MULTISPECIFIC BINDING MOIETIES COMPRISING PD-1 AND TGF-BRII BINDING DOMAINS
20230159645 · 2023-05-25
Assignee
Inventors
- Cecilia Anna Wilhelmina GEUIJEN (Utrecht, NL)
- Patrick MAYES (Wilmington, DE, US)
- Shaun M. STEWART (Wilmington, DE, US)
- Liang-Chuan Wang (Wilmington, DE, US)
Cpc classification
C07K16/2863
CHEMISTRY; METALLURGY
A61K2039/507
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/70
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present disclosure relates to a multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain blocks PD-1 mediated signaling and the TGF-βRII binding domain blocks TGF-βRII-mediated signaling. The present disclosure further relates to a pharmaceutical composition comprising such multispecific binding moiety, a method of treatment using such multispecific binding moiety, and a cell producing such multispecific binding moiety.
Claims
1. A multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain blocks PD-1 mediated signaling and the TGF-βRII binding domain blocks TGF-βRII-mediated signaling.
2. (canceled)
3. The multispecific binding moiety according to claim 1, wherein the multispecific binding moiety comprises a single Fab domain that binds to PD-1, a single Fab domain that binds to TGF-βRII, and an Fc region.
4. The multispecific binding moiety according to claim 1, wherein the multispecific binding moiety has a higher potency in blocking TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII than in cells expressing TGF-βRII and no, substantially no, or low levels of PD-1.
5. The multispecific binding moiety according to claim 4, wherein the cells expressing both PD-1 and TGF-βRII are Jurkat-PD-1.sup.+ cells and the cells expressing TGF-βRII and no, or substantially no, PD-1 are Jurkat-PD-1.sup.null cells, in particular wherein the potency in blocking TGF-βRII-mediated signaling is measured in a phospho-SMAD2/3 assay.
6. The multispecific binding moiety according to claim 4, wherein the cells expressing both PD-1 and TGF-βRII are activated CD4.sup.+ and/or CD8.sup.+ cells and the cells expressing TGF-βRII and no PD-1 are non-activated CD4.sup.+ and/or CD8.sup.+ cells, in particular wherein the potency in blocking TGF-βRII-mediated signaling is measured in a phospho-SMAD2/3 assay.
7. The multispecific binding moiety according to claim 4, wherein the cells expressing both PD-1 and TGF-βRII are HEK-Blue TGF-β-PD-1.sup.+ cells and the cells expressing TGF-βRII and no PD-1 are HEK-Blue TGF-β cells, in particular wherein the potency in blocking TGF-βRII-mediated signaling is measured in an isogenic PD-1-TGF-β reporter assay.
8-9. (canceled)
10. The multispecific binding moiety according to claim 4, wherein the potency in blocking TGF-βRII-mediated signaling in cells expressing both PD-1 and TGF-βRII is at least about 200 fold, preferably between about 200-30000 fold, higher than in cells expressing TGF-βRII and no, substantially no, or low levels of PD-1.
11. The multispecific binding moiety according to claim 4, wherein the potency of the multispecific binding moiety in blocking TGF-βRII-mediated signaling in cells expressing TGF-βRII and no PD-1 is lower than the potency of a reference anti-TGF-βRII antibody and the potency of the multispecific binding moiety in blocking TGF-βRII-mediated signaling in cells expressing both TGF-βRII and PD-1 is higher than the potency of the reference anti-TGF-βRII antibody, wherein the reference anti-TGF-βRII antibody is a bivalent monospecific antibody comprising a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 76 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 77.
12. The multispecific binding moiety according to claim 11, wherein the potency of the multispecific binding moiety in blocking TGF-βRII-mediated signaling in cells expressing both TGF-βRII and PD-1 is at least about 100 fold, preferably between about 100-20000 fold, higher than the potency of the reference anti-TGF-βRII antibody.
13. The multispecific binding moiety according to claim 1, wherein the multispecific binding moiety has a higher activity in reducing tumor volume than a combination of reference antibodies, wherein the combination of reference antibodies are two bivalent monospecific antibodies targeting PD-1 and TGF-βRII, wherein the bivalent monospecific antibody targeting PD-1 comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 78 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 79, and the bivalent monospecific antibody targeting TGF-βRII comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 76 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 77.
14. The multispecific binding moiety according to claim 13, wherein the activity in reducing tumor volume is determined by measuring tumor volume reduction in an in vivo mouse study, in particular in an in vivo mouse study using MDA-MB-231 xenograft huCD34 NSG mice.
15. The multispecific antibody according to claim 13, wherein a higher activity in reducing tumor volume is a tumor volume reduction of at least about 1.5 fold, preferably between about 1.5-100 fold, of the tumor volume reduction of the combination of reference antibodies.
16. A multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the PD-1 binding domain comprises a heavy chain variable region comprising: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; or e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; wherein each of the HCDRs may comprise at most three, two, or one amino acid variations.
17. The multispecific binding moiety according to claim 16, wherein the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NO: 1; 5; 9; 13; 14; 18; 19, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.
18-19. (canceled)
20. The multispecific binding moiety according to claim 16, wherein the TGF-βRII binding domain comprises a heavy chain variable region comprising: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; or g) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively, wherein each of the HCDRs may comprise at most three, two, or one amino acid variations.
21. The multispecific binding moiety according to claim 16, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NO: 23; 27; 31; 35; 39; 43; 47; 88; 89, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.
22. The multispecific binding moiety according to claim 16, wherein the PD-1 binding domain and/or TGF-βRII binding domain comprises a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having an amino acid sequence as set forth in SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively, or a variant thereof.
23. The multispecific binding moiety according to claim 16, wherein the PD-1 binding domain and/or TGF-βRII binding domain comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 48, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.
24. A multispecific binding moiety comprising a PD-1 binding domain and a TGF-βRII binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region comprising: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; or g) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively, wherein each of the HCDRs may comprise at most three, two, or one amino acid variations.
25. The multispecific binding moiety according to claim 24, wherein the TGF-βRII binding domain comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NO: 23; 27; 31; 35; 39; 43; 47; 88; 89, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.
26-27. (canceled)
28. The multispecific binding moiety according to claim 24, wherein the PD-1 binding domain comprises a heavy chain variable region comprising: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; or e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; wherein each of the HCDRs may comprise at most three, two, or one amino acid variations.
29. The multispecific binding moiety according to claim 28, wherein the PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NO: 1; 5; 9; 13; 14; 18; 19, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.
30. The multispecific binding moiety according to claim 28, wherein the PD-1 binding domain and/or TGF-βRII binding domain comprises a light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having an amino acid sequence as set forth in SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively, or a variant thereof.
31. The multispecific binding moiety according to claim 28, wherein the PD-1 binding domain and/or TGF-βRII binding domain comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 48, or having at least 80% sequence identity thereto.
32. (canceled)
33. A pharmaceutical composition comprising an effective amount of the multispecific binding moiety according to claim 1, and a pharmaceutically acceptable carrier.
34-35. (canceled)
36. A method for treating a disease, comprising administering an effective amount of a multispecific binding moiety according to claim 1, to a subject in need thereof.
37. A method for treating a disease associated with a suppressed immune system, in particular cancer, comprising administering an effective amount of a multispecific binding moiety according to claim 1, to a subject in need thereof.
38. A cell comprising a nucleic acid sequence encoding the heavy chain variable region of a PD-1 binding domain as defined in claim 16 and a nucleic acid sequence encoding a heavy chain variable region of a TGF-βRII binding domain, wherein the TGF-βRII binding domain comprises a heavy chain variable region comprising: a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; b) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively; c) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively; d) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; e) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; f) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; or g) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), having an amino acid sequence as set forth in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively, wherein each of the HCDRs may comprise at most three, two, or one amino acid variations.
39. The cell according to claim 38, wherein the cell further comprises a nucleic acid sequence encoding a CH1 region and preferably a hinge, CH2 and CH3 region.
40. The cell according to claim 38, wherein the cell further comprises at least one nucleic acid sequence encoding a light chain variable region wherein the light chain variable region comprising light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), having an amino acid sequence as set forth in SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, respectively, or a variant thereof, and preferably a CL region.
41. A cell producing a multispecific binding moiety as claimed in claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0461] The following naming conventions are used herein as follows. In the Figures, bivalent monospecific antibodies are indicated in the format SEQ ID NO: A/SEQ ID NO: B, where SEQ ID NO: A refers to the heavy chain of both binding domains and SEQ ID NO: B refers to the light chain of both binding domains.
[0462] Bivalent bispecific antibodies are indicated in the format SEQ ID NO: A×SEQ ID NO: B, where both SEQ ID NO: A and B refer to heavy chain variable sequences. Each binding domain of the bispecific antibodies comprises the same light chain.
[0463] Bivalent monospecific reference antibodies pembrolizumab, nivolumab, and TGF1 analog are indicated in the format SEQ ID NO: A/SEQ ID NO: B, where SEQ ID NO: A refers to the respective heavy chain sequence and SEQ ID NO: B refers to the respective light chain sequence. A combination of pembrolizumab and TGF1 analog is indicated in the format SEQ ID NO: A/SEQ ID NO: B+SEQ ID NO: C/SEQ ID NO: D, where SEQ ID NO: A refers to the heavy chain sequence and SEQ ID NO: B refers to the light chain sequence of either pembrolizumab or TGF1 analog, and SEQ ID NO: C to the heavy chain sequence and SEQ ID NO: D to the light chain sequence of the other.
[0464] Reference PD-L1-TGF-β TRAP molecule analog, which is an analog of Bintrafusp alfa, is indicated in the format SEQ ID NO: A/SEQ ID NO: B, where SEQ ID NO: A refers to the heavy chain sequence including a (G.sub.4S).sub.4G linker and extracellular domain of TGF-βRII, and SEQ ID NO: B refers to the light chain sequence. The reference PD-L1-TGF-β TRAP molecule analog comprises two PD-L1 binding domains and two TGF-βRII extracellular domains.
[0465]
[0466]
[0467]
[0468]
[0469]
[0470]
[0471]
[0472]
[0473]
[0474]
[0475]
EXAMPLES
[0476] In the Examples, which are used to illustrate the present disclosure but are not intended to limit the disclosure in any way, each binding domain of the bispecific antibodies comprises a light chain variable region variable region having an amino acid sequence as set forth in SEQ ID NO: 48 and a light chain constant region having an amino acid sequence as set forth in SEQ ID NO: 75. The bispecific antibodies preferably are IgG1 antibodies comprising a CH1, hinge, CH2, and CH3. In the Examples, which are used to illustrate the present disclosure but are not intended to limit the disclosure in any way, bispecific antibodies were screened in IgG1 format, wherein the PD-1 binding heavy chain comprises a CH1 having an amino acid sequence as set forth in SEQ ID NO: 69, a CH2 having an amino acid sequence as set forth in SEQ ID NO: 71, and a CH3 having an amino acid sequence as set forth in SEQ ID NO: 73; and the TGF-βRII binding heavy chain comprises a CH1 having an amino acid sequence as set forth in SEQ ID NO: 69, a CH2 having an amino acid sequence as set forth in SEQ ID NO: 71, and a CH3 having an amino acid sequence as set forth in SEQ ID NO: 74.
[0477] Reference antibodies and molecules, and control antibodies used in the Examples include: [0478] Reference PD-1 antibody pembrolizumab analog, which is a bivalent monospecific antibody comprising two heavy chains having an amino acid sequence as set forth in SEQ ID NO: 78 and two light chains having an amino acid sequence as set forth in SEQ ID NO: 79. [0479] Reference PD-1 antibody pembrolizumab (made by Merck, distributed by Myonex). [0480] Reference TGF-βRII antibody TGF1, which is a bivalent monospecific analog of TGF1 and comprises two heavy chains having an amino acid sequence as set forth in SEQ ID NO: 76 and two light chains having an amino acid sequence as set forth in SEQ ID NO: 77. [0481] Reference PD-L1-TGF-β TRAP molecule, which is an analog of Bintrafusp alfa bivalent monospecific for binding to PD-L1 and comprises two heavy chains having an amino acid sequence as set forth in SEQ ID NO: 80 and two light chains having an amino acid sequence as set forth in SEQ ID NO: 81, with an extracellular domain of TGF-βRII having an amino acid sequence as set forth in SEQ ID NO: 104 linked to the C-terminus of each heavy chain through a (G.sub.4S).sub.4G linker. [0482] Negative control IgG1 antibody (RSV-G), which is a bivalent monospecific antibody comprising two heavy chains having an amino acid sequence as set forth in SEQ ID NO: 86 and two light chains having an amino acid sequence as set forth in SEQ ID NO: 87. [0483] Control TGF-βRIIxRSV antibodies, which are bivalent bispecific antibodies comprising a TGF-βRII binding domain comprising a heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 23, 31, 39, 27, 35, or 43; a RSV binding domain comprising a heavy chain variable region amino acid sequence as set forth in SEQ ID NO: 86; and a common light chain comprising a light chain variable region amino acid sequence as set forth in SEQ ID NO: 48 and a light chain constant region amino acid sequence as set forth in SEQ ID NO: 75. [0484] Positive control LILRB2 (BioLegend; Cat. No. 338714). [0485] Commercial reference PD-1 antibody Opdivo (made by Bristol Myers Squibb (BMS).
Example 1—Generation of PD-1xTGF-βRII Bispecific Antibodies
[0486] Binding domains, antibodies and heavy chain variable regions with binding specificity to human PD-1 and heavy chain variable regions with binding specificity to human TGF-βRII were obtained by immunizing transgenic mice comprising a common IGKV1-39 light chain (MeMo® mice) with human PD-1 or TGF-βRII antigenic moieties, including the use of different forms of DNA, protein and cell-based antigen delivery.
[0487] Heavy chain variable regions with binding specificity to human PD-1 having an amino acid sequence as set forth in SEQ ID NO: 1; 5; 9; 13; 14; 18; and 19, and heavy chain variable regions with binding specificity to human TGF-βRII having an amino acid sequence as set forth in SEQ ID NO: 23; 27; 31; 35; 39; 43; 47; 88; and 89, were selected for the production of bispecific antibodies. The binding domain sequences herein, once characterized and sequenced through the techniques provided herein, can be subsequently obtained by any method known in the art.
TABLE-US-00001 TABLE 1 Combination ofPD-1 heavy chain variable regions and TGF-/3R11 heavy chain variable regions that can be used in the generation of bispecific antibodies. TGF-βRII SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID PD-1 NO: 23 NO: 27 NO: 31 NO: 35 NO: 39 NO: 43 NO: 47 NO: 88 NO: 89 SEQ ID PBI PB2 PB3 PB4 PB5 PB6 PB7 PB50 PB57 NO: 1 SEQ ID PB8 PB9 PB10 PB11 PB12 PB13 PB14 PB51 PB58 NO: 5 SEQ ID PB15 PB16 PB17 PB18 PB19 PB20 PB21 PB52 PB59 NO: 9 SEQ ID PB22 PB23 PB24 PB25 PB26 PB27 PB28 PB53 PB60 NO: 13 SEQ ID PB29 PB30 PB31 PB32 PB33 PB34 PB35 PB54 PB61 NO: 14 SEQ ID PB36 PB37 PB38 PB39 PB40 PB41 PB42 PB55 PB62 NO: 18 SEQ ID PB43 PB44 PB45 PB46 PB47 PB48 PB49 PB56 PB63 NO: 19
[0488] Bispecific IgG antibodies were generated by transient co-transfection of two plasmid vectors: one encoding an IgG heavy chain with a PD-1 binding VH region and the other encoding an IgG heavy chain with a TGF-βRII binding VH region. CH3 engineering technology as described in WO 2013/157954 and WO 2013/157953 was employed to ensure efficient hetero-dimerization and formation of bispecific antibodies. Both vectors further encode a common light chain comprising the IGKV1-39/Jk1 light chain variable region. Cell transfection, cell culture, and the harvesting and purification of antibodies was performed by methods known in the art.
Example 2—PD-1-SHP Recruitment Assay
[0489] Bispecific antibodies were characterized in a PD-1-SHP recruitment assay to determine their ability to block ligand binding to PD-1 and thereby inhibit PD-1/PD-L1 signaling in T cells. A PD-1-SHP recruitment assay involves a two cell system comprising U2OS cells engineered to express an Enzyme Donor (ED)-tagged PD-1 receptor (e.g. PD-L1 or PD-L2) and Jurkat T cells expressing PD-1 and engineered to express Enzyme Acceptor (EA)-fused SHP1. Ligand or agonistic antibody-induced PD-1 activation of the Jurkat T cells results in SHP1 recruitment, forcing ED and EA interaction and of reconstitution of an active β-gal enzyme. Reconstituted β-gal produces a chemiluminescent signal in the presence of substrate.
[0490] Antibody samples included several bispecific antibodies, negative control IgG1 antibody (RSV), reference PD-1 antibody pembrolizumab analog, an analog of reference TGF-βRII antibody TGF1, and an analog of reference PD-L1-TGF-β TRAP molecule.
[0491] U2OS/PD-L1 cells (DiscoveRx Corporation) were maintained in McCoy's 5A medium (Thermo Fisher Scientific) with addition of 10% FBS+0.25 μm/ml Puromycin (Thermo Fisher Scientific). Jurkat-PD-1-SHP cells (Src homology region 2 domain-containing phosphatase; DiscoveRx Corporation) were cultured in RPMI1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS, 250 μg/ml Hygromycin B (Thermo Fisher Scientific), and 500 μg/ml G418 (Thermo Fisher Scientific). Both U2OS/PD-L1 and Jurkat-PD-1-SHP cells were first centrifuged in a conical tube to remove the culture media, and then washed, and resuspended with assay medium (RPMI1640 medium with 1% FBS) before cell plating. The U2OS/PD-L1 cells were added in a 384-well black clear bottom assay plate (CELLCOAT® Tissue Culture Plates, Greiner Bio-One) at 5000 cells per well in 20 μL assay medium. Antibody samples were prepared by serial dilution in phosphate buffered saline (PBS) with 1% FBS, and 5 μL/well was transferred to the cell plate and incubated at 37° C., 5% CO.sub.2 for one hour. Jurkat-PD-1-SHP cells were subsequently added to the cell plate at 5000 cells per well in 20 μL assay medium and incubated at 37° C., 5% CO.sub.2 for two hours before the addition of 2.5 μL PathHunter reagent 1 (DiscoveRx Corporation) in each well. The assay plate was then shaken for 1 min at 350 rpm and kept in the dark for 15 minutes at room temperature followed by addition of 10 μL PathHunter reagent 2 (DiscoveRx Corporation). Chemiluminescent signal was recorded with TopCount reader (Perkin Elmer) after incubation at room temperature for one hour. Wells with PBS only served as the positive controls and wells containing no cells were used as negative controls. IC.sub.50 determination was performed by fitting the curve of percent control activity versus the log of the compound concentration using the GraphPad Prism 7.0 software.
[0492] Results are shown in Table 2 and
TABLE-US-00002 TABLE 2 Results of the PD-1-SHP Recruitment Assay. SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID IC.sub.50(ng/mL) NO: 86 NO: 23 NO: 31 NO: 39 NO: 27 NO: 35 NO: 43 SEQ ID NO: 86 — — — — — — — SEQ ID NO: 1 271 277 390 428 410 415 278 SEQ ID NO: 5 126 141 160 158 118 78 312 SEQ ID NO: 9 101 120 137 94 122 130 226 SEQ ID NO: 14 202 257 337 251 167 255 238 SEQ ID NO: 19 207 234 400 237 232 220 267
Example 3—PD-1-NFAT Reporter Assay
[0493] Bispecific antibodies were characterized in a PD-1-NFAT Reporter Assay to determine their ability to block PD-1/PD-L1 signaling in activated T cells. A PD-1-NFAT Reporter Assay involves a two transgenic cell line system with PD-L1.sup.+ aAPC/CHO-K1 cells co-expressing a TCR cognate protein and PD-1.sup.+ effector Jurkat T cells driving luciferase reporter under control of a NFAT-RE cis element. Effector Jurkat T cells activate TCR signaling in an antigen-independent manner. PD-1/PD-L1 interaction inhibits TCR-mediated luminescence. Blockade of the PD-1/PD-L1 interaction allows TCR signaling, thereby inducing luminescence detectable by adding substrate.
[0494] Antibody samples included several bispecific antibodies, negative control IgG1 antibody (RSV), reference PD-1 antibody pembrolizumab analog, an analog of reference TGF-βRII antibody TGF1, and an analog of reference PD-L1-TGF-β TRAP molecule.
[0495] PD-L1 aAPC/CHO-K1 cells (artificial Antigen Presenting Cell)/CHO (Chinese Hamster Ovary)-K1 cells; Promega) were maintained in F-12 medium (Thermo Fisher Scientific) with addition of 10% FBS, 200 μg/mL Hygromycin B (Thermo Fisher Scientific), and 250 μg/mL Geneticin (G418; Thermo Fisher Scientific). Jurkat-PD-1-NFAT effector cells (Nuclear Factor of Activated T cells; Promega) were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS, 100 μg/mL Hygromycin B (Thermo Fisher Scientific), and 500 μg/mL G418 (Thermo Fisher Scientific). Both PD-L1 aAPC/CHO-K1 cells and Jurkat-PD NFAT effector cells were centrifuged first to remove the culture media, then washed and resuspended with assay medium (RPMI1640 medium with 1% FBS) before cell plating. The PD-L1 aAPC/CHO-K1 cells were added to a 384-well white clear-bottom assay plate (CELLCOAT® Tissue Culture Plates, Greiner Bio-One) at 8000 cells per well in 10 μL assay medium. Antibody samples were prepared by serial dilution in phosphate buffered saline (PBS) with 1% FBS and 5 μL/well was transferred to the cell plate. Jurkat-PD-1-NFAT effector cells were then dispensed into each well at 10,000 cells per well in 5 μL assay medium. The assay plate was incubated at 37° C., 5% CO.sub.2 for 24 hours. After the assay plate was equilibrated to room temperature for 15 minutes, 20 μL/well of Bio-Glo™ reagent (Promega) was added. After 8 minutes of incubation at room temperature, luminescence was read out with a Pherastar microplate reader (BMG Labtech). Wells with PBS served as the negative controls (0% induction) and wells containing 12.5 ug/mL pembrolizumab analog were used as positive controls (100% induction). EC.sub.50 determination was performed by fitting the curve of percent control activity versus the log of the compound concentration using the GraphPad Prism 7.0 software.
[0496] Results are shown in Table 3 and
TABLE-US-00003 TABLE 3 Results of the PD-1-NFAT Reporter Assay. EC.sub.50 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (ng/mL) NO: 86 NO: 23 NO: 31 NO: 39 NO: 27 NO: 35 NO: 43 SEQ ID NO: 86 — — — — — — — SEQ ID NO: 1 1352 1828 3569 1775 756 2030 1236 SEQ ID NO: 5 545 444 535 299 369 533 555 SEQ ID NO: 9 346 448 325 287 317 664 257 SEQ ID NO: 14 1513 869 868 904 772 686 790 SEQID NO: 19 791 771 2900 1019 741 936 681
Example 4—Jurkat phosphoSMAD2/3 Assay
[0497] Bispecific antibodies were characterized in a Jurkat phosphoSMAD2/3 Assay to determine their ability to block TGF-βRII signaling in T cells. The Jurkat phosphoSMAD2/3 Assay involved a comparison of the activity of the bispecific antibodies on Jurkat-PD-1.sup.null cells and Jurkat-PD-1.sup.+ cells to determine if the bispecific antibodies inhibit TGF-β-induced SMAD2/3 phosphorylation in a PD-1 correlated manner. An analog of reference TGF-βRII antibody TGF1, a TGF-βRIIxRSV bispecific antibody, and a negative control bivalent monospecific IgG1 antibody comprising a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 86 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 87 were included as control antibodies.
[0498] Jurkat-PD-1.sup.null (ATCC Cat. no. TIB-152) and Jurkat-PD-1.sup.+ cells (Promega Cat. no. CS187105) in RPMI/10% FBS were seeded in 96-well flat-bottom plates. PD-1 expression was undetectable for the Jurkat-PD-1.sup.null cells; the number of PD-1 molecules on the Jurkat-PD-1.sup.+ cells was determined at about 4000, using quantibrite bead methodology. Bispecific and control antibodies were added in 6-step serial dilutions (100 μg/ml to 0.001 μg/ml), and the cells were incubated for one hour at 37° C./5% CO.sub.2. After one hour, human recombinant TGF-β1 (R&D Systems Cat. no. 7754-BH) was added at a final concentration of 10 ng/ml and the cells were incubated for two more hours at 37° C./5% CO.sub.2. After incubation, cells were washed gently with PBS. Cell lysates were prepared using lysis buffer (MSD #R60TX-2) containing phosphatase inhibitors (Sigma #P0044 and P-5726) and protease inhibitors (Pierce Biotechnology #87785). Samples were normalized to total protein using BCA protein assay kit (Pierce #23227). PhosphoSMAD2/3 levels were determined using ELISA (Cell Signaling #12001) according to manufacturer's instructions. GraphPad Prism (8.2.0) was used to plot the graphs.
[0499] Results are shown in Tables 4, 5 and 6, and
[0500] All bispecific antibodies inhibited TGF-βRII signaling in both Jurkat-PD-1.sup.null cells and Jurkat-PD-1.sup.+ cells. The bispecific antibodies are more potent in inhibiting TGF-βRII signaling in Jurkat-PD-1.sup.+ cells than in Jurkat-PD-1.sup.null cells, indicating that they inhibit TGF-β-induced SMAD2/3 phosphorylation in a manner correlated to PD-1 expression. All bispecific antibodies require a higher concentration to inhibit TGF-βRII signaling in Jurkat-PD-1.sup.null cells than the analog of reference TGF-βRII antibody TGF1. Many bispecific antibodies, that are monovalent for binding to TGF-βRII, are superior to the bivalent monospecific analog of reference TGF-βRII antibody TGF1 in inhibiting TGF-βRII signaling in Jurkat-PD-1.sup.+ cells.
TABLE-US-00004 TABLE 4 IC.sub.50 values of the Jurkat phosphoSMAD2/3 Assay in Jurkat-PD-1.sup.null cells. SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 86 NO: 23 NO: 31 NO: 39 NO: 27 NO: 35 NO: 43 SEQ ID — 54560 19170 14190 31510 15700 21600 NO: 86 SEQ ID — 60540 38700 17200 42960 23030 15240 NO: 1 SEQ ID — 41320 43600 30640 64230 24590 18080 NO: 5 SEQ ID — 70920 32920 29670 55380 16400 13100 NO: 9 SEQ ID — 55380 13620 21750 46580 19850 15380 NO: 14 SEQ ID — 41450 15250 8383 40960 26050 22760 NO: 19
TABLE-US-00005 TABLE 5 IC.sub.50 values of the Jurkat phosphoSMAD2/3 Assay in Jurkat-PD-1.sup.+ cells. IC.sub.50 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (ng/mL) NO: 86 NO: 23 NO: 31 NO: 39 NO: 27 NO: 35 NO: 43 SEQ ID — 37100 18160 10010 6118 31580 10130 NO: 86 SEQ ID — 182 27 9 7 577 16 NO: 1 SEQ ID — 3 15 24 35 10 7 NO: 5 SEQ ID — 128 10 101 7 13 17 NO: 9 SEQ ID — 4 1 10 10 11 53 NO: 14 SEQ ID — 3 11 56 6 21 11 NO: 19
TABLE-US-00006 TABLE 6 Fold difference in potency in inhibiting TGF-βRII signaling in Jurkat-PD-1.sup.null versus Jurkat-PD-1.sup.+ cells. Fold difference pSMAD TGF-βRII PD-1 PD-1.sup.null vs PD-1.sup.+ SEQ ID NO: 23 SEQ ID NO: 14 13845 SEQ ID NO: 23 SEQ ID NO: 19 13817 SEQ ID NO: 31 SEQ ID NO: 14 13620 SEQ ID NO: 39 SEQ ID NO: 9 294 SEQ ID NO: 35 SEQ ID NO: 9 1262 SEQ ID NO: 35 SEQ ID NO: 14 1805 SEQ ID NO: 35 SEQ ID NO: 19 1240 SEQ ID NO: 27 SEQ ID NO: 9 7911 SEQ ID NO: 43 SEQ ID NO: 9 771 SEQ ID NO: 43 SEQ ID NO: 19 2069 SEQ ID NO: 78/ NA SEQ ID NO: 79 SEQ ID NO: 1.3 76/SEQ ID NO: 77
Example 5—phosphoSMAD2 Assay with Stimulated and Unstimulated CD4.SUP.+ and CD8.SUP.+ T Cells
[0501] Bispecific antibodies were characterized in a phosphoSMAD2 assay to determine their specificity in blocking TGF-βRII signaling in PD-1 positive T cells. The phosphoSMAD2 assay involved a comparison of the activity of the bispecific antibodies on unstimulated T cells, expressing low levels of PD-1, and stimulated T cells, expressing high levels of PD-1, to determine if the bispecific antibodies inhibit TGF-β-induced SMAD2 phosphorylation in a manner correlated to PD-1 expression. An analog of reference TGF-βRII antibody TGF1, reference PD-1 antibody pembrolizumab, and a negative control bivalent monospecific IgG1 antibody comprising a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 86 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 87, were included as positive controls and a negative control, respectively.
