NOVEL PD-1 BINDING DOMAINS

Abstract

The present disclosure relates to novel PD-1 binding domains that have a higher binding affinity for human PD-1 than a reference PD-1 binding domain. The PD-1 binding domains of the present disclosure further provide a comparable, or equal or higher, potency in blocking ligand binding to human PD-1 than a reference PD-1 antibody. The present disclosure further relates to binding moieties comprising such PD-1 binding domains. Also provided is a method for treating a disease, in particular a disease associated with a suppressed immune system, such as cancer, with a PD-1 binding domain or binding moiety of the present disclosure. The present disclosure further relates to nucleic acids encoding the heavy chain variable region of the PD-1 binding domains, and a vector and cell comprising such nucleic acid.

Claims

1. An anti-human PD-1 binding domain having higher binding affinity for human PD-1 than a reference anti-human PD-1 binding domain, wherein the reference anti-human PD-1 binding domain comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 20 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 21.

2. An anti-human PD-1 binding domain, which when monovalently present in a bivalent antibody, provides comparable, or equal or higher, potency in blocking ligand binding to PD-1 than a reference anti-human PD-1 antibody, wherein the reference anti-human PD-1 antibody comprises two heavy chain variable regions having an amino acid sequence as set forth in SEQ ID NO: 20 and two light chain variable regions having an amino acid sequence as set forth in SEQ ID NO: 21.

3. The anti-human PD-1 binding domain according to claim 1, wherein the anti-human PD-1 binding domain comprises at least a heavy chain variable region and a light chain variable region, and wherein the light chain variable region preferably is a light chain variable region of a light chain that is capable of pairing with multiple heavy chains having different epitope specificities.

4. The anti-human PD-1 binding domain according to claim 1, wherein the binding affinity is measured by surface plasmon resonance.

5. The anti-human PD-1 binding domain according to claim 1, wherein the anti-human PD-1 binding domain has at least a ten-fold higher binding affinity for human PD-1 than the reference anti-human PD-1 binding domain.

6. The anti-human PD-1 binding domain according to claim 1, wherein the anti-human PD-1 binding domain has a ten-fold higher binding affinity for human PD-1 than the reference anti-human PD-1 binding domain.

7. The anti-human PD-1 binding domain according to claim 1, wherein the anti-human PD-1 binding domain has a binding affinity for human PD-1 in a range of about 0.1-1.0 nM, in particular in a range of about 0.3-0.8 nM, more in particular in a range of about 0.38-0.78 nM.

8. The anti-human PD-1 binding domain according to claim 1, wherein the binding affinity is measured with both the anti-human PD-1 binding domain and the reference anti-human PD-1 binding domain in a bivalent monospecific IgG format.

9. The anti-human PD-1 binding domain according to claim 1, wherein the binding affinity is measured with the anti-human PD-1 binding domain in a bivalent bispecific IgG format and the reference anti-human PD-1 binding domain in a bivalent monospecific IgG format.

10. The anti-human PD-1 binding domain according to claim 2, wherein the potency in blocking ligand binding to PD-1 is measured in a PD-1/PD-L1 reporter assay.

11. The anti-human PD-1 binding domain according to claim 2, wherein a comparable potency in blocking ligand binding to PD-1 activity is a potency within a 5 fold range of the potency in blocking ligand binding to PD-1 of the reference anti-human PD-1 antibody, including a 5, 4, 3, and 2 fold deviation from the potency in blocking ligand binding to PD-1 of the reference anti-human PD-1 antibody.

12. The anti-human PD-1 binding domain according to claim 1, wherein the heavy chain variable region comprises: 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: 22, SEQ ID NO: 23 and SEQ ID NO: 24, 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: 25, SEQ ID NO: 26, and SEQ ID NO: 27, 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: 28, SEQ ID NO: 29, and SEQ ID NO: 30, 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: 31, SEQ ID NO: 32, and SEQ ID NO: 33, 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: 34, SEQ ID NO: 35, and SEQ ID NO: 36, 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: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively; 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: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; h) 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: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively; or i) 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: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively; wherein each of the HCDRs may comprise at most three, two, or one amino acid substitution.

13. The anti-human PD-1 binding domain according to claim 1, comprising a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NO: 1-9, or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

14. The anti-human PD-1 binding domain according to claim 3, further comprising a CH1 and CL region.

15. An anti-human PD-1 binding domain comprising a heavy chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 (HCDR1) from a heavy chain variable region having an amino acid sequence from the group consisting of SEQ ID NO: 1-9, a heavy chain CDR2 (HCDR2) from a heavy chain variable region having an amino acid sequence from the group consisting of SEQ ID NO: 1-9, and a heavy chain CDR3 (HCDR3) from a heavy chain variable regions having an amino acid sequence from the group consisting of SEQ ID NO: 1-9.

16. The anti-human PD-1 binding domain according to claim 15, wherein the heavy chain variable region comprises: 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: 22, SEQ ID NO: 23 and SEQ ID NO: 24, 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: 25, SEQ ID NO: 26, and SEQ ID NO: 27, 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: 28, SEQ ID NO: 29, and SEQ ID NO: 30, 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: 31, SEQ ID NO: 32, and SEQ ID NO: 33, 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: 34, SEQ ID NO: 35, and SEQ ID NO: 36, 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: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively; 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: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; h) 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: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively; or i) 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: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively; wherein each of the HCDRs may comprise at most three, two, or one amino acid substitution.

17. The anti-human PD-1 binding domain according to claim 15, comprising a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NO: 1-9 or having at least 80%, preferably 85%, more preferably 90%, or most preferably 95% sequence identity thereto.

18. The anti-human PD-1 binding domain according to claim 15, further comprising a CH1 and CL region.

19. A binding moiety comprising an anti-PD-1 binding domain as claimed in claim 1.

20. The binding moiety according to claim 19, wherein the binding moiety is a monospecific binding moiety, preferably a bivalent monospecific antibody.

21. A pharmaceutical composition comprising an effective amount of the anti-human PD-1 binding domain according to claim 1, and a pharmaceutically acceptable carrier.

22-24. (canceled)

25. A method for treating a disease, comprising administering an effective amount of the anti-human PD-1 binding domain as claimed in claim 1 to an individual in need thereof.

26. A method for treating a disease associated with a suppressed immune system, comprising administering an effective amount of the anti-human PD-1 binding domain as claimed in claim 1 to an individual in need thereof.

27. A method for treating cancer, comprising administering an effective amount of the anti-human PD-1 binding domain as claimed in claim 1 to an individual in need thereof.

28. A nucleic acid sequence encoding the heavy chain variable region of the anti-human PD-1 binding domain as claimed in claim 1.

29. A vector comprising a nucleic acid sequence as claimed in claim 28.

30. The vector according to claim 29, wherein the vector further comprises a nucleic acid sequence encoding a CH1 region and preferably a hinge, CH2 and CH3 region.

31. The vector according to claim 29, wherein the vector further comprises at least one nucleic acid sequence encoding a light chain variable region, and preferably a CL region.

32. The vector according to claim 31, wherein the light chain variable region is a light chain variable region of a light chain that is capable of pairing with multiple heavy chains having different epitope specificities.

33. A cell comprising a nucleic acid sequence encoding the heavy chain variable region of the anti-human PD-1 binding domain as claimed in claim 1.

34. The cell according to claim 33, wherein the cell further comprises a nucleic acid sequence encoding a CH1 region and preferably a hinge, CH2 and CH3 region.

35. The cell according to claim 33, wherein the cell further comprises at least one nucleic acid sequence encoding a light chain variable region, and preferably a CL region.

36. A cell producing the anti-human PD-1 binding domain as claimed in claim 1.

37. The cell according to claim 36, wherein the cell is a recombinant cell, which has been transformed with a vector comprising a nucleic acid sequence encoding the heavy chain variable region of the anti-human PD-1 binding domain.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0168] In the Figures, bivalent monospecific antibodies are indicated in the format SEQ ID NO: A, where SEQ ID NO: A refers to the heavy chain variable sequence of both binding domains. Each binding domain of the monospecific antibodies comprises a light chain. 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 monospecific antibodies comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 16 and a light chain constant region having an amino acid sequence as set forth in SEQ ID NO: 60. The monospecific 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, monospecific antibodies were screened in IgG1 format, wherein the PD-1 binding heavy chains comprise a CH1 having an amino acid sequence as set forth in SEQ ID NO: 17, a CH2 having an amino acid sequence as set forth in SEQ ID NO: 18, and a CH3 having an amino acid sequence as set forth in SEQ ID NO: 19.

[0169] Bivalent monospecific nivolumab analog antibody is 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. This reference antibody analog is used in IgG1 or IgG4 format, and each binding domain comprises a light chain.

[0170] Bivalent bispecific antibodies are indicated in the format SEQ ID NO: A×Antigen A, where SEQ ID NO: A refers to the heavy chain variable sequence of the PD-1 binding domain and Antigen A refers to the heavy chain variable sequence of an unrelated; arbitrarily selected antigen. Each binding domain of the bispecific antibodies comprises a light chain. The bispecific antibodies are IgG1 antibodies, comprising a CH1, hinge, CH2, and CH3.

[0171] FIG. 1 shows the results of screening of affinity matured variants in a PD-1/PD-L1 reporter assay. A) IgG's comprising affinity matured heavy chain variable regions having an amino acid sequence as set forth in SEQ ID NO: 1. SEQ ID NO: 2, and SEQ ID NO: 6; were compared with parental antibody comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 9, a nivolumab analog (SEQ ID NO: 11/SEQ ID NO: 14) as a positive control, and a negative control (SEQ ID NO: 15/SEQ ID NO: 16). B) IgG's comprising affinity matured heavy chain variable regions having an amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5; were compared with parental antibody comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 10, nivolumab analogs (SEQ ID NO: 11/SEQ ID NO: 14 and SEQ ID NO: 12/SEQ ID NO: 14) as positive controls, and a negative control (SEQ ID NO: 15/SEQ ID NO: 16).

[0172] FIG. 2 shows the binding affinity of the PD-1 binding domains comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, SEQ NO: 6, or SEQ ID NO: 5, in bivalent monospecific format, compared with bivalent monospecific nivolumab analog 1 (SEQ ID NO: 11/SEQ ID NO: 14; in quadruplicate) and the PD-1 binding domain of nivolumab analog 1 as part of a bivalent bispecific antibody (SEQ ID NO: 11; SEQ ID NO: 14×SEQ ID NO: 15/SEQ ID NO: 16).

[0173] FIG. 3 shows the binding affinity of bispecific antibodies comprising SEQ ID NO: 7 and a binding domain against an arbitrarily selected antigen not impacting PD-1 binding tested in the assay, and SEQ ID NO: 8 and a binding domain against an arbitrarily selected antigen not impacting PD-1 binding tested in the assay, to human and cynomolgus PD-1, compared with a nivolumab analog (SEQ If) NO: 13/SEQ ID NO: 14).

[0174] The following Examples illustrate the present disclosure but are not intended to limit the disclosure in any way.

EXAMPLES

Example 1— Generation of Anti-Human PD-1 Binding Domains

[0175] Anti-human PD-1 binding domains can be obtained by methods known in the art, such as for instance as described in WO 2019/009728. A large panel of heavy chain variable regions were obtained by immunizing transgenic mice comprising a common IGKV1-39 light chain (MeMo® mice) with human PD-1 antigenic moieties, including the use of different forms of DNA, protein and cell-based antigen delivery. Heavy chain variable regions of SEQ ID NO: 9 and SEQ ID NO: 10 were selected for affinity maturation. This resulted in 202 affinity matured variants of which a number were selected for further characterization in a PD-1/PD-L1 reporter assay.

Example 2— Potency of PD-1 IgG

[0176] In order to confirm that the affinity matured PD-1 heavy chain variable regions in IgG format are at least as potent as their parental IgG's, affinity matured variants were screened in a PD-1/PD-L1 reporter assay. Also included in the assay were the parental anti-PD-1 IgG's, an anti-PD-1 antibody comprising the heavy chain variable region (SEQ ID NO: 11) and light chain variable region (SEQ ID NO: 14) of nivolumab (Fc-silenced IgG1 nivolumab analog 1), and an anti-PD-1 antibody comprising the heavy chain variable region (SEQ ID NO: 12) and light chain variable region (SEQ ID NO: 14) of nivolumab (IgG4 nivolumab analog 2) as positive controls, and an anti RSV-G antibody comprising the heavy chain variable region having SEQ ID NO: 0.15 and light chain variable region having SEQ ID NO: 16 as a negative control. The last 2 wells in this column were left without IgG as a basal level control.

[0177] The PD-1 PD-L1 reporter assay was performed according to manufacturer's protocol (Promega, cat. no. J1255), which uses two cell lines: PD-L1 aAPC/CHO-K1 which are CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate cognate TCRs in an antigen-independent manner (Promega, cat, no. J109A); and PD-1 effector cells: Jurkat T cells expressing human PD-1 and a luciferase reporter driven by an NFAT response element (NFAT-RE) (Promega, cat. no. J1115A).

[0178] On day 1, Cell Recovery Medium for PD-L1 cells was prepared at room temperature: 10% FBS (Sigma, cat. no. F2442) in DMEM/F12 (Life Technologies, cat. no. 21765). The required number of PD-L1 cell vials (J109A; 1 vial per 32 IgG's to be tested) were removed from the freezer, thawed quickly at 37° C. and cells transferred to a 50 nil tube. Cell Recovery Medium was slowly added to cells, 14.5 ml/vial, volume doubling per minute. Wells of ½-area plates (Corning, cat. no. 3688) were filled with this cell suspension at 50 μl/well or with 50 μl PBS (Invitrogen, cat. no. 10010). Assay plates were incubated overnight at 37° C., 5% CO.sub.2 and 95% Relative Humidity.

[0179] On the second day, 2× concentrated Assay Buffer was prepared: 4% FBS (Sigma, cat. no. F2442) in RPMI 1640 (Promega kit or Life Technologies, cat. no. 21875) at room temperature. 2× concentrated test and control IgG solutions were prepared in PBS. Serial dilutions of test and control IgG's were also made in PBS in U-bottom plates (Nunc, cat. no. 268152), starting with 10 μg/ml and performing 6-step 4-fold titration. Positive and negative control IgG serial dilutions were prepared in PBS on separate deep well plates (Greiner Bio-one, cat. no. 780270). Basal control, which is control without IgG was also prepared. IgG's of which activities need to be compared directly were incubated on same plate as much as possible, to avoid inter-plate variation.

[0180] Assay plates were taken out of the incubator and flicked to empty wells. 20 μl of IgG solution was added to assay plate, starting with transfer of lowest IgG concentration followed by higher concentration with same pipet tips.

[0181] Required number of PD-1 effector cells (J115A: 1 vial per 32 IgG's to be tested) were removed from freezer, thawed quickly at 37° C. and gently mixed by pipetting up and down, Cells from all vials were transferred to a 50 ml tube. 2× concentrated Assay Buffer (5.9 ml per vial of cells) was slowly added to cells such that volume doubled per minute. 20 μl of effector cell suspension was added to wells on assay plates. Plates were incubated for 6 hours at 37° C., 5% CO2 and 95% Relative Humidity. Following 6 hours incubation, plates were pre-incubated at room temperature for 10 min.

[0182] Luciferase activity was measured using the Bio-Glo™ luciferase Assay System (Promega, cat. no. G7941). Bio-Glo™ Luciferase Assay Buffer (protected from light) was equilibrated to room temperature overnight and thoroughly mixed with Bio-Glo™ Luciferase Assay Substrate. 40 μl of Bio-Glo luciferase was added to each well on the assay plate and luminescence measured after 5-10 min on EnVision plate reader (PerkinElmer, Model 2104-0040A Luminescence mode). Readout was obtained in Relative light unit (RLU) values. Fold Induction which is ratio of experimental activity to control activity was calculated as RLU value of IgG-X/RLU value of no IgG. Fold Induction was plotted against log IgG concentrations and the sigmoid curve fitted in GraphPad Prism using nonlinear regression and the log(inhibitor) vs. response (three parameters) equation.

[0183] Results are shown in FIG. 1. All controls displayed the expected activities and were consistent in different plates. The affinity matured variants were at least as potent as their parental IgG, and as potent or more potent than nivolumab analog 1. EC50 values of the affinity matured variants and parental antibodies are shown in Table 1.

TABLE-US-00001 TABLE 1 EC50 values of affinity matured variants and parental antibodies. IgG comprising EC50 (nM) a VH having amino nivolumab acid sequence: EC50 (nM) analog SEQ ID NO: 1 3.81 3.47 SEQ ID NO: 2 4.49 SEQ ID NO: 6 2.87 SEQ ID NO: 9 5.65 SEQ ID NO: 5 4.91 5.79 SEQ ID NO: 4 4.12 SEQ ID NO: 3 4.20 SEQ ID NO: 10 11.05

Example 3—Binding Characteristics

[0184] The binding affinity of selected affinity matured PD-1 binding domains was determined using SPR. The binding affinity for human PD-1 was determined in bivalent monospecific IgG format and compared with the binding affinity of bivalent monospecific analogs of reference antibody nivolumab and a bivalent bispecific antibody comprising a binding domain having the sequence of reference antibody nivolumab and a binding domain that binds an unrelated target.

[0185] SPR experiments were performed using a Biacore 8K instrument (GE Healthcare) at 25° C. The SPR running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% v/v Surfactant P20, pH 7.4) was prepared from 10×HBS-EP Buffer (GE Healthcare). Anti-human Fc antibodies (GE Healthcare) were immobilized via amine coupling on all sixteen flow cells of an S series sensor chip CM5 (GE Healthcare). The immobilization levels are ˜9000 RU for all flow cells. The desired capturing level (100-150 RU) of anti-PD-1 antibodies was achieved by flowing appropriate concentration of anti-PD-1 antibodies through the active flow cell of each channel for 60 seconds with 10 μL/min flow rate. Then, a PD-1 three-fold serial dilution concentration series (total 7 concentrations, highest at 300 nM) prepared from PD-1 stock (R&D 8986-PD) and running buffer (0 concentration) were injected for 240 seconds (association time) immediately followed by running buffer for 480 seconds (dissociation time) at a flow rate of 45 Surface was regenerated with 30-second injection of 3 M MgCl.sub.2 with 30 μL/min flow rate. Binding kinetics and affinity parameters were obtained from a global fit of the data to 1 to 1 binding model.

[0186] Data is shown in FIG. 2. IgG's comprising the PD-1 binding domains of the present disclosure have an at least ten-fold higher binding affinity (K.sub.D) than the analogs of the reference antibody.

[0187] Binding affinity was also determined in bispecific IgG format using SPR on a BIAcore-T200 instrument using an anti-huIgG antibody immobilized on a CM5 Series S sensor chip. It was also assessed if the two human proteins can be engaged simultaneously by the bispecific antibodies. The binding affinity of bispecific antibodies comprising a PD-1 binding domain comprising a heavy chain variable region having SEQ ID NO: 7 or a PD-1 binding domain comprising a heavy chain variable region having SEQ ID NO: 8 to human PD-1 and cynomolgus PD-1 was determined. The antibody format used is PD-1×Antigen A, wherein Antigen A is an arbitrarily selected antigen not reactive with PD-1 and not impacting PD-1 binding tested in the assay. Each binding domain of the bispecific antibodies comprises a heavy chain and a light chain. The binding affinity of the bispecific antibodies was compared with the binding affinity of an analog of reference antibody nivolumab, which comprises two anti-PD-1 binding domains.

[0188] Reference antibodies used were: nivolumab analog (SEQ ID NO: 13/SEQ ID NO: 14) and a reference antibody against antigen A. An antibody against an unrelated target was used as a negative control for binding. Test antibodies were SEQ ID NO: 7× Antigen A and SEQ ID NO: 8× Antigen A.

[0189] Monomeric recombinant antigens used were: hu-Antigen A, cy-Antigen A, huPD-1 (huPD-1-His, Sino Biological, cat, nr. 10377-H08H) and cyPD-1 (cyPD-1-His, R&D Systems, cat. nr. 8509-PD).

[0190] Immobilization:

[0191] Immobilization of goat anti-huIgG Fc (JIR, cat. nr. 109-005-098) on four flow channels of a CM5 sensor chip (GE Healthcare; Cat. Nr. BR-1005-30) was performed by amine coupling, using 40 μg/ml of the antibody diluted in 10 mM acetate pH 5.0. The following conditions were used: activation time of 420 seconds, deactivation time of 420 seconds, deactivation buffer: 1 M ethanolamine pH 8.5. A high density of immobilization was achieved, ranging from 9158 to 9428 RU.

[0192] Affinity Determination:

[0193] Fax affinity determination, test and control antibodies were captured by anti-huIgG antibody immobilized on the CM5 sensor chip at a flow rate of 30 μl/min for 60 seconds in only one flow cell. Captured antibody concentration was 20 nM for PD-1 affinity determination and 10 nM for Antigen A affinity determination. This was followed by a stabilization period of 60 seconds with buffer at a flow rate of 30 μl/min. Five step, two fold, serial dilutions of the antigens were injected, at 30 μl/min, for 60 seconds, in both the flow cell with the captured antibody and a reference flow cell (no captured antibody). Antigen concentrations were 80 nM down to 2.5 nM for huPD-1 and cyPD-1, and 40 to 1.25 nM for hu-Antigen A and cy-Antigen A. Background correction for buffer effects was performed by injection with buffer alone and the reference flow cell was used for background subtraction.

[0194] Following antibody—antigen interaction, an off-rate wash of 300 seconds, at 30 μl/min was done. Regeneration between cycles was done using two 15 μl injections of 10 mM Glycine pH 1.5 at 30 μl/min, followed by a stabilization step of 90 seconds at 90 μl/min. To confirm total regeneration and assay consistency, a repeat run of the reference antibody with all the tested antigen concentrations was performed at the end of the assay and for all antigens tested.

[0195] HBS-EP+ buffer was used for PD-1 affinity determination, while, for Antigen A, HBS-EP+ was supplemented with NaCl to a final concentration of 500 nM NaCl, in order to avoid unspecific binding.

[0196] Results were analyzed in Biacore T200 Evaluation Software. The raw RU signal were blank subtracted (channel with no captured antibody) and background corrected for buffer effects (subtraction of the run with captured antibody but with buffer in the second injection, instead of antigen). 1:1 binding Langmuir fitting was applied to the set of sample curves, using the simultaneous fitting option of the Biacore T200 Evaluation Software to calculate association rate (ka), dissociation rate (kd) and affinity (KD).

[0197] The captured bispecific and reference antibodies showed binding to the respective recombinant antigens. No binding of the antigen to the negative control antibody was observed.

[0198] An overview of the data is provided in FIG. 3. For huPD-1, the two bispecific antibodies had similar affinity, both showing more than 10 fold improvement in KD over the reference antibody nivolumab analog, mainly due to the slower dissociation observed. For cyPD-1-His, SEQ ID NO: 7×Antigen A showed approximately 10 fold improvement over nivolumab analog mainly due to the slower dissociation.

[0199] Simultaneous Binding:

[0200] Simultaneous binding of the bispecific antibodies to hu-Antigen A and huPD-1 was assayed with a similar set-up as for affinity determination. An immobilized anti-huIgG was used to capture the bispecific antibodies. A mix of nivolumab analog and Antigen A reference antibody was included as a positive control and an antibody against an unrelated target was included as negative control. Then, one of the antigens was injected at a saturating concentration (80 nM for huPD-1 and 40 nM for hu-Antigen A) for 300 sec, to occupy all antigen binding sites. The second antigen was injected sequentially at the same concentration used in injection 1, either alone or in combination with the first antigen (to ensure that all binding sites remained occupied). High salt buffer was used during the whole process, to prevent hu-Antigen A unspecific binding.

TABLE-US-00002 SEQUENCES SEQ ID NO: 1 - Heavy chain variable region - CDRs indicated in bold and underlined according to Rabat QVQLQESGPGLVKPSETLSLTCTVSNGSLGFDFWSWIRQPPGRGLEWIGYIYYSGSW SLNPSFKGRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPW GQGTLVTVSS SEQ ID NO: 2- Heavy chain variable region - CDRs indicated in bold and underlined according to Kabat QVQLQESGPGLVKPSETLSLTCTVSNGSLGFEFWSWIRQPPGRGLEWIGYIVYSGSH SVSPSLKTRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGHGGDWFDTW GQGTLVTVSS SEQ ID NO: 3- Heavy chain variable region - CDRs indicated in bold and underlined according to Kabat QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALSWVRQAPGQGLEWMGWIDPNT GTPTYAQDFTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCGSDICYPN GILDNWGQGTLVTVSS SEQ ID NO: 4- Heavy chain variable region - CDRs indicated in bold and underlined according to Kabat QVQLVQSGSELKKPGASVKVSCKASGYTFTRFAVNWVRQAPGQGLEWMGWIDPN TGTPTYAQGVTNRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCSSDICYP NLIFDNWGQGTLVTVSS SEQ ID NO: 5 - Heavy chain variable region - CDRs indicated in bold and underlined according to Kabat QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPN TGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYP NWIFDNWGQGTLVTVSS SEQ ID NO: 6- Heavy chain variable region - CDRs indicated in bold and underlined according to Kabat QVQLQESGPGLVKPSETLSLTCTVSDGSIGYHFWSWIRQPPGRGLEWIGYIVYSGSY NVNPSLKTRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDP WGQGTLVTVSS SEQ ID NO: 7- Heavy chain variable region - CDRs indicated in bold and underlined according to Kabat QVQLQESGPGLVKPSETLSLTCTVSEGSIGYHFWSWIRQPPGRGLEWIGYIVYSGSY NVNPSLKTRVTMS VDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPWGQGTLVTVSS SEQ ID NO: 8- Heavy chain variable region - CDRs indicated in bold and underlined according to Kabat QVQLVQSGSELKKPGASVKVSCKASGYTFTRFALHWVRQAPGQGLEWMGWIDPN TGTPTFAQGVTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARSLGYCDSDICYP NWIFDNWGQGTLVTVSS SEQ ID NO: 9- Heavy chain variable region - CDRs indicated in bold and underlined according to Kabat QVQLQESGPGLVKPSETLSLTCTVSNGSLGFYFWSWIRQPPGRGLEWIGYIYYSGST SFNPSLKSRVTMSVDTSKNQFSLNLRSVTAADTAVYYCARGGYTGYGGDWFDPW GQGTLVTVSS SEQ ID NO: 10 - Heavy chain variable region - CDRs indicated in bold and underlined according to Kabat QVQLVQSGSELKKPGASVKVSCKASGYTFTRFTMSWVRQAPGQGLEWMGWINPN TGNPTYAQDFTGRFVFSLDTSVTTAYLQISSLKAEDTAVYYCARILGYCNTDNCYP NWIFDYWGQGTLVTVSS SEQ ID NO: 11 - Heavy chain nivolumab analog 1 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDG SKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELGRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 12 - Heavy chain nivolumab analog 2 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDG SKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 13 - Heavy chain nivolumab analog 4 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDG SKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLWDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 14 - Light chain nivolumab EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 15 - Heavy chain variable region EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDG STKYSADSLKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKEGWSFDSSGYRSW FDSWGQGTLVT SEQ ID NO: 16 - Light chain variable region - CDRs indicated in bold and underlined according to IMGT DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK SEQ ID NO: 17-CH1 WT ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV SEQ ID NO: 18 - CH2 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID NO: 19 - CH3 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 20 - Nivolumab analog heavy chain variable region QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDG SKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVT VSS SEQ ID NO: 21 - Nivolumab analog light chain variable region EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK SEQ ID NO: 22 - HCDR1 according to Kabat FYFWS SEQ ID NO: 23 - HCDR2 according to Kabat YIYYSGSTSFNPSLKS SEQ ID NO: 24 - HCDR3 according to Kabat GGYTGYGGDWFDP SEQ ID NO: 25 - HCDR1 according to Kabat FDFWS SEQ ID NO: 26 - HCDR2 according to Kabat YIYYSGSWSLNPSFKG SEQ ID NO: 27 - HCDR3 according to Kabat GGYTGYGGDWFDP SEQ ID NO: 28 - HCDRI according to Kabat FEFWS SEQ ID NO: 29 - HCDR2 according to Kabat YIVYSGSHSVSPSLKT SEQ ID NO: 30 - HCDR3 according to Kabat GGYTGHGGDWFDT SEQ ID NO: 31 - HCDR1 according to Kabat RFALS SEQ ID NO: 32 - HCDR2 according to Kabat WIDPNTGTPTYAQDFTG SEQ ID NO: 33 - HCDR3 according to Kabat SLGYCGSDICYPNGILDN SEQ ID NO: 34 - HCDR1 according to Rabat RFAVN SEQ ID NO: 35 - HCDR2 according to Kabat WIDPNTGTPTYAQGVTN SEQ ID NO: 36 - HCDR3 according to Kabat SLGYCSSDICYPNLIFDN SEQ ID NO: 37 - HCDRI according to Kabat RFALH SEQ ID NO: 38 - HCDR2 according to Kabat WIDPNTGTPTFAQGVTG SEQ ID NO: 39 - HCDR3 according to Kabat SLGYCDSDICYPNWIFDN SEQ ID NO: 40 - HCDR1 according to Kabat YHFWS SEQ ID NO: 41 - HCDR2 according to Kabat YIVYSGSYNVNPSLKT SEQ ID NO: 42 - HCDR3 according to Kabat GGYTGYGGDWFDP SEQ ID NO: 43 - HCDR1 according to Kabat YHFWS SEQ ID NO: 44 - HCDR2 according to Kabat YIVYSGSYNVNPSLKT SEQ ID NO: 45 - HCDR3 according to Kabat GGYTGYGGDWFDP SEQ ID NO: 46 - HCDR1 according to Kabat RFALH SEQ ID NO: 47 - HCDR2 according to Kabat. WIDPNTGTPTFAQGVTG SEQ ID NO: 48 - HCDR3 according to Kabat SLGYCDSDICYPNWIFDN SEQ ID NO: 49 LCDR1 according to IMGT QSISSY SEQ ID NO: 50 LCDR2 according to IMGT AAS SEQ ID NO: 51 LCDR3 according to IMGT QQSYSTPPT SEQ ID NO: 52 Light chain variable region - CDRs indicated in bold and underlined according to IMGT DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK SEQ ID NO: 53 Light chain variable region - CDRs indicated in bold and underlined according to IMGT EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGI PARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK SEQ ID NO: 54 Light chain variable region - CDRs indicated in bold and underlined according to IMGT EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK SEQ ID NO: 55 Light chain variable region - CDRs indicated in bold and underlined according to IMGT SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGI PERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL SEQ ID NO: 56 V region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP SEQ ID NO: 57 V region EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGI PARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP SEQ ID NO: 58 V region EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP SEQ ID NO: 59 V region SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIP ERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDH SEQ ID NO: 60 Light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC