of B7-H4×4-1BB Bispecific Antibody in BALB/c-hCD137/CT26-B7-H4 Mouse Model
[0282] To evaluate the in vivo anti-tumor effect of the B7-H4×4-1BB bispecific antibody, 6-8 week-old female BALB/c-hCD137 mice were used, and each was subcutaneously inoculated with 5×10.sup.5 CT26/hB7-H4 cells on the day of tumor cell inoculation. When the mean tumor volume reached 80 mm.sup.3, the mice were randomized into groups of 6. After grouping, drugs diluted with PBS at specific concentrations were administered by intraperitoneal injection (i.p.) twice a week, for a total of 6 doses (BIW×3). PBS was used as a blank control group. Tumor volume and body weight of mice were measured on the day of the first administration and on days 4, 7, 11, 14 and 18 thereafter. Tumor size calculation formula: tumor volume (mm.sup.3)=0.5×(long diameter of tumor×short diameter of tumor.sup.2).
[0283]
Example 22. Evaluation of Anti-tumor Effect of B7-H4×4-1BB Bispecific Antibody in MDA-MB-468 Xenograft Mouse Model
[0284] To evaluate the in vivo anti-tumor effect of the B7-H4×4-1BB bispecific antibody, 6-8 week-old female NCG mice were used, and each was subcutaneously inoculated with 5×10.sup.6 MDA-MB-468 cells on the day of tumor cell inoculation. When the mean tumor volume reached 120 mm.sup.3, the mice were randomized into groups of 6. After grouping, the mice were inoculated with 5×10.sup.6 human PBMCs. The next day, drugs diluted with PBS at specific concentrations were administered by intraperitoneal injection (i.p.). The administration was performed twice a week, for a total of 6 doses (BIW×3). An isotype control IgG was used as a control group. Tumor volume and body weight of mice were measured on the day of the first administration and on days 7, 12, 15, 19, 22, 26 and 29 thereafter. Tumor size calculation formula: tumor volume (mm.sup.3)=0.5×(long diameter of tumor×short diameter of tumor.sup.2).
[0285]
Example 23. Evaluation of Anti-tumor Effect of B7-H4×4-1BB Bispecific Antibody in OVCAR3 Xenograft Mouse Model
[0286] To evaluate the in vivo anti-tumor effect of the B7-H4×4-1BB bispecific antibody, 6-8 week-old female NCG mice were used, and each was subcutaneously inoculated with 5×10.sup.6 OVCAR3 cells on the day of tumor cell inoculation. When the mean tumor volume reached 120 mm.sup.3, the mice were randomized into groups of 6. After grouping, the mice were inoculated with 5×10.sup.6 human PBMCs. The next day, drugs diluted with PBS at specific concentrations were administered by intraperitoneal injection (i.p.). The administration was performed twice a week, for a total of 6 doses (BIW×3). An isotype control IgG was used as a control group. Tumor volume and body weight of mice were measured on the day of the first administration and on days 7, 12, 15, 19, 22, 26 and 29 thereafter. Tumor size calculation formula: tumor volume (mm.sup.3)=0.5×(long diameter of tumor×short diameter of tumor.sup.2).
[0287]
Example 24. Evaluation of Anti-tumor Effect and Memory Immune Effect of B7-H4×4-1BB Bispecific Antibody in BALB/c-hCD137/CT26-hB7-H4 Mouse Model
[0288] To evaluate the in vivo anti-tumor effect of the B7-H4×4-1BB bispecific antibody, 6-8 week-old female BALB/c-hCD137 mice were used, and each was subcutaneously inoculated with 5×10.sup.5 CT26-B7-H4 cells on the day of tumor cell inoculation. When the mean tumor volume reached 80 mm.sup.3, the mice were randomized into groups of 6. After grouping, drugs diluted with PBS at specific concentrations were administered by intraperitoneal injection (i.p.) twice a week, for a total of 6 doses (BIW×3). PBS was used as a blank control group, and urelumab as a positive control group. Tumor volume and body weight of mice were measured on the day of the first administration and three times a week over the following 45 days. Tumor size calculation formula: tumor volume (mm.sup.3)=0.5×(long diameter of tumor×short diameter of tumor.sup.2). Mice with tumor elimination were inoculated subcutaneously with 5×10.sup.5 CT26/hB7H4 cells on the other side 45 days after the administration, and a mouse of the same age was used as a new inoculation control group. Tumor volume and body weight of mice were measured three times a week, and the mice were sacrificed on day 66.
[0289]
Example 25. Specificity Evaluation of Anti-tumor Effect of B7-H4×4-1BB Bispecific Antibody
[0290] 25.1 Specificity Evaluation of Anti-Tumor Effect of B7-H4×4-1BB Bispecific Antibody in JIMT-1 Xenograft Mouse Model
[0291] 6-8 week-old female NCG mice were used, and each was subcutaneously inoculated with 5×10.sup.6 JIMT-1 cells on the day of tumor cell inoculation. When the mean tumor volume reached 120 mm.sup.3, the mice were randomized into groups of 6. After grouping, the mice were inoculated with 5×10.sup.6 human PBMCs. The next day, drugs diluted with PBS at specific concentrations were administered by intraperitoneal injection (i.p.). The administration was performed twice a week, for a total of 6 doses (BIW×3). An isotype control IgG was used as a control group. Tumor volume and body weight of mice were measured on the day of the first administration and on days 7, 12, 15, 19, 22, 26 and 29 thereafter. Tumor size calculation formula: tumor volume (mm.sup.3)=0.5×(long diameter of tumor×short diameter of tumor.sup.2).
[0292]
[0293] 25.2 Specificity Evaluation of Anti-Tumor Effect of B7-H4×4-1BB Bispecific Antibody in BALB/c-hCD137/CT26 Mouse Model
[0294] To evaluate the specificity of the in vivo anti-tumor effect of the B7-H4×4-1BB bispecific antibody, 6-8-week-old female BALB/c-hCD137 mice were used, and each was subcutaneously inoculated with 5×10.sup.5 CT26 wild-type cells on the day of tumor cell inoculation. When the mean tumor volume reached 80 mm.sup.3, the mice were randomized into groups of 6. After grouping, drugs diluted with PBS at specific concentrations were administered by intraperitoneal injection (i.p.) twice a week, for a total of 6 doses (BIW×3). PBS was used as a blank control group. Tumor volume and body weight of mice were measured on the day of the first administration and on days 4, 7, 11, 14 and 18 thereafter. Tumor size calculation formula: tumor volume (mm.sup.3)=0.5×(long diameter of tumor×short diameter of tumor.sup.2).
[0295]
[0296] Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these embodiments are merely illustrative and that many changes or modifications can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of protection of the present invention is therefore defined by the appended claims.