[0502] PBMCs from healthy donors were stimulated with 1 μg/ml anti-CD3 antibody (BD Biosciences #555336) for 48 hours followed by 16 hour serum deprivation (0.1% FBS). The number of PD-1 molecules on activated CD4.sup.+ and CD8.sup.+ T cells was determined at about 1000-2000, using quantibrite bead methodology. Stimulated and unstimulated PBMCs were then incubated with bispecific and control antibodies for 30 min at room temperature. Recombinant human TGF-β1 (Cell Signaling #7754-BH) was added at a final concentration of 1 ng/ml and the cells were incubated for another 30 min. Finally, cells were washed twice with PBS and stained for cell surface markers followed by intracellular phosphoSMAD2 staining.
[0503] The following antibodies were used in staining for flow cytometry: antibodies against human CD45 (clone: HI30; cat557748, BD Biosciences), human CD11b (clone: M1/70; cat #563015, BD Biosciences), human CD3 (Clone: UCHT1; cat #565491, BD Biosciences), human CD4 (Clone: SK3; cat #563550, BD Biosciences), human CD8 (clone #SK1, cat #344714, Biolegend) and human phospho-SMAD2 (cat #56532, Cell Signaling). Viability dye (Biolegend #423114) was used to exclude dead cells during analysis. Cell acquisition was performed under FACSymphony A3 using DIVA software.
[0504] PBMCs were gated based on size and granularity using FSC-A vs SSC-A to exclude debris. Dead cells were then excluded using fixable viability dye. CD45 positive cells were selected followed by a CD11b negative and CD3 positive T cell selection. Finally, CD4 and CD8 positive subsets were gated and phospho-SMAD2 signal was measured in geo mean fluorescence intensity (GMFI) on these subsets. Data analysis was performed using FlowJo software and GrapPad Prism (8.2.0) was used to plot the graphs.
[0505] Results are shown in
Example 6—Exhausted Mixed Lymphocyte Reaction (MLR) Assay
[0506] Bispecific antibodies were characterized in an Exhausted Mixed Lymphocyte Reaction (MLR) Assay to determine their potency in inducing cytokine production by exhausted T cells.
[0507] Antibody samples included several bispecific antibodies, negative control IgG1 antibody (RSV), reference PD-1 antibody pembrolizumab, an analog of reference TGF-βRII antibody TGF1, and a combination of reference PD-1 antibody pembrolizumab and an analog of reference TGF-βRII antibody TGF1.
[0508] Human PBMCs were isolated from healthy donors. In vitro T cell exhaustion was carried out by repeated activation of PBMCs by Staphylococcal enterotoxin B (SEB) for 6 days. Total T cells were isolated using T cell isolation kit (StemCell Technologies, cat #17951) according to manufacturer's instructions. T cells were mixed with dendritic cells (DC) from different donors at 10:1 T cell DC ratio in 96-well U bottom plates. Serial dilutions of antibody samples were added and the plates were incubated for six more days at 37° C./5% CO.sub.2. After six days, IFN-γ, IL-2, or TNF-α cytokine level in the supernatant was measured using custom MSD kit. GraphPad Prism (8.2.0) was used to plot the graphs.
[0509] Representative results from two experiments with five donors each are shown in
TABLE-US-00007 TABLE 7 IC.sub.50 values of the Exhausted MLR Assay AUC IFNg AUC IL2 AUC TNFa (% of (% of (% of Combi- Combi- Combi- TGF-βRII PD-1 nation) nation) nation) SEQ ID NO: 23 SEQID NO: 14 95.3 97.1 92 SEQ ID NO: 23 SEQID NO: 19 121.3 98.5 93.2 SEQ ID NO: 31 SEQID NO: 14 137.2 105.4 100.4 SEQ ID NO: 39 SEQID NO: 9 139.3 105.9 101 SEQ ID NO: 35 SEQID NO: 9 132.5 101.9 96 SEQ ID NO: 35 SEQID NO: 14 121.2 104.3 94.5 SEQ ID NO: 35 SEQID NO: 19 94.1 91.8 78 SEQ ID NO: 27 SEQID NO: 9 90.1 86.7 76.4 SEQ ID NO: 43 SEQID NO: 9 98.5 104.3 83.1 SEQ ID NO: 43 SEQID NO: 19 95.7 89 77.5
Example 7—HEK-BLUE-PD-1 TGF-β Reporter Assay
[0510] Bispecific antibodies were characterized in an HEK-BLUE-PD-1 TGF-β Reporter Assay to determine their potency in inhibiting TGF-β-induced signaling in T cells. Stimulation of HEK-Blue™ TGF-β cells or HEK-Blue™ TGF-β-PD-1.sup.+ cells with TGF-β induces the activation of the TGF-β/Smad signaling pathway, leading to the formation of a Smad3/Smad4 complex inducing the production of SEAP.
[0511] Reagents used: Recombinant Human TGF-beta 1 (Human Cell-expressed) Protein, R&D, Cat #7754-BH. Growth Medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS; 30 min at 56° C.), 100 μg/ml Normocin™, Pen-Strep (100 U/ml-100 μg/ml). Growth Medium with puromycin 0.4 ug/mL for HEK-Blue™ TGF-β-PD-1.sup.+ cells. Test Medium: DMEM 4.5 g/l glucose, 2 mM L-glutamine, 0.1% heat-inactivated FBS, Pen-Strep (100 U/ml-100 μg/ml) without Normocin™, Blasticidin, Hygromycin B, and Zeocin™.
[0512] A stable PD-1-expressing HEK-Blue™ TGF-β cell line was generated as follows: the full length cds for PD-1 was inserted into the mammalian expression vector pD2529-EFM (ATUM) which contains the gene for puromycin resistance. The promoter is a modified EF1a. The vector construction was done by ATUM and the sequence was confirmed. See
[0513] Antibody samples included several bispecific antibodies, negative control IgG1 antibody (RSV), reference PD-1 antibody pembrolizumab, and an analog of reference TGF-βRII antibody TGF1.
[0514] HEK-Blue™ TGF-β cells (Invivogen, Catalog code: hkb-tgfb), expressing TGF-βRII, or HEK-Blue™ TGF-β-PD-1.sup.+ cells were seeded in a 96-well flat-bottom plate, 25000 cells per well in test medium. Serial dilutions of antibody samples were added and the cells were incubated for one hour at room temperature; followed by the addition of human recombinant TGF-β1 at a final concentration of 1 ng/ml. Cells were incubated at 37° C./5% CO.sub.2 overnight. After incubation, 40 ul of supernatants were transferred from each well into a fresh flat-bottom 96-well plate; and 160 ul of re-suspended QUANTI-Blue™ Solution added to the supernatant. The plate was incubated at 37° C./5% CO.sub.2 for 40 minutes. The quantity of SEAP secreted in the supernatant was assessed using QUANTI-Blue™ Solution, a SEAP detection reagent. SEAP levels were determined using a spectrophotometer at 650 nm. GraphPad Prism (8.2.0) was used to plot the graphs.
[0515] Results are shown in
TABLE-US-00008 TABLE 8 Results from the HEK-BLUE-PD-1 TGF-β Reporter Assay. HEK-Blue ™ HEK-Blue ™ Fold Antibody TGF-β TGF-β -PD-1+ difference SEQ ID NO: 86/ >100 >100 — SEQ ID NO: 87 SEQ ID NO: 78/ >100 >100 — SEQ ID NO: 79 SEQ ID NO: 76/ 0.8056 0.6551 — SEQ ID NO: 77 SEQ ID NO: 23 × >100 (142) 0.005606 25330 SEQ ID NO: 14 SEQ ID NO: 23 × >100 (177) 0.001747 101317 SEQ ID NO: 19 SEQ ID NO: 31 × 9.7 7.404 1.3101 SEQ ID NO: 14 SEQ ID NO: 39 × 38 0.000193 197198 SEQ ID NO: 9 SEQ ID NO: 35 × 18.5 0.001533 12068 SEQ ID NO: 9 SEQ ID NO: 35 × 20.45 0.000687 20450 SEQ ID NO: 14 SEQ ID NO: 35 × 23.79 0.001215 19580 SEQ ID NO: 19 SEQ ID NO: 27 × 65 0.00141 46099 SEQ ID NO: 9 SEQ ID NO: 43 × 8.2 <0.0001 NA SEQ ID NO: 9 SEQ ID NO: 43 × 4.1 <0.0001 NA SEQ ID NO: 19
Example 8—Treg Suppression Assay
[0516] Bispecific antibodies were characterized in a Treg Suppression Assay to determine their capability to eliminate or reduce the suppressive effect of regulatory T cells and thereby to induce cytokine production by T cells.
[0517] Antibody samples included several bispecific antibodies, negative control IgG1 antibody (RSV), reference PD-1 antibody pembrolizumab, an analog of reference TGF-βRII antibody TGF1, and a combination of reference PD-1 antibody pembrolizumab and an analog of reference TGF-βRII antibody TGF1.
[0518] Human PBMCs were isolated from healthy donors by Ficoll-Paque gradient centrifugation. Tregs were isolated using EasySep treg isolation kit (StemCell Technologies, #18063) according to manufacturer's instructions. Tregs were mixed with PBMCs from the same donor. Anti-CD3 ab (BD Biosciences #555336) and anti-CD28 an (BD Biosciences, #555725) were added to the coculture. Finally, serial dilutions of bispecific antibodies were added and the plates were incubated for three days at 37° C./5% CO.sub.2. After three days of incubation, IFN-γ and TNF-α cytokine levels in the supernatant were measured using MSD kit (Mesoscale). GraphPad Prism (8.2.0) was used to plot the graphs.
[0519] Results are shown in
TABLE-US-00009 TABLE 9 Results of the Treg Suppression Assay. AUC IFN-γ AUC TNF-α % of % of combination combination of reference of reference TGF-βRII PD-1 antibodies antibodies SEQ ID NO: 23 SEQ ID NO: 14 95.1 134.5 SEQ ID NO: 23 SEQ ID NO: 19 122.5 179.4 SEQ ID NO: 31 SEQ ID NO: 14 81.8 101 SEQ ID NO: 39 SEQ ID NO: 9 107.2 138.9 SEQ ID NO: 35 SEQ ID NO: 9 99.9 121.5 SEQ ID NO: 35 SEQ ID NO: 14 123.9 121.7 SEQ ID NO: 35 SEQ ID NO: 19 88.3 115.8 SEQ ID NO: 27 SEQ ID NO: 9 103.8 127.2 SEQ ID NO: 43 SEQ ID NO: 9 109 114 SEQ ID NO: 43 SEQ ID NO: 19 128.6 107.2
Example 9—Macrophage Suppression Assay
[0520] Tumor-associated macrophages of the M2 phenotype inhibit T cell proliferation and cytokine production. Bispecific antibodies were characterized in an M2 macrophage suppression assay to test if they can reverse the inhibitory effect of M2 macrophages on T cell proliferation and IFN-γ production.
[0521] Bispecific antibodies were tested along with negative control IgG1 antibody (RSV), an analog of reference TGF-βRII antibody TGF1, an analog of reference PD-L1-TGF-β TRAP molecule, an analog of reference PD-1 antibody nivolumab, commercial reference PD-1 antibody Opdivo (BMS), positive control antibody anti-LILRB2 (Biolegend), rat IgG2a isotype control (Biolegend) and huIgG4 isotype control (Biolegend). In addition, a co-culture of stimulated CD4.sup.+ T cells and M2 macrophages without the addition of test or control antibodies or CD4.sup.+ T cells alone (unstimulated versus stimulated), were included as control conditions in the assay.
[0522] PBMC-isolated monocytes from three different healthy donors were differentiated into macrophages with M-CSF for six days and polarized using a specific cocktail of the cytokines IL-4 (20 ng/ml), IL-10 (20 ng/ml) and TGF-β (20 ng/ml) (+M-CSF) to obtain M2 macrophages. To confirm the phenotype, the expression of CD163 (Miltenye Biotec), CD209 (Miltenye Biotec), CD206 (BD Bioscience) and CD86 (Miltenye Biotec) was measured by flow cytometry (
[0523] Next, the M2 macrophages were activated with LPS (100 ng/ml) for 4 hours. The macrophages were harvested, washed, and seeded in 5-plicates in 96-well plates with autologous CD4.sup.+ activated T cells (activated by CD3/CD28 ImmunoCult™ from StemCell technologies) in a 1:5 ratio in the presence of the test or control antibodies at 10 μg/ml concentration. On day 5 of the co-culture, the concentration of secreted IFN-γ was measured by ELISA (LEGEND MAX™ Human IFN-γ ELISA Kit, Biolegend). Data was analyzed in GraphPad Prism 7.0 using multi-way ANOVA.
[0524] Results are shown in
Example 10—In Vivo Humanized NSG MDA-MB-231 Mouse Model
[0525] Bispecific antibodies were characterized in vivo in a humanized NSG MDA-MB-231 mouse model to determine their potency in reducing tumor volume. This mouse model was validated using 10 mg/kg of a negative control bivalent monospecific IgG1 antibody comprising a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 86 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 87; an analog of reference TGF-βRII antibody TGF1 (10 mg/kg); reference PD-1 antibody pembrolizumab (10 mg/kg); a combination of analog of reference TGF-βRII antibody TGF1 (10 mg/kg) and reference PD-1 antibody pembrolizumab (10 mg/kg); and an analog of reference PD-L1-TGF-β TRAP molecule (10 mg/kg). Results are shown in
[0526] Humanized CD34 NSG mice were inoculated subcutaneously with a total of 3×10.sup.6 MDA-MB-231 tumor cells suspended in 100 μl of serum-free culture medium and matrigel matrix (Corning) in equal volumes. After tumors were established (80-100 mm.sup.3), the mice were randomized into the following treatment groups:
[0527] 1) Negative control bivalent monospecific IgG1 antibody comprising a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 86 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 87 (10 mg/kg);
[0528] 2) An analog of reference TGF-βRII antibody TGF1 (10 mg/kg);
[0529] 3) Reference PD-1 antibody pembrolizumab (10 mg/kg);
[0530] 4) An analog of reference TGF-βRII antibody TGF1 (10 mg/kg)+reference PD-1 antibody pembrolizumab (10 mg/kg);
[0531] 5) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 43 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 9 (1 mg/kg);
[0532] 6) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 43 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 9 (10 mg/kg);
[0533] 7) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:43 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 19 (1 mg/kg);
[0534] 8) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 43 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 19 (10 mg/kg);
[0535] 9) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 23 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 14 (1 mg/kg);
[0536] 10) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 23 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 14 (10 mg/kg);
[0537] 11) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 23 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 19 (1 mg/kg);
[0538] 12) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 23 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 19 (10 mg/kg);
[0539] 13) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 39 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 9 (10 mg/kg);
[0540] 14) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 27 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 9 (10 mg/kg);
[0541] 15) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 23 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 18 (10 mg/kg);
[0542] 16) Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 47 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 13 (10 mg/kg).
[0543] Each group had 8-9 mice. Animals were dosed intraperitoneally every five days for a period of 27 or 30 days. Tumors were measured using calipers, and the tumor volume was calculated by assimilating them to an ellipsoid using the formula: 1(length)×w.sup.2 (width)×½. Body weights were also monitored all through the study. Tumors were harvested (24 hours post last dosing) for tumor immune profiling and receptor occupancy post termination of the study.
[0544] Results are shown in
[0545] Along with efficacy, similar receptor coverage of both PD-1 and TGF-βRII receptors upon bispecific antibody treatment was observed as with the combination treatment of the analog of reference TGF-βRII antibody TGF1 and reference PD-1 antibody pembrolizumab on T cells analyzed 24 hours post last dose (
Example 11—Testing Different Doses in an In Vivo Humanized NSG MDA-MB-231 Mouse Model
[0546] Bispecific antibody comprising a TGF-βRII binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 23 and a PD-1 binding domain with a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 18 was characterized at two different dose levels, 1 mg/kg and 10 mg/kg, in vivo in a humanized NSG MDA-MB-231 mouse model, as described in Example 10. Negative control bivalent monospecific IgG1 antibody comprising a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 86 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 87 was included at 10 mg/kg.
[0547] Results are shown in
[0548] Sequences
TABLE-US-00010 Heavy chain variable region SEQ ID NO: 1 EVQLVQSGAEVKKPGSSMKVSCKASGGTFSSYVISWVRQAPGQGLEWMGMIIPVFDTSSYEKKFQGRITIIADKS TSTVYLELSSLRSEDAAVYYCARGTVEATLLFDFWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 2 SYVIS Heavy chain CDR2 SEQ ID NO: 3 MIIPVFDTSSYEKKFQG Heavy chain CDR3 SEQ ID NO: 4 GTVEATLLFDF Heavy chain variable region SEQ ID NO: 5 QVQLQESGPGLVKPSETLSLTCTVSNGSLGFDFWSWIRQPPGRGLEWIGYIYYSGSWSLNPSFKGRVTMSVDTSK NQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 6 FDFWS Heavy chain CDR2 SEQ ID NO: 7 YIYYSGSWSLNPSFKG Heavy chain CDR3 SEQ ID NO: 8 GGYTGYGGDWFDP Heavy chain variable region SEQ ID NO: 9 QVQLQESGPGLVKPSETLSLTCTVSDGSIGYHFWSWIRQPPGRGLEWIGYIVYSGSYNVNPSLKTRVTMSVDTSK NQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 10 YHFWS Heavy chain CDR2 SEQ ID NO: 11 YIVYSGSYNVNPSLKT Heavy chain CDR3 SEQ ID NO: 12 GGYTGYGGDWFDP Heavy chain variable region SEQ ID NO: 13 QVQLQESGPGLVKPSETLSLTCTVSEGSIGYHFWSWIRQPPGRGLEWIGYIVYSGSYNVNPSLKTRVTMSVDTSK NQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS Heavy chain variable region SEQ ID NO: 14 QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTS VTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 15 RFALH Heavy chain CDR2 SEQ ID NO: 16 WIDPNTGTPTFAQGVTG Heavy chain CDR3 SEQ ID NO: 17 SLGYCDSDICYPNWIFDN Heavy chain variable region SEQ ID NO: 18 QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPNTGTPTFAQGVTGRFVFSLDTS VTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYPNWIFDNWGQGTLVTVSS Heavy chain variable region SEQ ID NO: 19 QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALSWVRQAPGQGLEWMGWIDPNTGTPTYAQDFTGRFVFSLDTS VTTAYLQISSLKAEDTAVYYCARSLGYCGSDICYPNGILDNWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 20 RFALS Heavy chain CDR2 SEQ ID NO: 21 WIDPNTGTPTYAQDFTG Heavy chain CDR3 SEQ ID NO: 22 SLGYCGSDICYPNGILDN Heavy chain variable region SEQ ID NO: 23 EVQLVESGGGLVQPGGSLRLSCAASGFTFDIYAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 24 IYAMT Heavy chain CDR2 SEQ ID NO: 25 VISGSGGTTYYADSVKG Heavy chain CDR3 SEQ ID NO: 26 RGQYRDIVGATDY Heavy chain variable region SEQ ID NO: 27 EVQLVESGGGLVQPGGSLRLSCAASGFTFDINAMTWVRQAPGKGLEWVSVISGSGGTTYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCARRGQYRDIVGATDYWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 28 INAMT Heavy chain CDR2 SEQ ID NO: 29 VISGSGGTTYYADSVKG Heavy chain CDR3 SEQ ID NO: 30 RGQYRDIVGATDY Heavy chain variable region SEQ ID NO: 31 QVQLVESGGGLVEPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATDFAAPVKGRFTISRD DSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 32 NAWMS Heavy chain CDR2 SEQ ID NO: 33 RIKTTISGGATDFAAPVKG Heavy chain CDR3 SEQ ID NO: 34 DLRDY Heavy chain variable region SEQ ID NO: 35 QVQLVESGGGLVEPGGSLRLSCAASGFKFSNAWMSWVRQAPGKGLEWVGRIKTTISGGATQFAAPVKGRFTISRD DSKNTLYLQMNSLKTEDTAVYYCTLDLRDYWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 36 NAWMS Heavy chain CDR2 SEQ ID NO: 37 RIKTTISGGATQFAAPVKG Heavy chain CDR3 SEQ ID NO: 38 DLRDY Heavy chain variable region SEQ ID NO: 39 QVQLVESGGGLVQPGGSLRLSCAVSGFTFRRYAMSWVRQAPGKGLEWVSAISASGDRTHNTDSVKGRFSISRDNS KNTLYLQMNSLRAEDTAVYFCAKGIAASGKNYFDPWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 40 RYAMS Heavy chain CDR2 SEQ ID NO: 41 AISASGDRTHNTDSVKG Heavy chain CDR3 SEQ ID NO: 42 GIAASGKNYFDP Heavy chain variable region SEQ ID NO: 43 QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKNTDSVKGRFSISRDNS KNTLYLQMNSLRAEDTAVYFCAKGTAAAGKNYFDPWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 44 RYAMS Heavy chain CDR2 SEQ ID NO: 45 AISASGDRTKNTDSVKG Heavy chain CDR3 SEQ ID NO: 46 GTAAAGKNYFDP Heavy chain variable region SEQ ID NO: 47 QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKYTDSVKGRFSISRDNS KNTLYLQMNSLRAEDTAVYFCAKGTAAAGKNYFDPWGQGTLVTVSS Light chain variable region SEQ ID NO: 48 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK Light chain CDR1 according to IMGT SEQ ID NO: 49 QSISSY Light chain CDR2 according to IMGT SEQ ID NO: 50 AAS Light chain CDR3 according to IMGT SEQ ID NO: 51 QQSYSTPPT Light chain variable region SEQ ID NO: 52 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK Light chain CDR1 according to IMGT SEQ ID NO: 53 QSISSY Light chain CDR2 according to IMGT SEQ ID NO: 54 AAS Light chain CDR3 according to IMGT SEQ ID NO: 55 QQSYSTPPIT Light chain variable region SEQ ID NO: 56 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTI SSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK Light chain CDR1 according to IMGT SEQ ID NO: 57 QSVSSN Light chain CDR2 according to IMGT SEQ ID NO: 58 GAS Light chain CDR3 according to IMGT SEQ ID NO: 59 QQYNNWPWT Light chain variable region SEQ ID NO: 60 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLT ISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK Light chain CDR1 according to IMGT SEQ ID NO: 61 QSVSSSY Light chain CDR2 according to IMGT SEQ ID NO: 62 GAS Light chain CDR3 according to IMGT SEQ ID NO: 63 QQYGSSPWT Light chain variable region SEQ ID NO: 64 SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL Light chain CDR1 according to IMGT SEQ ID NO: 65 NIGRKS Light chain CDR2 according to IMGT SEQ ID NO: 66 YDS Light chain CDR3 according to IMGT SEQ ID NO: 67 QVWDGSSDHWV hinge region SEQ ID NO: 68 EPKSCDKTHTCPPCP CH1 region SEQ ID NO: 69 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKRV CH2 region SEQ ID NO: 70 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK CH2-DM region SEQ ID NO: 71 APELGRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK CH3 region SEQ ID NO: 72 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG CH3-DE region SEQ ID NO: 73 GQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG CH3-KK region SEQ ID NO: 74 GQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG CL region SEQ ID NO: 75 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain reference anti-TGF-βRII antibody TGF1 analog SEQ ID NO: 76 QLQVQESGPGLVKPSETLSLTCTVSGGSISNSYFSWGWIRQPPGKGLEWIGSFYYGEKTYYNPSLKSRATISIDT SKSQFSLKLSSVTAADTAVYYCPRGPTMIRGVIDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K Light chain reference anti-TGF-βRII antibody TGF1 analog SEQ ID NO: 77 EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain reference PD-1 antibody pembrolizumab SEQ ID NO: 78 QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSS TTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Light chain reference PD-1 antibody pembrolizumab SEQ ID NO: 79 EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDF TLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain reference PD-L1-TGF-β TRAP molecule analog SEQ ID NO: 80 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA GGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC EKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEE YNTSNPD Light chain reference PD-L1TGF-β TRAP molecule analog SEQ ID NO: 81 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASL TISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS human TGF-βRII isoform A SEQ ID NO: 82 MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITS ICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS EEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRS DISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLK HENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPI VHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSM ALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDP EARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK extracellular domain of human TGFβRII isoform A SEQ ID NO: 83 TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITL ETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ human TGF-βRII isoform B SEQ ID NO: 84 MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQL CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSST WETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVA VKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKL GSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYM APEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEI PSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGSLNTTK extracellular domain of isoform B of human TGF-βRII SEQ ID NO: 85 TIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCS ITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDLLLVIFQ Heavy chain negative control RSV IgG1 antibody SEQ ID NO: 86 EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSTKYSADSLKGRFTISRDNS KNTLYLQMNSLRADDTAVYYCAKEGWSFDSSGYRSWFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG Light chain SEQ ID NO: 87 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain variable region SEQ ID NO: 88 QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKNTDSVKGRFSISRDNS KNTLYLQMNSLRAEDTAVYYCAKGTAAAGKNYFDPWGQGTLVTVSS Heavy chain variable region SEQ ID NO: 89 QVQLVESGGGLVQPGGSLRLSCAVSGFTFSRYAMSWVRQAPGKGLEWVSAISASGDRTKYTDSVKGRFSISRDNS KNTLYLQMNSLRAEDTAVYYCAKGTAAAGKNYFDPWGQGTLVTVSS Heavy chain CDR1 SEQ ID NO: 90 RYAMS Heavy chain CDR2 SEQ ID NO: 91 AISASGDRTKYTDSVKG Heavy chain CDR3 SEQ ID NO: 92 GTAAAGKNYFDP V region VK1-39 SEQ ID NO: 93 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQSYSTP VK1-39/JK1 SEQ ID NO: 94 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK VK1-39/JK5 SEQ ID NO: 95 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK Heavy chain reference nivolumab analog antibody SEQ ID NO: 96: QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNS KNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Light chain reference nivolumab analog antibody SEQ ID NO: 97 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC V region VK3-15 SEQ ID NO: 98 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP VK3-15/JK1 SEQ ID NO: 99 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTI SSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK V region VK3-20 SEQ ID NO: 100 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLT ISRLEPEDFAVYYCQQYGSSP VK3-20/JK1 SEQ ID NO: 101 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLT ISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK V region VL3-21 SEQ ID NO: 102 SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGSSDH VL3-21/JL3 SEQ ID NO: 103 SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTIS RVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL Extracellular domain TGF-βRII SEQ ID NO: 104 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLE TVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD