PD1 and VEGFR2 dual binding agents

Abstract

The invention relates to a PD1 and VEGFR2 dual binding agent. Specifically, the invention relates to an antibody molecule specifically binding to PD1 and VEGFR2, a related nucleic acid molecule, a vector and a host cell. Also provided herein are medical uses of such antibody molecules.

Description

Cross-references to related applications

[0001] This application is a divisional application of Chinese invention patent application filed on October 9, 2020, entitled "PD1 and VEGFR2 dual binder" with application number 202080075802.2.

[0002] This application claims the benefit of UK Patent Application No. 2013180.1, filed on 24 August 2020, and UK Patent Application No. 1914747.9, filed on 11 October 2019, the disclosure of each of which is incorporated herein by reference in its entirety. Description of text files submitted electronically

[0003] The contents of the text file submitted electronically in this article are incorporated herein in their entirety by reference: a computer-readable copy of the sequence list (filename: UHEL_002_02WO_SeqList_ST25.txt, date of record: October 9, 2020, file size approximately 86,707 bytes). Technical Field

[0004] This invention relates to antibody molecules that specifically bind to both PD1 (also known as programmed cell death protein 1, PDCD1, CD279, PD-1, SLEB2, PD-1, SLE1) and VEGFR2 (also known as KDR, CD309, FLK1, kinase insertion domain receptor) and their medical uses. Background Technology

[0005] PD1 is a cell surface receptor that has been shown to be an immune "checkpoint" mediator. PD1 checkpoint activity minimizes the risk of autoimmunity by promoting apoptosis (programmed cell death) in lymph node-resident T cells that are reactive to self-antigens and by promoting the survival of regulatory (anti-inflammatory) T cells. Antagonism of PD1 activity by human or humanized monoclonal antibodies has proven to be a successful treatment for many forms of cancer because it can lead to the reactivation of T cells in the tumor. This clinical success has led to the proliferation of anti-PD1 antibody molecules examined in clinical trials, but most patients still do not initiate a durable anti-cancer response when treated with anti-PD1 agents alone.

[0006] Increasing the number of patients responding to anti-PD1 antibody therapy is a significant challenge. A possible strategy to improve the clinical response rate of anti-PD1 antibodies is to combine them with previously validated cancer therapies such as anti-angiogenic agents. One such class of agents are antibodies that block the VEGF signaling pathway, such as anti-VEGFR2 or anti-VEGF antibodies. However, such combination therapies require the use of two separate, expensive agents, each with its own toxicity risks. There remains a need for single-agent therapy that offers the benefits of combination therapy without the drawbacks of combining two separate agents. Summary of the Invention

[0007] This invention provides a number of anti-PD1 and anti-VEGFR2 biantagonistic antibodies and their medical uses.

[0008] According to one aspect of the invention, an antibody molecule or its antigen-binding portion is provided that specifically binds to human PD1 and human VEGFR2, and optionally also to cynomolgus monkey PD1 and cynomolgus monkey VEGFR2.

[0009] In some aspects, the present invention provides an antibody or an antigen-binding portion of said antibody that specifically binds to both PD1 and VEGFR2, wherein said antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein (a) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (b) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (c) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQESGIWLS (SEQ ID NO:8), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (d) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVYSIPWT (SEQ ID NO:6); (e) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVYSIPWT (SEQ ID NO:6); (f) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of LASQESGIWLS (SEQ ID NO:8), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVYSIPWT (SEQ ID NO:6); (g) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQTIGTWLT (SEQ ID NO:9), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVAELPFG (SEQ ID NO:10); (h) The amino acid sequence of the VH region includes HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region includes LCDR1 of LASQTIGTWLT (SEQ ID NO:9), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (i) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVAELPFG (SEQ ID NO:10); (j) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVAELPFG (SEQ ID NO:10); (k) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQESGIWLG (SEQ ID NO:12), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVAELPFG (SEQ ID NO:10); (l) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (m) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (n) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (o) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TASSLAD (SEQ ID NO:40) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (p) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLAD (SEQ ID NO:41) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (q) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (r) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TASSLAD (SEQ ID NO:40) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (s) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLAD (SEQ ID NO:41), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); or (t) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42) and HCDR3 of QVYYFDY (SEQ ID NO:64); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43) and LCDR3 of QQVSVTPFT (SEQ ID NO:11).

[0010] In some respects, this document discloses anti-PD1 antibodies or their antigen-binding moieties, wherein said antibodies comprise a heavy chain variable (VH) region and a light chain variable (VL) region, wherein The amino acid sequence of the VH region includes: (a) HCDR1 of SEQ ID NO:1; (b) HCDR2 of SEQ ID NO:2, SEQ ID NO:38 or SEQ ID NO:42, and (c) HCDR3 of SEQ ID NO:3 or SEQ ID NO:64; and The VL region amino acid sequence includes: (aʹ) LCDR1 of SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12 or SEQ ID NO:39; (bʹ) LCDR2 of SEQ ID NO:5, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:43; and (cʹ) LCDR3 of SEQ ID NO:6, SEQ ID NO:10 or SEQ ID NO:11.

[0011] In some respects, this document discloses antibodies or antigen-binding moieties of said antibodies that specifically bind to both PD1 and VEGFR2, wherein said antibodies comprise a heavy chain variable (VH) region and a light chain variable (VL) region, wherein (a) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:44; (b) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:47; (c) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:24; (d) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:14; (e) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:15; (f) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:16; (g) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:17; (h) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:18; (i) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:19; (j) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:20; (k) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:21; (l) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:22; (m) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:23; (n) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:44; (o) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:45; (p) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:46; (q) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:47; (r) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:45; (s) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:46; or (t) The amino acid sequence of the VH region contains SEQ ID NO:73, and the amino acid sequence of the VL region contains SEQ ID NO:47.

[0012] In some aspects of the invention, the HCDR1 of the antibody molecule or antigen-binding portion may exclude the sequence GFTFSSYMMS (SEQ ID NO:1; MAb005 mouse / humanized antibody HCDR1 disclosed in WO2015 / 085847A1; US2016 / 376367A1), and / or the HCDR2 of the antibody molecule or antigen-binding portion may exclude the sequence TISGGGANTYYPDSVKG (SEQ ID NO:2; MAb005 mouse / humanized antibody HCDR2 disclosed in WO2015 / 085847A1; US2016 / 376367A1), and / or the HCDR3 of the antibody molecule or antigen-binding portion may exclude the sequence QLYYFDY (SEQ ID NO:3; MAb005 mouse / humanized antibody HCDR3 disclosed in WO2015 / 085847A1; US2016 / 376367A1).

[0013] In some aspects of the invention, the LCDR1 of the antibody molecule or antigen-binding portion may exclude the sequence LASQTIGTWLT (SEQ ID NO: 9; MAb005 mouse / humanized antibody LCDR1 disclosed in WO2015 / 085847A1; US2016 / 376367A1), and / or the LCDR2 of the antibody molecule or antigen-binding portion may exclude the sequence TATSLAD (SEQ ID NO: 5; MAb005 mouse / humanized antibody LCDR2 disclosed in WO2015 / 085847A1; US2016 / 376367A1), and / or the LCDR3 of the antibody molecule or antigen-binding portion may exclude the sequence QQVYSIPWT (SEQ ID NO: 6; MAb005 mouse / humanized antibody LCDR3 disclosed in WO2015 / 085847A1; US2016 / 376367A1).

[0014] The invention also provides immunoconjugates comprising an antibody molecule or its antigen-binding portion as defined herein, which is linked, fused to, or conjugated with a therapeutic agent.

[0015] In another aspect, the present invention provides nucleic acid molecules encoding antibody molecules or their antigen-binding portions as defined herein.

[0016] A carrier containing the nucleic acid molecules of the present invention is also provided.

[0017] Host cells comprising nucleic acid molecules or vectors of the present invention as defined herein are also provided.

[0018] In another aspect, a method is provided for generating anti-PD1 / VEGFR2 antibodies and / or their antigen-binding portions, the method comprising culturing host cells of the invention under conditions that result in the expression and / or generation of the antibodies and / or their antigen-binding portions, and isolating the antibodies and / or their antigen-binding portions from the host cells or cultures.

[0019] In another aspect of the invention, pharmaceutical compositions are provided comprising an antibody molecule or its antigen-binding portion as defined herein, a nucleic acid molecule as defined herein, or a carrier as defined herein.

[0020] Methods for enhancing the immune response of subjects are also provided, the methods comprising administering an effective amount of an antibody molecule or its antigen-binding portion as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a carrier as defined herein, or a pharmaceutical composition as defined herein.

[0021] In another aspect, methods for treating or preventing cancer in a subject are provided, the methods comprising administering an effective amount of an antibody molecule or its antigen-binding portion as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a carrier as defined herein, or a pharmaceutical composition as defined herein.

[0022] This invention also provides antibody molecules or antigen-binding moieties thereof as defined herein, or immunoconjugates as defined herein, or nucleic acid molecules as defined herein, or carriers as defined herein, or pharmaceutical compositions as defined herein, for use as pharmaceuticals. The invention further provides antibody molecules or antigen-binding moieties thereof as defined herein, or immunoconjugates as defined herein, or nucleic acid molecules as defined herein, or carriers as defined herein, or pharmaceutical compositions as defined herein, for the treatment of cancer.

[0023] In another aspect, the present invention provides an antibody molecule or its antigen-binding portion, or an immunoconjugate, or a nucleic acid molecule or carrier, or a treatment method of the present invention as defined herein, for use alone, sequentially or simultaneously in combination with a second therapeutic agent (e.g., an anticancer agent).

[0024] In another aspect, the use of antibody molecules or antigen-binding portions thereof of the present invention as defined herein, or immunoconjugates of the present invention as defined herein, or nucleic acid molecules of the present invention as defined herein, or carriers of the present invention as defined herein, or pharmaceutical compositions of the present invention as defined herein, in the manufacture of medicaments for treating cancer is provided.

[0025] The present invention also provides a method for treating or preventing an infectious disease in a subject, the method comprising administering an effective amount of an antibody molecule or its antigen-binding portion as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a carrier as defined herein, or a pharmaceutical composition as defined herein.

[0026] In all respects, the infectious disease can be selected from the group consisting of viruses, bacteria, fungi, or parasites. In one embodiment, the infectious disease is human immunodeficiency virus (HIV).

[0027] It also provides antibody molecules or their antigen-binding portions as defined herein, or immune conjugates as defined herein, or nucleic acid molecules as defined herein, or carriers as defined herein, or pharmaceutical compositions as defined herein, for the treatment of infectious diseases.

[0028] It also provides the use of antibody molecules or their antigen-binding portions as defined herein, or immune conjugates as defined herein, or nucleic acid molecules as defined herein, or carriers as defined herein, or pharmaceutical compositions as defined herein in the manufacture of medicaments for the treatment of infectious diseases.

[0029] The present invention also provides a method for treating or preventing an infectious disease in a subject, the method comprising administering an effective amount of an antibody molecule or its antigen-binding portion as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a carrier as defined herein, or a pharmaceutical composition as defined herein.

[0030] The present invention also provides a method for generating antibody molecules or antigen-binding portions thereof that specifically bind to human PD1 and human VEGFR2, and optionally also to cynomolgus monkey PD1 and cynomolgus monkey VEGFR2, the method comprising the following steps.

[0031] (1) Transplanting anti-PD1 CDRs from non-human sources into a human v domain framework to generate humanized anti-PD1 antibody molecules or their antigen-binding portions; (2) Generate a clonal library containing one or more mutations in the CDR, or the antigen-binding portion thereof; (3) The library was screened for binding to human PD1 and human VEGFR2, and optionally also to cynomolgus monkey PD1 and rhesus monkey VEGFR2; (4) Select clones from the screening step (3) that exhibit binding specificity to human PD1 and human VEGFR2, and optionally also to cynomolgus monkey PD1 and rhesus monkey VEGFR2; and (5) Generate an antibody molecule or its antigen-binding portion that specifically binds to human PD1 and human VEGFR2, and optionally also to cynomolgus monkey PD1 and rhesus monkey VEGFR2, from the clone selected from step (4).

[0032] The method may further include the following steps: generating additional clones based on the clone selected in step (4) (e.g., based on further exploratory mutagenesis at a specific location in the CDR of the clone selected in step (4)) to enhance humanization and / or minimize human T cell epitope content and / or improve the manufacturing characteristics of the antibody molecule or its antigen-binding portion generated in step (5). Attached Figure Description

[0033] Figure 1ELISA for direct binding of library-derived anti-PD1 Fab to human cyno PD1 and human rhesus monkey VEGFR2 proteins. Clones were derived from multiple phage library selection branches, with phage populations selected against biotinylated targets in each round. After each round of selection, library-derived clones were screened based on the expression of Fab protein in the periplasm for both human (hu) and cyno (cy) PD1 and human (hu) and rhesus monkey (rh) VEGFR2 (black circles). The hMAb005v domain of human IgG1 Fab was expressed on each plate as a positive control (gray diamond).

[0034] Figure 2 Epitope competition analysis of Fab protein in AlphaScreen The anti-PD1 clone was expressed as Fab in E. coli, and a periprep was used in an epitope competition assay using AlphaScreen technology. In this assay, the relative affinity of the library-derived Fab and the retention of the parental hMAb005 epitope were analyzed by competition for ineffective binding of hMAb005 IgG1 to human PD1 protein.

[0035] Figure 3A – Figure 3B A direct titration ELISA for the binding of purified IgG1 ineffective to human cyno PD1-Fc and human rhesus monkey VEGFR2 protein. Clones derived from a library containing hMab005, isotype control IgG1, and human IgG1 ineffective form were used to target human cyno PD1 protein. Figure 3A ) and human and rhesus monkey VEGFR2 protein ( Figure 3B Titration (in nM) is performed in direct combination with ELISA.

[0036] Figure 4 Cell-based VEGFR2 antagonism assay. Clones derived from a library containing hMab005, isotype control IgG1, and a human IgG1 ineffective form were titrated (in nM) in a human VEGFR2 signaling assay, to which human VEGF-165 protein was added to induce VEGFR2 signaling. Neither hMab005 nor the isotype IgG1 control protein showed any concentration-dependent VEGFR2 antagonism. All 11 lead clones and the positive control anti-VEGFR2 IgG1 ramucirumab showed potent antagonism in the nM range.

[0037] Figure 5A – Figure 5KCell-based VEGFR2 antagonism assay – monoclonal analysis. Reanalysis on a clone-by-clone basis. Figure 4 The VEGFR2 antagonism of the lead clones is shown. Although all 11 lead clones showed strong antagonism, only one clone with a mutated library source was found in each clone's CDR. Figure 5A – Figure 5E It was found to be less potent than clones that combined mutations in both LCDR1 and LCDR3. Figure 5F - Figure 5K ).

[0038] Figure 6 Cell-based PD1 antagonism assay. hMab005 (SHR-1210 IgG1-3M), isotype control IgG1, nivolumab IgG4, and clones MAB06.1-MAB06.8 (ineffective form of human IgG1) were titrated in a human PD1 signal transduction assay (in which human PD1+ and human PD-L1+ cells were mixed and antagonism of the PD1 / PD-L1 interaction resulted in increased signal). The isotype IgG1 control protein showed no concentration-dependent PD1 antagonism. Clones MAB06.1-MAB06.8, hMab005, and the positive control anti-PD1 IgG4 nivolumab showed potent antagonism in the nM range.

[0039] Figure 7 Cell-based VEGFR2 antagonism assay. hMab005 (SHR-1210 IgG1-3M), isotype control IgG1, nivolumab IgG4, and clones MAB06.1-MAB06.8 (ineffective form of human IgG1) were titrated in a human VEGFR2 signaling assay, to which human VEGF-165 protein was added to induce VEGFR2 signaling. Neither hMab005 nor the isotype IgG1 control protein showed any concentration-dependent VEGFR2 antagonism. Clones MAB06.1-MAB06.8 and the positive control anti-VEGFR2 IgG1 ramucirumab showed potent antagonism in the nM range.

[0040] Figure 8A – Figure 8H Cellular PD1 and VEGFR2 antagonism assay – monoclonal analysis. Reanalysis on a clone-by-clone basis. Figure 7 The PD1 and VEGFR2 antagonism of the lead clone shown is illustrated. Clone MAB06.5 ( Figure 8A , Figure 8B MAB06.6 Figure 8C , Figure 8D MAB06.7 Figure 8E , Figure 8F ) and MAB06.8 ( Figure 8G , Figure 8H Comparative analysis of the PD1 and VEGFR2 antagonistic effects of the MAB06 clone demonstrated that the sequence of the MAB06 clone can accept mutations in multiple residues in LCDR1, LCDR2 and HCDR2 while retaining the ability to antagonize the signal transduction of both receptors.

[0041] Figure 9 Dual PD1-VEGFR2 antagonism. Tumor-infiltrating immune cells can express both PD1 and VEGFR2. Cancer cells (or stromal cells, other immune cells, etc.) within a tumor can express PD-L1 and / or VEGF. These signals work together to suppress immune function in the tumor microenvironment. Therefore, dual antagonist antibodies that can effectively block the signaling of both PD1 and VEGFR2 (on the same or different cells) may have improved antitumor potency compared to PD1 or VEGFR2 blocking antibodies alone.

[0042] Figure 10 Cell-based VEGFR2 agonist assay. Ramucirumab, isotype control human IgG1, MAB06.5, MAB06.8, and VEGF-165 proteins were titrated in a human VEGFR2 signaling assay. Ramucirumab, isotype and clone MAB06.5, and MAB06.8 did not show any concentration-dependent VEGFR2 agonist activity. The positive control VEGF-165 elicited a potent antagonistic effect in the nM range.

[0043] Figure 11A - Figure 11F Single-channel human DC:T cell mixed lymphocyte reaction (MLR) assay. MAB06.5, MAB06.8, and nivolumab (anti-PD1) were titrated (nM) in the human MLR assay using three separate human donor pairs in two replicate runs. MAB6.5 and MAB6.8 showed concentration-dependent potency equivalent to nivolumab in PD1 blockade-driven IFN-γ signaling. For donor pair 1 ( Figure 11A , Figure 11B ), donor pair 2 ( Figure 11C , Figure 11D ) and donor pair 3 ( Figure 11E , Figure 11F This finding was observed in both runs.

[0044] Figure 12A – Figure 12DA direct titration ELISA targeting purified IgG1 that is ineffective in binding to human cyno PD1-Fc and human cynomolgus monkey VEGFR2 proteins. In targeting human PD1 protein (… Figure 12A , Figure 12B ) and human and cynomolgus monkey VEGFR2 protein ( Figure 12C , Figure 12D The direct binding ELISA titrated (in nM) SHR-1210 IgG1-3M, isotype control IgG1, and seven third-generation clones in the ineffective form of human IgG1.

[0045] Figure 13A – Figure 13B Assay for PD1 and VEGFR2 antagonism based on cells. Clones SHR-1210 IgG1-3M, nivolumab, isotype control IgG1, and seven third-generation clones of PD1 ineffective against human IgG1 were used. Figure 13A ) and VEGFR2 ( Figure 13B Comparative analysis of antagonistic effects. Detailed Implementation

[0046] This invention is based on the discovery that antibodies unexpectedly antagonize both the PD1 and VEGF pathways. Inhibition of the VEGF signaling pathway can provide an effective combination with inhibition of the PD1 pathway. VEGF-VEGFR2 signaling not only drives angiogenesis but has also been shown to enhance immunosuppression in the tumor microenvironment, where VEGFR2 is expressed on multiple immune cell types. However, generating dual-inhibitor antibodies is a complex and unpredictable task because it involves producing a single antibody with potent regulation of two target proteins (e.g., PD1 and VEGFR2) that are highly different in amino acid sequence and structure. This sequence and structural difference between the two targets is a barrier to the successful generation of such antibodies.

[0047] WO2015 / 085847A1 describes an antagonistic mouse anti-PD1 IgG molecule called "MAb005" and the preparation of a humanized form of MAb005. Clinical trials have shown that humanized MAb005 (hMAb005) has a highly unusual side effect of inducing hemangiomas. in vitro The study successfully traced this issue back to off-target antibody reactivity, including VEGFR2 binding (Finlay). et al. (2019) mAbs, 11:1, 26-44). Importantly, the binding of hMAb005 (also known as SHR-1210) to VEGFR2 has been shown to result in potent receptor agonism (activation), which may be a major driver of hemangioma side effects. For the reasons described above, this humanized form of MAb005 described in WO2015 / 085847A1 is not ideal. Furthermore, it is impossible to advance Predicting which changes in molecular characteristics could convert an antagonist / agonist antibody, such as Mab005, into a dual antagonist of both the PD1 and VEGFR2 pathways. This invention provides antibodies that maintain potent PD1 antagonism but also potently antagonize VEGFR2. Such antibodies can provide synergistic clinical benefits.

[0048] This document provides a method for generating an antibody derived from hMAb005 that specifically binds to PD1 and antagonizes VEGF-VEGFR2 signaling. For example, such an antibody can be generated by introducing a mutation into one or more CDR sequences of antibody hMAb005. In some embodiments, the mutation is introduced into the LCDR1 and / or LCDR2 and / or HCDR2 sequences of antibody hMAb005.

[0049] In some aspects, antibodies or antigen-binding portions of said antibodies that specifically bind to both human PD1 and human VEGFR2 are provided. In some embodiments, the antibody or its antigen-binding portion is capable of antagonizing both the PD1-PDL1 signaling pathway and the VEGFR2-VEGF signaling pathway. In some embodiments, the antibody or its antigen-binding portion antagonizes the binding of human PD1 to human PD-L1 and antagonizes human VEGFR2 signaling in response to human VEGF.

[0050] In some aspects, the anti-PD1 and anti-VEGFR2 antibody or antigen-binding moieties provided herein specifically bind to PD1 proteins comprising or consisting of SEQ ID NO:32 or SEQ ID NO:33. In some aspects, the anti-PD1 and anti-VEGFR2 antibody or antigen-binding moieties provided herein specifically bind to PD1 proteins having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:32 or SEQ ID NO:33.

[0051] In some aspects, the anti-PD1 and anti-VEGFR2 antibody or antigen-binding moieties provided herein specifically bind to VEGFR2 proteins comprising or consisting of SEQ ID NO:34 or SEQ ID NO:35. In some aspects, the anti-PD1 and anti-VEGFR2 antibody or antigen-binding moieties provided herein specifically bind to VEGFR2 proteins having an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:34 or SEQ ID NO:35.

[0052] In some aspects of the invention, the HCDR1 of the antibody molecule or antigen-binding portion may exclude the sequence GFTFSSYMMS (SEQ ID NO:1; MAb005 mouse / humanized antibody HCDR1 disclosed in WO2015 / 085847A1; US2016 / 376367A1), and / or the HCDR2 of the antibody molecule or antigen-binding portion may exclude the sequence TISGGGANTYYPDSVKG (SEQ ID NO:2; MAb005 mouse / humanized antibody HCDR2 disclosed in WO2015 / 085847A1; US2016 / 376367A1), and / or the HCDR3 of the antibody molecule or antigen-binding portion may exclude the sequence QLYYFDY (SEQ ID NO:3; MAb005 mouse / humanized antibody HCDR3 disclosed in WO2015 / 085847A1; US2016 / 376367A1).

[0053] In some aspects of the invention, the LCDR1 of the antibody molecule or antigen-binding portion may exclude the sequence LASQTIGTWLT (SEQ ID NO: 9; MAb005 mouse / humanized antibody LCDR1 disclosed in WO2015 / 085847A1; US2016 / 376367A1), and / or the LCDR2 of the antibody molecule or antigen-binding portion may exclude the sequence TATSLAD (SEQ ID NO: 5; MAb005 mouse / humanized antibody LCDR2 disclosed in WO2015 / 085847A1; US2016 / 376367A1), and / or the LCDR3 of the antibody molecule or antigen-binding portion may exclude the sequence QQVYSIPWT (SEQ ID NO: 6; MAb005 mouse / humanized antibody LCDR3 disclosed in WO2015 / 085847A1; US2016 / 376367A1).

[0054] In some embodiments, the anti-PD1 antibodies of the present invention have been selected to be equivalent in binding specificity and affinity to both human PD1 and cynomolgus monkey PD1 (to facilitate the most accurate primate toxicology and pharmacokinetic studies). Further optimization of the optimized antibody molecules as described herein has provided improved binding to cynomolgus monkey orthologs of PD1 and / or maintained or improved potency in neutralizing PD1 / PD-L1 signaling. Crucially, these antibodies also significantly enhance their potential clinical activity and reduce their risk of inducing hemangiomas by becoming potent antagonists of the human receptor VEGFR2 compared to MAb005 (WO2015 / 085847A1; US2016 / 376367A1).

[0055] In some aspects, the anti-PD1 and anti-VEGFR2 antibody molecules of the present invention do not necessarily have the maximum number of human substitutions at the corresponding mouse CDR or other (such as framework) amino acid positions. In some embodiments, the “maximally humanized” antibody molecule is not necessarily “maximally optimized” in terms of anti-PD1 or anti-VEGFR2 binding characteristics and / or other desired characteristics.

[0056] This invention covers modifications to the amino acid sequence of antibody molecules or their antigen-binding moieties as defined herein. For example, the invention includes antibody molecules and their corresponding antigen-binding moieties comprising functionally equivalent variable regions and CDRs that do not significantly affect their properties, as well as variants with enhanced or reduced activity and / or affinity. For example, amino acid sequences can be mutated to obtain antibodies with desired binding affinity for PD1 and VEGFR2. Insertions including amino and / or carboxyl-terminal fusions ranging in length from one residue to peptides containing one hundred or more residues are contemplated, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibody molecules having an N-terminal methionyl residue or antibody molecules fused to an epitope tag. Other insertion variants of antibody molecules include enzymes or peptides that increase the half-life of the antibody in circulation, fused to the N-terminus or C-terminus of the antibody.

[0057] The antibody molecule or antigen-binding portion of the present invention may include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as glycosylation, acetylation, and phosphorylation with different sugars. Such post-translational modifications can be altered by mutating the antibody molecule or antigen-binding portion of the present invention, for example by adding, removing, or replacing one or more amino acid residues to form or remove glycosylation sites.

[0058] The antibody molecule or antigen-binding portion of the present invention can be modified, for example, by amino acid substitution, to remove potential proteolytic sites in the antibody.

[0059] In some embodiments of the present invention, antibodies or antigen-binding portions of said antibodies that specifically bind to both PD1 and VEGFR2 are provided, wherein said antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein (a) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVYSIPWT (SEQ ID NO:6); [MAB02A03] (b) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVYSIPWT (SEQ ID NO:6); [MAB02B03] (c) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQESGIWLS (SEQ ID NO:8), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVYSIPWT (SEQ ID NO:6); [MAB02D08] (d) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQTIGTWLT (SEQ ID NO:9), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVAELPFG (SEQ ID NO:10); [MAB05G03] (e) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQTIGTWLT (SEQ ID NO:9), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB05E08] (f) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVAELPFG (SEQ ID NO:10); [MAB01] (g) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVAELPFG (SEQ ID NO:10); [MAB02] (h) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQESGIWLG (SEQ ID NO:12), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVAELPFG (SEQ ID NO:10); [MAB03] (i) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB04] (j) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB05] or (k) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQESGIWLS (SEQ ID NO:8), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06] or (l) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.1] or (m) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TASSLAD (SEQ ID NO:40), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.2] or (n) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLAD (SEQ ID NO:41), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.3] or (l) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.4] or (p) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.5] or (q) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TASSLAD (SEQ ID NO:40), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.6] or (r) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLAD (SEQ ID NO:41), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.7] or (s) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.8] or (t) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYLMS (SEQ ID NO:61), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.8.1] or (u) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYLMS (SEQ ID NO:61), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QVYYFDY (SEQ ID NO:64); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.8.2] or (v) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYLMS (SEQ ID NO:61), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYGFDY (SEQ ID NO:65); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.8.3] or (w) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYLMS (SEQ ID NO:61), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYADY (SEQ ID NO:66); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.8.4] or (x) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYLMS (SEQ ID NO:61), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYFFDY (SEQ ID NO:62); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.8.5] or (y) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYLMS (SEQ ID NO:61), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYYDY (SEQ ID NO:63); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.8.6] or (z) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QVYYFDY (SEQ ID NO:64); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.8.7] or (aa) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYADY (SEQ ID NO:66); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); [MAB06.8.8] or (bb) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYYDY (SEQ ID NO:63); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11). [MAB06.8.9] In some respects, this document discloses anti-PD1 and anti-VEGFR2 antibodies or antigen-binding moieties thereof, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the amino acid sequence of the VH region comprises or consists of EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSS (SEQ ID NO:13).

[0060] In some respects, this document discloses anti-PD1 and anti-VEGFR2 antibodies or antigen-binding portions thereof, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises or is composed of SEQ ID NO:13, and the VL region comprises or is composed of any of the VL region amino acid sequences in Table 5 or Table 8.

[0061] In some respects, this document discloses antibodies or antigen-binding moieties of said antibodies that specifically bind to both PD1 and VEGFR2, wherein said antibodies comprise a heavy chain variable (VH) region and a light chain variable (VL) region, wherein (a) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:14; (b) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:15; (c) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:16; (d) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:17; (e) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:18; (f) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:19; (g) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:20; (h) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:21; (i) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:22; (j) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:23; (k) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:24; (l) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:44; (m) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:45; (n) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:46; (o) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:47; (p) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:44; (q) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:45; (r) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:46; (s) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:47; (t) The amino acid sequence of the VH region contains SEQ ID NO:67, and the amino acid sequence of the VL region contains SEQ ID NO:47; (u) The amino acid sequence of the VH region contains SEQ ID NO:68, and the amino acid sequence of the VL region contains SEQ ID NO:47; (v) The amino acid sequence of the VH region contains SEQ ID NO:69, and the amino acid sequence of the VL region contains SEQ ID NO:47; (w) The amino acid sequence of the VH region contains SEQ ID NO:70, and the amino acid sequence of the VL region contains SEQ ID NO:47; (x) The amino acid sequence of the VH region contains SEQ ID NO:71, and the amino acid sequence of the VL region contains SEQ ID NO:47; (y) The amino acid sequence of the VH region contains SEQ ID NO:72, and the amino acid sequence of the VL region contains SEQ ID NO:47; (z) The amino acid sequence of the VH region contains SEQ ID NO:73, and the amino acid sequence of the VL region contains SEQ ID NO:47; (aa) The amino acid sequence of the VH region contains SEQ ID NO:74, and the amino acid sequence of the VL region contains SEQ ID NO:47; or (bb) The amino acid sequence of the VH region contains SEQ ID NO:75, and the amino acid sequence of the VL region contains SEQ ID NO:47.

[0062] In some respects, this document discloses anti-PD1 and anti-VEGFR2 antibodies or their antigen-binding moieties, wherein said antibodies comprise a heavy chain variable (VH) region and a light chain variable (VL) region, wherein (a) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:13, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:14; (b) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:13, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:15; (c) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:13, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:16; (d) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:13, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:17; (e) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:13, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:18; (f) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:13, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:19; (g) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:13, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:20; (h) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:13, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:21; (i) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:13, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:22; (j) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:13, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:23; (k) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:13, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:24; (l) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:48, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:44; (m) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:48, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:45; (n) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:48, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:46; (o) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:48, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:47; (p) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:49, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:44; (q) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:49, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:45; (r) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:49, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:46; or (s) The amino acid sequence of the VH region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:49, and the amino acid sequence of the VL region is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to that of SEQ ID NO:47. In some aspects, the CDR amino acid sequence of the anti-PD1 and anti-VEGFR2 antibodies is 100% identical to the CDR amino acid sequence in the described sequence, while the FR amino acid sequence is less than 100% identical to the FR amino acid sequence in the described sequence.

[0063] In some respects, antibody or antigen-binding moieties, as defined herein, can be isolated.

[0064] Antibody molecules or antigen-binding moieties as defined herein can cross-competitively bind to PD1 and VEGFR2 with antibodies or antigen-binding moieties comprising the CDR set disclosed herein. In some embodiments, the present invention provides anti-PD1 and anti-VEGFR2 antibodies or antigen-binding moieties thereof, wherein said antibody or antigen-binding moieties cross-competitively bind to PD1 and VEGFR2 with antibodies or antigen-binding moieties comprising the CDR set disclosed herein; and (a) comprises a complete germline human framework amino acid sequence; (b) specifically binds to human PD1, cynomolgus monkey PD1, human VEGFR2, and rhesus monkey VEGFR2; and (c) antagonizes the binding of human PD1 to human PD-L1 and antagonizes human VEGFR2 signaling in response to human VEGF.

[0065] In some embodiments, the anti-PD1 and anti-VEGFR2 antibody or antigen-binding moieties have minimal immunogenicity. In some cases, the antibody or antigen-binding moieties exhibit reduced immunogenicity compared to anti-PD1 antibodies comprising HCDR1 of SEQ ID NO:1, HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3, LCDR1 of SEQ ID NO:9, LCDR2 of SEQ ID NO:5, and LCDR3 of SEQ ID NO:6. In some instances, the immunogenicity risk of the antibody or antigen-binding moieties can be determined by identifying the location of T-cell epitopes in the antibody or moieties (e.g., in the variable region of the antibody or moieties). Computer simulation Sure.

[0066] For example, by using iTope TM To identify T-cell epitopes in antibody or antigen-binding regions. (iTope) TM This method can be used to analyze the VL and VH region sequences of peptides that incidentally bind with high affinity to human MHC class II. It is believed that incidentally high-affinity MHC class II binding peptides are associated with the presence of T-cell epitopes, which are high-risk indicators of the clinical immunogenicity of drug proteins. TM The software predicted favorable interactions between the amino acid side chains of peptides and specific binding pockets (specifically pocket positions: p1, p4, p6, p7, and p9) within the open-terminal binding grooves of 34 human MHC class II alleles. These alleles represent the most common HLA-DR alleles found worldwide, without being weighted towards those most prevalent in any particular ethnic group. Twenty alleles contained the 'open' p1 conformation, and 14 alleles contained the 'closed' conformation, where glycine at position 83 was replaced by valine. The localization of key binding residues was determined by… Computer simulation This is achieved by generating a 9-mer peptide that overlaps with the test protein sequence by eight amino acids. This process successfully distinguishes peptides that bind to or do not bind to MHC class II molecules with high accuracy.

[0067] By using TCED TM (T cell epitope database) TM Analyzing the VL and VH region sequences to identify T-cell epitopes in the antibody or antigen-binding moiety, in order to search for previously identified epitopes via other protein sequences. in vitro Matching of T cell epitopes identified by human T cell epitope mapping analysis. Using TCED. TM Search a large database of >10,000 peptides for any test sequence from peptides derived from unrelated protein and antibody sequences.

[0068] In some implementations, the anti-PD1 and anti-VEGFR2 antibody or antigen-binding moieties may exhibit low immunogenicity because the antibody or moieties have a low number of one or more of the following peptides in their sequence: High Affinity Foreign ('HAF' – high immunogenicity risk), Low Affinity Foreign ('LAF' – low immunogenicity risk), and / or TCED+ (previously in TCED) TM (Tabletops identified in the database).

[0069] In some implementations, the anti-PD1 and anti-VEGFR2 antibody or antigen-binding moieties may have a high germline epitope (GE) content in their sequences. In some instances, the anti-PD1 antibody or antigen-binding moieties have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or more) germline epitopes in their sequences (e.g., in the VL and / or VH regions). Germline epitopes can be defined as human germline peptide sequences with high MHC class II binding affinity. As previously validated by studies using a broad range of germline peptides, germline epitope 9-meric peptides are unlikely to have immunogenic potential due to T-cell tolerance. Importantly, these germline v-domain epitopes (further aided by similar sequences in the constant region of human antibodies) also compete for MHC class II occupancy at the membrane of antigen-presenting cells, thereby reducing the risk that exogenous peptide presentation is sufficient to achieve the "activation threshold" required for T cell stimulation. Therefore, high GE content is a beneficial quality in the clinical development of antibody therapeutics and can provide low immunogenicity.

[0070] In some embodiments, compared to anti-PD1 antibodies containing the variable domain sequence of antibody Mab005, anti-PD1 and anti-VEGFR2 antibody or antigen-binding moieties may have a reduced number of HAF, LAF, and / or TCED+ epitopes found in both the heavy and light chain variable regions (Tables 1 and 2). In some embodiments, HAF, LAF, and / or TCED+ epitopes are not present in the VL and / or VH region sequences of the anti-PD1 antibody or antigen-binding moieties.

[0071] The terms “cross-compete” and “cross-competition” and “cross-block” are used interchangeably herein to refer to the ability of an antibody or a portion thereof to directly or indirectly interfere with binding to the anti-PD1 and anti-VEGFR2 antibodies of the present invention against targets PD1 and VEGFR2 (e.g., human PD1 and human VEGFR2) through allosteric modulation. A competitive binding assay can be used to determine the extent to which an antibody or a portion thereof can interfere with the binding of another antibody to the target, and therefore whether it can be referred to as cross-blocking or cross-competition according to the present invention. An example of a binding competition assay is homogeneous time-resolved fluorescence (HTRF). A particularly suitable quantitative cross-competition assay uses FACS-based or AlphaScreen-based methods to measure the competition between labeled (e.g., His-labeled, biotinylated, or radiolabeled) antibodies or portions thereof and other antibodies or portions thereof for binding to the target. Generally, a cross-competitive antibody or a portion thereof is, for example, an antibody or portion thereof that binds to a target in a cross-competitive assay such that, during said assay and in the presence of a second antibody or a portion thereof, the recorded shift of the immunoglobulin single variable domain or polypeptide according to the invention is up to 100% (e.g., in a FACS-based competitive assay) of the maximum theoretical shift of a potential cross-blocking antibody or fragment thereof present in a given amount (e.g., by the shift of a cold (e.g., unlabeled) antibody or fragment thereof requiring cross-blocking). Preferably, the cross-competitive antibody or a portion thereof has a recorded shift between 10% and 100% or between 50% and 100%.

[0072] As defined herein, an antibody molecule or antigen-binding moiety may contain one or more removal of post-translational modification (PTM) sites, such as substitutions, deletions, and / or insertions of glycosylation sites (N-linked or O-linked), deamination sites, phosphorylation sites, or isomerization / fragmentation sites.

[0073] There are over 350 known types of PTMs. Key forms of PTMs include phosphorylation and glycosylation. N -and O - Linked), ubiquitination (sumoylation), palmitoylation, acetylation, sulfation, myristoylation, preacylation, and methylation (K and R residues). Statistical methods for identifying the putative amino acid site responsible for a specific PTM are well known in the art (see Zhou). wait people(2016, Nature Protocols 1: 1318-1321). Imagine removing such sites, for example, by substitution, deletion and / or insertion and then optionally testing (experimentally and / or theoretically) (a) binding activity and / or (b) loss of PTM.

[0074] Antibody molecules or their antigen-binding portions can be human, humanized, or chimeric.

[0075] Antibody molecules or their antigen-binding portions may contain one or more human variable domain frameworks with inserted CDRs. For example, the VH region, VL region, or both VH and VL regions may contain one or more human framework amino acid sequences.

[0076] The antibody molecule or its antigen-binding portion may contain an IGHV3-7 human scaffold with the corresponding HCDR sequence inserted. The antibody molecule or its antigen-binding portion may contain a VH region containing an IGHV3-7 human scaffold amino acid sequence with a set of corresponding HCDR1, HCDR2, and HCDR3 amino acid sequences inserted.

[0077] The antibody molecule or its antigen-binding portion may contain an IGKV1-39 human germline scaffold with the corresponding LCDR sequence inserted. The antibody molecule or its antigen-binding portion may contain a VL region containing an IGKV1-39 human germline scaffold amino acid sequence with a set of corresponding LCDR1, LCDR2, and LCDR3 amino acid sequences inserted.

[0078] The antibody molecule or its antigen-binding portion may comprise an IGHV3-7 human germline scaffold with the corresponding HCDR sequence inserted and an IGKV1-39 human germline scaffold with the corresponding LCDR sequence inserted. The antibody molecule or its antigen-binding portion may comprise a VH region and a VL region, wherein the VH region comprises an IGHV3-7 human germline scaffold amino acid sequence with a set of corresponding HCDR1, HCDR2, and HCDR3 amino acid sequences inserted, and the VL region comprises an IGKV1-39 human germline scaffold amino acid sequence with a set of corresponding LCDR1, LCDR2, and LCDR3 amino acid sequences inserted. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 can be any of the following clones (MAB02A03, MAB02B03, MAB02D08, MAB05G03, MAB05E08, MAB01, MAB02, MAB03, MAB04, MAB05, MAB06, MAB06.1, MAB06.2, MAB06.3, MAB06.4, MAB06.5, M...). The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of AB06.6, MAB06.7, MAB06.8, MAB06.8.1, MAB06.8.2, MAB06.8.3, MAB06.8.4, MAB06.8.5, MAB06.8.6, MAB06.8.7, MAB06.8.8, or MAB06.8.9 (where all six CDR sequences are from the same clone).

[0079] In some aspects, antibody molecules or their antigen-binding portions may include immunoglobulin constant regions. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In other embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. Antibody molecules or their antigen-binding portions may include immune-inert constant regions. In some aspects, anti-PD1 and anti-VEGFR2 antibodies or their antigen-binding portions may include immunoglobulin constant regions, said immunoglobulin constant regions comprising wild-type human IgG1 constant regions, human IgG1 constant regions containing amino acid substitutions for L234A, L235A, and G237A, or human IgG1 constant regions containing amino acid substitutions for L234A, L235A, G237A, and P331S. In some aspects, anti-PD1 and anti-VEGFR2 antibodies, or their antigen-binding portions, may include immunoglobulin constant regions comprising either the wild-type human IgG2 constant region or the wild-type human IgG4 constant region. In some aspects, anti-PD1 and anti-VEGFR2 antibodies may include immunoglobulin constant regions comprising any of the amino acid sequences in Table 6. The Fc region sequences in Table 6 begin at the CH1 domain. In some aspects, anti-PD1 and anti-VEGFR2 antibodies may include immunoglobulin constant regions comprising the amino acid sequence of the Fc region of human IgG4, human IgG4 (S228P), human IgG2, human IgG1, human IgG1-3M, or human IgG1-4M. For example, compared to the wild-type human IgG4 Fc region, the human IgG4 (S228P) Fc region contains the following substitution: S228P. For example, compared to the wild-type human IgG1 Fc region, the human IgG1-3M Fc region contains the following substitutions: L234A, L235A, and G237A, while the human IgG1-4M Fc region contains the following substitutions: L234A, L235A, G237A, and P331S. In some respects, the positions of amino acid residues in the constant regions of immunoglobulin molecules are numbered according to EU nomenclature (Ward). et al. , 1995 Therap. Immunol.2:77-94). In some respects, the immunoglobulin constant region may contain the RDELT (SEQ ID NO:36) motif or the REEM (SEQ ID NO:37) motif (underlined in Table 6). The REEM (SEQ ID NO:37) allotype is found in a smaller human population than the RDELT (SEQ ID NO:36) allotype. In some respects, anti-PD1 and anti-VEGFR2 antibodies may contain the immunoglobulin constant region containing any of SEQ ID NO:25-31. In some respects, anti-PD1 and anti-VEGFR2 antibodies may comprise any of the following clones (MAB02A03, MAB02B03, MAB02D08, MAB05G03, MAB05E08, MAB01, MAB02, MAB03, MAB04, MAB05, MAB06, MAB06.1, MAB06.2, MAB06.3, MAB06.4, MAB06.5, MAB06.6, MAB06.7, MAB06.8, MAB06.8.1, MAB06.8.2, MAB06.8.3, MAB06.8.4, MAB06.8.5, MAB06.8.6, MAB06.8.7, MAB06.8.8, or... The six CDR amino acid sequences of MAB06.8.9 (where all six CDR sequences are from the same clone) and any of the Fc region amino acid sequences in Table 6. In some aspects, anti-PD1 and anti-VEGFR2 antibodies may comprise immunoglobulin heavy chain constant regions and immunoglobulin light chain constant regions, wherein the immunoglobulin heavy chain constant regions comprise any of the Fc region amino acid sequences in Table 6, and the immunoglobulin light chain constant regions are κ light chain constant regions or λ light chain constant regions.

[0080] In some respects, this document discloses anti-PD1 and anti-VEGFR2 antibodies or their antigen-binding moieties, wherein said antibodies comprise a heavy chain variable (VH) region, a light chain variable (VL) region, and a heavy chain constant region, wherein (a) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVYSIPWT (SEQ ID NO:6); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (b) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVYSIPWT (SEQ ID NO:6); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (c) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQESGIWLS (SEQ ID NO:8), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVYSIPWT (SEQ ID NO:6); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (d) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQTIGTWLT (SEQ ID NO:9), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVAELPFG (SEQ ID NO:10); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (e) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQTIGTWLT (SEQ ID NO:9), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (f) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVAELPFG (SEQ ID NO:10); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (g) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVAELPFG (SEQ ID NO:10); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (h) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQESGIWLG (SEQ ID NO:12), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVAELPFG (SEQ ID NO:10); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (i) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (j) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (k) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQESGIWLS (SEQ ID NO:8), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (l) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (m) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TASSLAD (SEQ ID NO:40), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (n) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLAD (SEQ ID NO:41), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (o) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (p) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TATSLAD (SEQ ID NO:5), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (q) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TASSLAD (SEQ ID NO:40), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31; (r) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLAD (SEQ ID NO:41), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises SEQ ID NO:25-31; or (s) The VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the VL region amino acid sequence comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); and the heavy chain constant region comprises any one of SEQ ID NO: 25-31. In some embodiments, the antibody further comprises an immunoglobulin light chain constant region, which is a κ light chain constant region or a λ light chain constant region.

[0081] In some respects, this document discloses anti-PD1 and anti-VEGFR2 antibodies or their antigen-binding moieties, wherein said antibodies comprise a heavy chain variable (VH) region, a light chain variable (VL) region, and a heavy chain constant region, wherein a) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:14; and the heavy chain constant region comprises: a wild-type human IgG4 constant region; a human IgG4 constant region containing amino acid substitution S228P; a wild-type human IgG2 constant region; a wild-type human IgG1 constant region; a human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region containing amino acid substitutions L234A, L235A, G237A, and P331S. (b) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:13; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:15; and the heavy chain constant region comprises: a wild-type human IgG4 constant region; a human IgG4 constant region containing amino acid substitution S228P; a wild-type human IgG2 constant region; a wild-type human IgG1 constant region; a human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region containing amino acid substitutions L234A, L235A, G237A, and P331S; (c) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:13; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:16; and the heavy chain constant region comprises the wild-type human IgG4 constant region; the human IgG4 constant region containing the amino acid substitution S228P; the wild-type human IgG2 constant region; the wild-type human IgG1 constant region; the human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A; or the human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S; (d) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:13; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:17; and the heavy chain constant region comprises the wild-type human IgG4 constant region; the human IgG4 constant region containing the amino acid substitution S228P; the wild-type human IgG2 constant region; the wild-type human IgG1 constant region; the human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A; or the human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S; (e) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:13; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:18; and the heavy chain constant region comprises the wild-type human IgG4 constant region; the human IgG4 constant region containing the amino acid substitution S228P; the wild-type human IgG2 constant region; the wild-type human IgG1 constant region; the human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A; or the human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S; (f) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:13; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:19; and the heavy chain constant region comprises the wild-type human IgG4 constant region; the human IgG4 constant region containing the amino acid substitution S228P; the wild-type human IgG2 constant region; the wild-type human IgG1 constant region; the human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A; or the human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S; (g) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:13; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:20; and the heavy chain constant region comprises: a wild-type human IgG4 constant region; a human IgG4 constant region containing amino acid substitution S228P; a wild-type human IgG2 constant region; a wild-type human IgG1 constant region; a human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region containing amino acid substitutions L234A, L235A, G237A, and P331S. (h) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:13; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:21; and the heavy chain constant region comprises the wild-type human IgG4 constant region; the human IgG4 constant region containing the amino acid substitution S228P; the wild-type human IgG2 constant region; the wild-type human IgG1 constant region; the human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A; or the human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S; (i) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:13; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:22; and the heavy chain constant region comprises the wild-type human IgG4 constant region; the human IgG4 constant region containing the amino acid substitution S228P; the wild-type human IgG2 constant region; the wild-type human IgG1 constant region; the human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A; or the human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S; (j) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:13; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:23; and the heavy chain constant region comprises the wild-type human IgG4 constant region; the human IgG4 constant region containing the amino acid substitution S228P; the wild-type human IgG2 constant region; the wild-type human IgG1 constant region; the human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A; or the human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S; (k) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:13; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:24; and the heavy chain constant region comprises: a wild-type human IgG4 constant region; a human IgG4 constant region containing amino acid substitution S228P; a wild-type human IgG2 constant region; a wild-type human IgG1 constant region; a human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region containing amino acid substitutions L234A, L235A, G237A, and P331S; (l) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:48; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:44; and the heavy chain constant region comprises the wild-type human IgG4 constant region; the human IgG4 constant region containing the amino acid substitution S228P; the wild-type human IgG2 constant region; the wild-type human IgG1 constant region; the human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A; or the human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S; (m) The amino acid sequence of the VH region comprises or consists of SEQ ID NO:48; the amino acid sequence of the VL region comprises or consists of SEQ ID NO:45; and the heavy chain constant region comprises: a wild-type human IgG4 constant region; a human IgG4 constant region containing amino acid substitution S228P; a wild-type human IgG2 constant region; a wild-type human IgG1 constant region; a human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region containing amino acid substitutions L234A, L235A, G237A, and P331S. (n) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:48; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:46; and the heavy chain constant region comprises the wild-type human IgG4 constant region; the human IgG4 constant region containing the amino acid substitution S228P; the wild-type human IgG2 constant region; the wild-type human IgG1 constant region; the human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A; or the human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S; (o) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:48; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:47; and the heavy chain constant region comprises the wild-type human IgG4 constant region; the human IgG4 constant region containing the amino acid substitution S228P; the wild-type human IgG2 constant region; the wild-type human IgG1 constant region; the human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A; or the human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S; (p) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:49; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:44; and the heavy chain constant region comprises: a wild-type human IgG4 constant region; a human IgG4 constant region containing amino acid substitution S228P; a wild-type human IgG2 constant region; a wild-type human IgG1 constant region; a human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region containing amino acid substitutions L234A, L235A, G237A, and P331S. (q) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:49; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:45; and the heavy chain constant region comprises: a wild-type human IgG4 constant region; a human IgG4 constant region containing amino acid substitution S228P; a wild-type human IgG2 constant region; a wild-type human IgG1 constant region; a human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region containing amino acid substitutions L234A, L235A, G237A, and P331S. (r) The VH region amino acid sequence comprises or is composed of SEQ ID NO:49; the VL region amino acid sequence comprises or is composed of SEQ ID NO:46; and the heavy chain constant region comprises: a wild-type human IgG4 constant region; a human IgG4 constant region containing an amino acid substitution for S228P; a wild-type human IgG2 constant region; a wild-type human IgG1 constant region; a human IgG1 constant region containing amino acid substitutions for L234A, L235A, and G237A; or a human IgG1 constant region containing amino acid substitutions for L234A, L235A, G237A, and P331S; or (s) The amino acid sequence of the VH region comprises or is composed of SEQ ID NO:49; the amino acid sequence of the VL region comprises or is composed of SEQ ID NO:47; and the heavy chain constant region comprises the wild-type human IgG4 constant region; the human IgG4 constant region containing the amino acid substitution S228P; the wild-type human IgG2 constant region; the wild-type human IgG1 constant region; the human IgG1 constant region containing the amino acid substitutions L234A, L235A and G237A; or the human IgG1 constant region containing the amino acid substitutions L234A, L235A, G237A and P331S.

[0082] In some respects, this document discloses anti-PD1 and anti-VEGFR2 antibodies or their antigen-binding moieties, wherein said antibodies comprise a heavy chain variable (VH) region, a light chain variable (VL) region, and a heavy chain constant region, wherein a) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:14; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (b) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:15; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (c) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:16; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (d) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:17; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (e) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:18; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (f) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:19; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (g) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:20; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (h) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:21; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (i) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:22; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (j) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:23; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (k) The VH region amino acid sequence comprises or consists of SEQ ID NO:13; the VL region amino acid sequence comprises or consists of SEQ ID NO:24; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (l) The VH region amino acid sequence comprises or is composed of SEQ ID NO:48; the VL region amino acid sequence comprises or is composed of SEQ ID NO:44; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (m) The VH region amino acid sequence comprises or is composed of SEQ ID NO:48; the VL region amino acid sequence comprises or is composed of SEQ ID NO:45; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (n) The VH region amino acid sequence comprises or consists of SEQ ID NO:48; the VL region amino acid sequence comprises or consists of SEQ ID NO:46; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (o) The VH region amino acid sequence comprises or is composed of SEQ ID NO:48; the VL region amino acid sequence comprises or is composed of SEQ ID NO:47; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (p) The VH region amino acid sequence comprises or is composed of SEQ ID NO:49; the VL region amino acid sequence comprises or is composed of SEQ ID NO:44; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (q) The VH region amino acid sequence comprises or is composed of SEQ ID NO:49; the VL region amino acid sequence comprises or is composed of SEQ ID NO:45; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; (r) The VH region amino acid sequence comprises or is composed of SEQ ID NO:49; the VL region amino acid sequence comprises or is composed of SEQ ID NO:46; and the heavy chain constant region comprises any one of SEQ ID NO:25-31; or (s) The VH region amino acid sequence comprises or consists of SEQ ID NO:49; the VL region amino acid sequence comprises or consists of SEQ ID NO:47; and the heavy chain constant region comprises any one of SEQ ID NO:25-31.

[0083] In some respects, anti-PD1 and anti-VEGFR2 antibodies may be immune effector ineffective. In some respects, anti-PD1 and anti-VEGFR2 antibodies, or their antigen-binding portions, do not induce immune effector function and optionally inhibit it. In some respects, anti-PD1 and anti-VEGFR2 antibodies may lack measurable binding to human FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb receptors, but maintain binding to the human FcγRIIb receptor and optionally maintain binding to the human FcRn receptor. FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb are examples of activating receptors. FcγRIIb is an example of an inhibitory receptor. FcRn is an example of a circulating receptor. In some respects, the binding affinity of anti-PD1 and anti-VEGFR2 antibodies, or their antigen-binding portions, to human Fc receptors can be measured by BIACORE. ® Analysis is used to measure these effects. In some respects, homogeneous time-resolved fluorescence (HTRF) can be used to study the binding of anti-PD1 and anti-VEGFR2 antibodies to human Fc receptors. In one example of HTRF, human IgG1 (wild-type) is labeled, as with the entire Fcγ receptor suite, and then antibodies with engineered Fc fragments are used for titration competition. In some respects, PD1-positive and / or VEGFR2-positive cells can be mixed with human leukocytes and anti-PD1 and anti-VEGFR2 antibodies, and cell killing via CDC, ADCC, and / or ADCP can be measured. In some respects, anti-PD1 and anti-VEGFR2 antibodies containing the Fc region amino acid sequence of human IgG1-3M (see Table 6) are effector-ineffective. In some respects, anti-PD1 and anti-VEGFR2 antibodies containing the Fc region amino acid sequence of human IgG1-3M (see Table 6) are not effector-ineffective.

[0084] Antibody molecules or their antigen-binding portions can be Fab fragments, F(ab)2 fragments, Fv fragments, tetrameric antibodies, tetravalent antibodies, multispecific antibodies (e.g., bivalent antibodies), domain-specific antibodies, single-domain antibodies, monoclonal antibodies, or fusion proteins. In one embodiment, the antibody can be a multispecific antibody comprising two or more antigen-binding domains. In some embodiments, a first antigen-binding domain specifically binds to PD1 and VEGFR2, and a second antigen-binding domain specifically binds to an antigen that is neither PD1 nor VEGFR2. Antibody molecules and methods of their construction and use are described, for example, in Holliger and Hudson (2005, Nature Biotechnol. 23(9): 1126-1136).

[0085] In another aspect of the invention, an immunoconjugate is provided comprising an antibody molecule or its antigen-binding portion thereof, as defined herein, conjugated to a therapeutic agent.

[0086] Examples of suitable therapeutic agents include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, antiproliferative agents, pro-apoptotic agents, and cell-inhibiting and cell-lysing enzymes (e.g., RNase). Other therapeutic agents include therapeutic nucleic acids, such as genes encoding immunomodulators, anti-angiogenic agents, antiproliferative agents, or pro-apoptotic agents. These drug descriptors are not mutually exclusive, and therefore one or more of the terms mentioned above can be used to describe a therapeutic agent.

[0087] Examples of suitable therapeutic agents for immunoconjugates include taxanes, maytansine, CC-1065 and docalmicin, cazithromycin and other enediynes, and auristatin. Other examples include antifolate, vinca alkaloids, and anthracyclines. Phytotoxins, other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes, and photosensitizers can also be used in immunoconjugates. Additionally, secondary carriers (such as liposomes or polymers) can be used as cytotoxic agents to prepare conjugates. Suitable cytotoxins include agents that inhibit or prevent cell function and / or cause cell destruction. Representative cytotoxins include antibiotics, microtubule polymerization inhibitors, alkylating agents that bind to and destroy DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins (such as protein kinases, phosphatases, topoisomerases, enzymes, and cyclins).

[0088] Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, azarubicin, zorubicin, mitoxantrone, epirubicin, carrarubicin, nogamycin, menorubicin, pirarubicin, pentorubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enoxatabine, azacitidine, doxifluhdine, pentostatin, broxuhdine, capecitabine, cladhbine, decitabine, fluxuhdine, fludarabine, oryzanol, puromycin, tegafur, and tiazofuran. Hn), adhamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vincristine, mitoxantrone, bleomycin, nitrogen mustard, prednisone, procarbazine, methotrexate, fluorouracil, etoposide, paclitaxel, paclitaxel analogs, platinum such as cisplatin and carboplatin, mitomycin, thiotepa, taxane, vincristine, daunorubicin, epirubicin, actinomycin D, autramycin, azoserine, bleomycin, tamoxifen, idarubicin, dolasstatin / auresstatin, hemiasterlin, esporamycin, and maytansine.

[0089] Suitable immunomodulators include anti-hormonal agents that block the effects of hormones on tumors and immunosuppressants that inhibit cytokine production, downregulate self-antigen expression, or mask MHC antigens.

[0090] Also provided are nucleic acid molecules encoding antibody molecules or their antigen-binding portions as defined herein. The nucleic acid molecules may encode (a) the VH region amino acid sequence of the anti-PD1 and anti-VEGFR2 antibodies or their antigen-binding portions as described herein; (b) the VL region amino acid sequence; or (c) both the VH and VL region amino acid sequences. In some aspects, nucleic acid molecules as defined herein can be isolated.

[0091] Vectors comprising nucleic acid molecules of the present invention as defined herein are also provided. The vector may be an expression vector.

[0092] Host cells comprising nucleic acid molecules or vectors of the present invention as defined herein are also provided. The host cell may be a recombinant host cell. In some aspects, the host cell may comprise a vector containing a nucleic acid molecule encoding both the VH region amino acid sequence and the VL region amino acid sequence of the anti-PD1 and anti-VEGFR2 antibodies or their antigen-binding portions as described herein. In some aspects, the host cell may comprise a first vector containing a nucleic acid molecule encoding the VH region amino acid sequence of the anti-PD1 and anti-VEGFR2 antibodies or their antigen-binding portions as described herein, and a second vector containing a nucleic acid molecule encoding the VL region amino acid sequence.

[0093] In another aspect, a method is provided for generating anti-PD1 and anti-VEGFR2 antibodies and / or their antigen-binding portions, the method comprising culturing host cells of the invention under conditions that result in the expression and / or generation of the antibodies and / or their antigen-binding portions, and isolating the antibodies and / or their antigen-binding portions from the host cells or culture.

[0094] In another aspect of the invention, pharmaceutical compositions are provided comprising an antibody molecule or its antigen-binding portion as defined herein, a nucleic acid molecule as defined herein, or a carrier as defined herein.

[0095] A method for enhancing the immune response of a subject is also provided, the method comprising administering to the subject an effective amount of an antibody molecule or antigen-binding portion thereof as defined herein, or an immunoconjugate of the present invention as defined herein, or a nucleic acid molecule of the present invention as defined herein, or a carrier of the present invention as defined herein, or a pharmaceutical composition of the present invention as defined herein.

[0096] In another aspect, methods for treating or preventing cancer in a subject are provided, the methods comprising administering to the subject an effective amount of an antibody molecule or its antigen-binding portion as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a carrier as defined herein, or a pharmaceutical composition as defined herein. In some embodiments, the cancer is associated with the expression or overexpression of PD1 and / or VEGFR2.

[0097] For example, cancer can be pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.

[0098] The present invention also provides antibody molecules or antigen-binding portions thereof as defined herein, or immunoconjugates as defined herein, or nucleic acid molecules as defined herein, or carriers as defined herein, or pharmaceutical compositions as defined herein, for the treatment of cancer.

[0099] In another aspect, the present invention provides an antibody molecule or its antigen-binding portion, or an immunoconjugate, or a nucleic acid molecule or carrier, or a treatment method of the present invention as defined herein, for use alone, sequentially or simultaneously in combination with a second therapeutic agent (e.g., an anticancer agent).

[0100] In another aspect, the use of antibody molecules or antigen-binding portions thereof of the present invention as defined herein, or immunoconjugates of the present invention as defined herein, or nucleic acid molecules of the present invention as defined herein, or carriers of the present invention as defined herein, or pharmaceutical compositions of the present invention as defined herein, in the manufacture of medicaments for treating cancer is provided.

[0101] The present invention also provides a method for treating or preventing an infectious or immune disease in a subject, the method comprising administering to the subject an effective amount of an antibody molecule or its antigen-binding portion as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a carrier as defined herein, or a pharmaceutical composition as defined herein.

[0102] In one embodiment, the present invention provides anti-PD1 and anti-VEGFR2 antibodies or antigen-binding portions thereof comprising the amino acid sequences disclosed herein, for use in therapy.

[0103] The pharmaceutical compositions of the present invention may comprise pharmaceutically acceptable excipients, carriers, or diluents. Pharmaceutically acceptable excipients may be compounds or combinations of compounds incorporated into the pharmaceutical composition that do not initiate secondary reactions and allow, for example, increased administration of anti-PD1 and anti-VEGFR2 antibody molecules, increased duration of presence and / or efficacy of said anti-PD1 and anti-VEGFR2 antibody molecules in vivo, or increased solubility of said anti-PD1 and anti-VEGFR2 antibody molecules in solution. These pharmaceutically acceptable carriers are well known and will be adapted by those skilled in the art to vary the administration modality of the anti-PD1 and anti-VEGFR2 antibody molecules.

[0104] In some implementations, anti-PD1 and anti-VEGFR2 antibody molecules can be provided in a lyophilized form that is reconstituted before administration. For example, the lyophilized antibody molecules can be reconstituted in sterile water and mixed with saline before being administered to an individual.

[0105] Anti-PD1 and anti-VEGFR2 antibody molecules are typically administered in the form of pharmaceutical compositions, which may contain at least one component in addition to the antibody molecules. Therefore, in addition to anti-PD1 and anti-VEGFR2 antibody molecules, the pharmaceutical composition may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the anti-PD1 and anti-VEGFR2 antibody molecules. The precise properties of the carrier or other materials will depend on the route of administration, which may be by bolus, infusion, injection, or any other suitable route, as discussed below.

[0106] For parenteral (e.g., subcutaneous or intravenous) administration (e.g., by injection), pharmaceutical compositions containing anti-PD1 and anti-VEGFR2 antibody molecules can be in the form of parenteral-acceptable aqueous solutions that are pyrogen-free and have suitable pH, isotonicity, and stability. Those skilled in the art are fully capable of preparing suitable solutions using isotonic media such as sodium chloride injection, Ringer's solution, lactated Ringer's solution, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be used as needed, including buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexachlorocyclohexane quaternary ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); and low molecular weight peptides. Proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG).

[0107] Pharmaceutical compositions containing anti-PD1 and anti-VEGFR2 antibody molecules can be administered alone or in combination with other treatments (simultaneously or sequentially) depending on the condition to be treated.

[0108] The anti-PD1 and anti-VEGFR2 antibody molecules described herein can be used in methods of treating the human or animal body, including prophylactic or preventative treatment (e.g., treating an individual before the onset of symptoms to reduce the risk of developing symptoms; delaying their onset; or reducing their severity after onset). Treatment methods may include administering anti-PD1 and anti-VEGFR2 antibody molecules to individuals in need.

[0109] Administration is typically performed at a “therapeuticly effective dose,” sufficient to demonstrate benefit to the patient. Such benefit may be an improvement in at least one symptom. The actual dosage, as well as the rate and timing of administration, will depend on the nature and severity of the disease being treated, the specific mammal being treated, the individual patient’s clinical condition, the cause of the condition, the site of delivery of the composition, the method of administration, the timing of administration, and other factors known to the physician. Treatment prescriptions (e.g., decisions regarding dosage) are the responsibility of general practitioners and other physicians and may depend on the severity and / or progression of the symptoms of the disease being treated. Appropriate doses of antibody molecules are well known in the art (Ledermann JA). et al. , 1991, Int. J. Cancer 47:659-664; Bagshawe KD et al. (1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). This article or Physician's Desk Reference (2003) may specify a specific dosage applicable to the type of drug being administered. This can be compared with that in animal models. in vitro Activity and in vivo Activity determines the therapeutically effective amount or appropriate dose of an antibody molecule. Methods for extrapolating effective doses from mice and other test animals to humans are known. The precise dose will depend on many factors, including whether the antibody is for prophylaxis or treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g., intact antibody, fragment), and the nature of any detectable markers or other molecules attached to the antibody.

[0110] For systemic application, typical antibody doses range from 100 µg to 1 g, and for topical application, from 1 µg to 1 mg. An initial higher loading dose may be administered, followed by one or more lower doses. Typically, the antibody will be a whole antibody, such as an IgG1 or IgG4 isotype. This is the dose for a single treatment in adult patients, which can be scaled up for children and infants, and can also be scaled up for other antibody forms according to molecular weight. Treatment can be repeated daily, twice weekly, once weekly, or monthly, depending on the physician's decision. An individual's treatment schedule may depend on the pharmacokinetic and pharmacodynamic properties of the antibody composition, the route of administration, and the nature of the condition being treated.

[0111] Treatment can be periodic, and the intervals between applications can be approximately two weeks or longer, such as approximately three weeks or longer, approximately four weeks or longer, approximately one month or longer, approximately five weeks or longer, or approximately six weeks or longer. For example, treatment can be administered every two to four weeks or every four to eight weeks. Treatment can be given before and / or after surgery, and / or can be applied directly or via the anatomical site of the surgical treatment or invasive procedure. Suitable formulations and routes of administration are as described above.

[0112] In some implementations, the anti-PD1 and anti-VEGF antibody molecules, as described herein, can be administered subcutaneously. Subcutaneous injection can be administered using an autoinjector, for example, for long-term prevention / treatment.

[0113] In some implementations, the therapeutic effects of anti-PD1 and anti-VEGF antibody molecules can last for several half-lives, depending on the dose. For example, the therapeutic effects of a single dose of anti-PD1 and anti-VEGF antibody molecules can last for 1 month or longer, 2 months or longer, 3 months or longer, 4 months or longer, 5 months or longer, or 6 months or longer in an individual.

[0114] The present invention also provides a method for generating antibody molecules or antigen-binding portions thereof that specifically bind to human PD1 and human VEGFR2, and optionally also to cynomolgus monkey PD1 and cynomolgus monkey VEGFR2, the method comprising the following steps.

[0115] (1) Transplanting anti-PD1 CDRs from non-human sources into a human v domain framework to generate humanized anti-PD1 antibody molecules or their antigen-binding portions; (2) Generate a clonal library containing one or more mutations in the CDR, or the antigen-binding portion thereof; (3) The library was screened based on its binding to human PD1 and optionally also to cynomolgus monkey PD1, human VEGFR2 and rhesus monkey VEGFR2; (4) Select clones from the screening step (3) that exhibit binding specificity to human PD1 and optionally also to cynomolgus monkey PD1, human VEGFR2, and rhesus monkey VEGFR2; and (5) Generate an antibody molecule or its antigen-binding portion that specifically binds to human PD1 and optionally also to cynomolgus monkey PD1, human VEGFR2 and rhesus monkey VEGFR2 from a clone selected from step (4). (6) Perform germline mutagenesis tests in CDR to determine the potential for further improvement of the molecular properties of the antibody.

[0116] The method may further include the following steps: generating additional clones based on the clone selected in step (4) (e.g., based on further exploratory mutagenesis at a specific location in the CDR of the clone selected in step (4)) to enhance humanization and / or minimize human T cell epitope content and / or improve the manufacturing characteristics of the antibody molecule or its antigen-binding portion generated in step (5).

[0117] As used herein, the term "PD1" refers to programmed cell death protein 1 and its variants that retain at least a portion of the biological activity of PD1. As used herein, PD1 can include the natural sequence PD1 of all species, including human, rat, mouse, and chicken. The term "PD1" can be used to include variants, isotypes, and species homologs of human PD1.

[0118] In some implementations, "PD1" refers to the wild-type human form of PD1. The antibodies of this invention can be synthesized with PD1 from species other than humans (particularly from cynomolgus monkeys). Macaca fascicularis Cross-reactivity occurs with PD1. Examples of human and cynomolgus monkey PD1 amino acid sequences are provided in Table 7. In some embodiments, the antibody may be completely specific to human PD1 and may not exhibit non-human cross-reactivity.

[0119] As used herein, the term "VEGFR2" refers to vascular endothelial growth factor receptor 2 (also known as KDR or FLK1) and its variants that retain at least a portion of the biological activity of VEGFR2. As used herein, VEGFR2 can include naturally occurring sequences of VEGFR2 from all species, including humans, rats, mice, and chickens. The term "VEGFR2" can be used to include variants, isotypes, and species homologs of human VEGFR2. In some embodiments, "VEGFR2" refers to the wild-type human form of VEGFR2. The antibodies of the present invention can be used with VEGFR2 from species other than humans (particularly from rhesus monkeys). Macaca mulatta Cross-reactivity occurs between the antibody and human VEGFR2. Examples of human and rhesus monkey VEGFR2 amino acid sequences are provided in Table 7. In some embodiments, the antibody may be completely specific to human VEGFR2 and may not exhibit non-human cross-reactivity.

[0120] As used herein, in the context of the antibodies of this invention or "anti-PD1 and anti-VEGFR2 antagonist antibodies" (which may be interchangeably referred to as "anti-PD1 and anti-VEGFR2 antibodies"), "antagonist" means an antibody capable of binding to PD1 and VEGFR2 and inhibiting the biological activity of PD1 and VEGFR2 and / or downstream pathways mediated by PD1 and / or VEGFR2 signaling. Anti-PD1 and anti-VEGFR2 antagonist antibodies encompass antibodies that can block, antagonize, inhibit, or reduce (including significantly) the biological activity of PD1 and VEGFR2, including downstream pathways mediated by PD1 and / or VEGFR2 signaling, such as receptor binding and / or triggering cellular responses to PD1 and VEGFR2. For the purposes of this invention, it will be clearly understood that the term “anti-PD1 and anti-VEGFR2 antagonist antibody” encompasses all terms, titles, and functional states and characteristics whereby PD1 and VEGFR2, in themselves and in their biological activities (including, but not limited to, their ability to inhibit the activation of anti-tumor cell activity of T cells), or the consequences thereof, are substantially ineffective, reduced, or neutralized to any meaningful degree.

[0121] If an antibody binds to PD1 or VEGFR2 with greater affinity, easier binding and / or longer duration than it binds to other receptors, then the antibody "specifically binds", "specifically interacts" with PD1 or VEGFR2, "preferentially binds", "binds" or "interacts" with PD1 or VEGFR2.

[0122] An "antibody molecule" is an immunoglobulin molecule capable of specifically binding to a target (such as carbohydrates, polynucleotides, lipids, peptides, etc.) through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term "antibody molecule" encompasses not only complete polyclonal or monoclonal antibodies, but also any antigen-binding fragment (e.g., "antigen-binding moiety") or its single chain, fusion proteins containing antibodies, and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site, including, but not limited to, scFvs, single-domain antibodies (e.g., shark and camel antibodies), large antibodies, microantibodies, intracellular antibodies, biantibodies, triantibodies, tetraantibodies, v-NARs, and biscFvs.

[0123] The term "antibody molecule" encompasses any type of antibody, such as IgG, IgA, or IgM (or their subclasses), and antibodies do not necessarily belong to any specific type. Immunoglobulins can be classified into different types based on the antibody amino acid sequence of the antibody in the heavy chain constant region. There are five main types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different types of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well known.

[0124] As used herein, the term "antigen-binding moiety" of an antibody molecule refers to one or more fragments of an intact antibody that retain the ability to bind specifically to PD1. The antigen-binding function of an antibody molecule can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding moiety" of an antibody molecule include Fab; Fab'; F(ab')2; the Fd fragment consisting of VH and CH1 domains; the Fv fragment consisting of the VL and VH domains of a single arm of the antibody; single-domain antibody (dAb) fragments; and isolated complementarity-determining regions (CDRs).

[0125] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its C-terminus. The residues in the Fc region are numbered using the EU index as described in Kabat. The Fc region of an immunoglobulin generally contains two constant domains, CH2 and CH3. As is known in the art, the Fc region can exist in dimer or monomeric form.

[0126] The “variable region” of an antibody refers to the variable region of the antibody light chain, either alone or in combination, or the variable region of the antibody heavy chain. As is known in the art, the variable regions of both the heavy and light chains are each composed of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions, which contribute to the formation of the antigen-binding site of the antibody. When FRs are selected to be flanked by CDRs, such as when humanizing or optimizing antibodies, FRs from antibodies containing CDR sequences within the same canonical class are preferred.

[0127] The CDR definition used in this application combines the domains used in many different (often contradictory) schemes that have emerged in the art, and the CDR definition is based on a combination of immunoglobulin library analysis and structural analysis of isolated and co-crystallized antibodies with antigens (see Swindells). et al. (A review of abYsis: Integrated Antibody Sequence and Structure-Management, Analysis, and Prediction. J MolBiol. [PMID: 27561707; published electronically on August 22, 2016]). The CDR definition (“unified” definition) used in this paper incorporates the lessons learned from all such previous insights and includes all appropriate loop locations required to sample the full residue landscape of potential target binding complementarity.

[0128] Table 1 shows the amino acid sequence of the VL domain of the MAb005 humanized anti-PD1 antibody having the CDR as defined herein. Table 2 shows the amino acid sequence of the VH domain of the MAb005 humanized anti-PD1 antibody having the CDR as defined herein.

[0129] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid that does not significantly and harmfully alter its functional activity. A preferred example of "conservative substitution" is the replacement of one amino acid with another amino acid having a value ≥ 0 in the following BLOSUM 62 substitution matrix (see Henikoff and Henikoff, 1992, PNAS 89: 10915-10919): ARNDCQEGHILKMFPSTWYV A 4 -1 -2 -2 0 -1 -1 0 -2 -1 -1 -1 -1 -2 -1 1 0 -3 -2 0 R -1 5 0 -2 -3 1 0 -2 0 -3 -2 2 -1 -3 -2 -1 -1 -3 -2 -3 N -2 0 6 1 -3 0 0 0 1 -3 -3 0 -2 -3 -2 1 0 -4 -2 -3 D -2 -2 1 6 -3 0 2 -1 -1 -3 -4 -1 -3 -3 -1 0 -1 -4 -3 -3 C 0 -3 -3 -3 9 -3 -4 -3 -3 -1 -1 -3 -1 -2 -3 -1 -1 -2 -2 -1 Q -1 1 0 0 -3 5 2 -2 0 -3 -2 1 0 -3 -1 0 -1 -2 -1 -2 E -1 0 0 2 -4 2 5 -2 0 -3 -3 1 -2 -3 -1 0 -1 -3 -2 -2 G 0 -2 0 -1 -3 -2 -2 6 -2 -4 -4 -2 -3 -3 -2 0 -2 -2 -3 -3 H -2 0 1 -1 -3 0 0 -2 8 -3 -3 -1 -2 -1 -2 -1 -2 -2 2 -3 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 2 -3 1 0 -3 -2 -1 -3 -1 3 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 -2 2 0 -3 -2 -1 -2 -1 1 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 -1 -3 -1 0 -1 -3 -2 -2 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 0 -2 -1 -1 -1 -1 1 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 -4 -2 -2 1 3 -1 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 -1 -1 -4 -3 -2 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 1 -3 -2 -2 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 -2 -2 0 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 2 -3 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 -1 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4. The term "monoclonal antibody" (Mab) refers to an antibody or its antigen-binding moiety derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone) regardless of the method of its production. Preferably, the monoclonal antibodies of the present invention exist in a homogeneous or substantially homogeneous population.

[0130] "Humanized" antibody molecules refer to the form of non-human (e.g., mouse) antibody molecules or their antigen-binding portions, which are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) containing minimal sequences derived from non-human immunoglobulins. Humanized antibodies can be human immunoglobulins (recipient antibodies) in which residues from the receptor CDR are replaced by residues from the non-human species (donor antibody) (such as mouse, rat, or rabbit) CDRs with the desired specificity, affinity, and ability.

[0131] "Human antibody or fully human antibody" refers to antibody molecules or their antigen-binding portions derived from transgenic mice carrying human antibody genes or from human cells.

[0132] The term "chimeric antibody" is intended to refer to an antibody molecule or its antigen-binding portion in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody molecule in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0133] "Antibody-drug conjugates" and "immunoconjugates" refer to antibody molecules or their antigen-binding portions, including antibody derivatives that bind to PD1 and VEGFR2 and are conjugated to cytotoxic agents, cell inhibitors, and / or therapeutic agents.

[0134] The antibody molecules or their antigen-binding portions of the present invention can be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or combinations of such techniques, or other techniques readily understood in the art.

[0135] The term "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide, or antibody) is defined as a molecule that, due to its origin or derived source, (1) is unrelated to the naturally associated components that accompany it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) is not present in nature. Therefore, a chemically synthesized molecule or expressed in a cellular system different from the cell of its natural origin will be "isolated" from its naturally associated components. A molecule can also be isolated substantially free of its naturally associated components using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a variety of methods well known in the art. For example, the purity of a polypeptide sample can be determined by using polyacrylamide gel electrophoresis and gel staining to visualize the polypeptide using techniques well known in the art. For certain purposes, purification using HPLC or other methods well known in the art can provide higher resolution.

[0136] The term "epitope" refers to a molecular moiety that can be recognized and bound by an antibody molecule or its antigen-binding moiety at one or more sites within the antigen-binding region of an antibody molecule. An epitope can consist of a defined region of a primary, secondary, or tertiary protein structure and includes a combination of secondary structural units or domains of a target recognized by the antigen-binding region of the antibody or its antigen-binding moiety. Similarly, an epitope can consist of a defined grouping of chemically active surfaces of a molecule (such as amino acids or sugar side chains) and possess specific three-dimensional structural features and specific charge characteristics. As used herein, the term "antigen epitope" is defined as a portion of a polypeptide that an antibody molecule can specifically bind to, as determined by any method well known in the art (e.g., by conventional immunoassay, antibody competitive binding assay, or by X-ray crystallography or related structural determination methods (e.g., NMR)).

[0137] The term "binding affinity" or "KD" refers to the rate of dissociation of a specific antigen-antibody interaction. KD is the rate of dissociation (also known as the "off-rate") off ")" and association rate (or "on-rate") (k on The ratio of K to K. D equals k off / k on And expressed as molar concentration (M). Therefore, K D The smaller the value, the stronger the binding affinity. Therefore, with 1 nM K D Compared to 1 μM K DIndicating weak binding affinity. The KD value of an antibody can be determined using methods well-established in the art. One method for determining the KD of an antibody is to use surface plasmon resonance (SPR), typically using biosensor systems such as Biacore. ® system).

[0138] The term "potency" is a measure of biological activity and can be specified as IC50. 50 Or conjugate with antigen PD1 or VEGFR2 to inhibit 50% of the effective concentration of the antibody or antibody drug of the activity measured in the PD1 or VEGFR2 activity assay as described herein.

[0139] As used herein, the phrase “effective amount” or “therapeutic effective amount” refers to the amount (in terms of dosage and time period or means of administration) necessary to achieve the desired therapeutic outcome. An effective amount is at least the minimum amount of active agent necessary to impart a therapeutic benefit to a subject, but less than a toxic amount.

[0140] As used herein with respect to the bioactivity of the antibody molecules of the present invention, the terms “inhibition” or “neutralization” mean the ability of an antibody to substantially antagonize, prohibit, prevent, limit, slow down, destroy, eliminate, stop, reduce, or reverse, for example, the progression or severity of the inhibited object (including, but not limited to, the bioactivity or binding interaction of the antibody molecule with PD1 or VEGFR2).

[0141] "Host cell" includes a single cell or cell culture that can serve as, or has become, a recipient of a vector for incorporating polynucleotide intercalations. Host cells include the offspring of a single host cell, and due to natural, accidental, or intentional mutations, the offspring may not necessarily be identical to the original parent cell (morphologically or in terms of genomic DNA complement). Host cells include cells transfected in vivo with the polynucleotides of the present invention.

[0142] As used herein, “vector” means a construct capable of being delivered into a host cell and preferably expressing one or more genes or sequences of interest. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (such as producer cells).

[0143] Unless otherwise specified, as used herein, the term "treatment" means reversing or alleviating the condition or symptom to which such term applies, or one or more symptoms of such condition or symptom, inhibiting their progression, delaying their progression, delaying their onset, or preventing them. Unless otherwise specified, as used herein, the term "treatment" means the therapeutic act as defined above. The term "treatment" also includes adjunctive and neoadjunctive treatments for the subject. For the avoidance of doubt, as used herein, "treatment" includes references to therapeutic treatment, palliative treatment, and preventative treatment. For the avoidance of doubt, as used herein, "treatment" also includes references to therapeutic treatment, palliative treatment, and preventative treatment.

[0144] It should be understood that when this document uses the word "comprising" to describe an implementation scheme, similar implementation schemes described as "consisting of" and / or "substantially consisting of" are also provided.

[0145] When aspects or embodiments of the invention are described using the Markush group or other alternative groupings, the invention covers not only the entire group listed in its entirety, but also each individual member of that group and all possible subgroups of the main group, as well as the main group that has no one or more members. The invention also contemplates the explicit exclusion of one or more members of the invention protected by the claims.

[0146] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of any conflict, this specification and its included definitions shall prevail. Throughout this specification and claims, the word “comprising” or its variations, such as “comprising” or “having,” shall be understood to mean that the stated integer or group of integers is included, but does not exclude any other integer or group of integers. Unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Any examples of the terms “such as” or “for example” hereinafter shall not be intended to be exhaustive or limiting.

[0147] Unless otherwise stated, the practice of this invention will employ conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art.

[0148] Specific non-limiting embodiments of the invention will now be described with reference to the accompanying drawings.

[0149] Example 1. Optimized generation of anti-PD1 therapeutic antibodies introduction In this embodiment, we successfully generated a set of anti-PD1 antibodies that are also optimized for antagonism against VEGFR2 antagonists. These anti-PD1 antibodies are well expressed in preferred human strains, are biophysically stable, highly soluble, and exhibit maximized identity.

[0150] Materials and methods PD1 library generation and selection PD1 Fab libraries were assembled via mass oligo synthesis and PCR. Mutagenesis was applied to the CDR and CDR-adjacent regions of the VL (Table 1) and VH (Table 2) domains. The amplified Fab libraries were then cloned into phage vectors via restriction ligation and transformed into... E. coli In TG-1 cells, and essentially as described in previous descriptions, the phage repositories (Finlay) were rescued. et al. , 2011, Methods Mol Biol 681: 383-401).

[0151] Phage selection was performed by coating streptoacidin magnetic beads with biotinylated PD1 or VEGFR2 target proteins (human or monkey), washing the beads three times with PBS, and resuspending them in PBS at pH 7.4 with 5% skim milk protein. The beads were coated with 100 nM target protein in the first round of selection, followed by three consecutive rounds with decreasing antigen concentrations. In each round, the phages were washed with trypsin before reinfection into TG1 cells.

[0152] Periplasmic extract production (small scale) In a single E. coli The production of soluble Fab is carried out in cloning. With isopropyl 1-thio-β-D-galactopyran Glycoside-induced Escherichia coli in logarithmic growth phase TG1 cells. Periplasmic extract containing soluble Fab was produced by freeze / thaw cycling: the bacterial cell pellet was frozen overnight at -20°C, then thawed at room temperature and resuspended in PBS pH 7.4. The supernatant containing soluble Fab was collected after shaking and centrifugation at room temperature.

[0153] IgG expression and purification A mammalian codon-optimized synthetic gene encoding a leader anti-PD1 antibody with variable heavy and light chain domains of Mab005 was cloned into a mammalian expression vector containing both an effector-ineffective human IgG1 ('IgG1 ineffective'; containing L234A, L235A, G237A mutations in the lower hinge that eliminate the functions of normal immunoglobulins ADCC, ADCP, and CDC) and a human Cκ domain. Co-transfection of the vector containing both heavy and light chains was performed in the mammalian expression system, followed by protein A-based purification, quantification, and QC on denaturing and non-denaturing SDS-PAGE of IgG.

[0154] Direct binding ELISA of Fab and IgG First, the binding and cross-reactivity of the pilot plate with the recombinant protein were assessed using a combined ELISA. Human PD1-human Fc-labeled recombinant protein and cynomolgus monkey PD1-human Fc-labeled recombinant protein were coated onto the surface of a MaxiSorp™ flat-bottom 96-well plate at 1 µg / ml. Purified Fab or IgG samples were titrated in serially diluted buffers from 500 nM to 0.98 nM, allowing them to bind to the coated antigens. Fab was detected using mouse anti-c-myc antibody, followed by donkey anti-mouse IgG conjugated with horseradish peroxidase. IgG was detected using mouse anti-human IgG conjugated with horseradish peroxidase. The binding signal was visualized using 3,3',5,5'-tetramethylbenzidine substrate solution (TMB), and absorbance was measured at 450 nm.

[0155] AlphaScreen epitope competition determination of Fab periphery products AlphaScreen assays (Perkin Elmer) were performed in a final volume of 25 µl in a 384-well white microtiter plate (Greiner). The reaction buffer contained 1 x PBS pH 7.3 (Oxoid, catalog number BR0014G) and 0.05% (v / v) Tween® 20 (Sigma, catalog number P9416). Periplasmic sample diluted in the reaction buffer was incubated at room temperature for 20 min with biotinylated human PD1-His / AviTag at a final concentration of 0.6 nM. 0.3 nM of hMAb005 IgG and 20 µg / ml (final concentration) of anti-human IgG1 receptor beads were added, and the mixture was incubated at room temperature for 1 h, followed by the addition of 20 µg / ml (final concentration) of streptoacidin donor beads and incubation at room temperature for 30 min. Light emission was measured in an EnVision multi-label plate reader (Perkin Elmer) and analyzed using EnVision management software. The value is reported as counts per second (CPS) and corrected for crosstalk. The percentage reduction in signal is calculated relative to uncorrelated samples.

[0156] Assay based on PD1 / PD-L1 cell antagonistic effect A PD1 / PD-L1-based cell-based bioassay (Promega) was used to measure the potency of antibodies blocking PD1 / PD-L1 interaction. One day prior to the assay, PD-L1 aAPC / CHO-K1 cells were thawed and transferred to cell recovery medium (90% Ham's F12 / 10% FBS). The cell suspension was dispensed at 100 µl / well into each of the inner 60 wells of two 96-well white flat-bottom assay plates. Cell recovery medium was added to each outer well of the assay plate, and the plates were incubated overnight at 37°C / 5% CO2. On the day of the assay, sample IgG was diluted 4-fold from 300 nM to 0.04 nM in assay buffer (99% RPMI 1640 / 1% FBS), and 40 µl of each dilution was added to the assay plate containing PD-L1 aAPC / CHO-K1 cells. Positive inhibition controls included mAb005 in an IgG1-ineffective form and nivolumab IgG4. As a negative inhibition control, unrelated IgG was included. PD1 effector cells were then thawed in assay buffer (99% RPMI 1640 / 1% FBS), and the cell suspension was added to the wells of an assay plate containing PD-L1 aAPC / CHO-K1 cells and IgG titration samples. The assay plate was incubated at 37°C / 5% CO2 for six hours, allowing equilibration to ambient temperature for 5–10 minutes, followed by the addition of 80 µl of Bio-Glo™ reagent (Promega). The assay plate was then incubated at ambient temperature for another 5–30 minutes, and the luminescence signal was subsequently measured at 10, 20, and 30 minutes.

[0157] Unidirectional human DC:T cell mixed lymphocyte reaction (MLR) assay CD14+ monocytes were isolated from PBMCs from three donors and cultured in vitro in the presence of GM-CSF and IL-4 to generate immature monocyte-derived dendritic cells (mo-DCs). mo-DCs were further matured in the cultures by adding TNFα. Pan-T cells were isolated from allogeneic PBMC donors. A one-way MLR was established by co-culturing mature DCs with freshly isolated pan-T cells at a 10:1 T cell:DC ratio for approximately 5 days in the presence of titrant. MLR activity was measured by quantifying IFN-γ production on day 5 using ELISA.

[0158] Results and discussion Document generation and filtering The variable domain of the antagonistic anti-PD1 IgG hMAb005 was cloned into a phage display vector in the form of human IgG1-κ Fab. Oligonucleotide mutagenesis was applied to 5–6 residue extensions of each sub-library using NNK randomization, resulting in 5 VL sub-libraries (Table 1) and 6 VH sub-libraries (Table 2). These libraries were transformed into… E. coli In the process, the phage populations from each library were rescued, and four rounds of selection were performed on human cyno PD1 and human rhesus monkey VEGFR2 proteins.

[0159] Then, Fab samples in the form of peritoneal articles were screened by ELISA to check the binding of individual clones selected from each library to human PD1, cyno PD1, human VEGFR2, and cyno VEGFR2. Figure 1 These analyses demonstrated that individual clones in all rounds of selection exhibited binding to both PD1 orthologs, with binding signals similar to the positive control hMAb005Fab. Importantly, the binding analyses also demonstrated that several libraries had been enriched with clonal populations that not only retained PD1 activity but also exhibited significantly improved binding to human and / or cyno VEGFR2. 168 clones from these selection populations were cherry-picked and further characterized in both AlphaScreen PD1 epitope competition with hMAb005 IgG and DNA sequencing of the VL and VH domains. AlphaScreen analysis revealed unique sequences in both the LCDR1 and LCDR3 libraries exhibiting improved binding to VEGFR2 (…). Figure 1 It maintained epitope competition for hMAb005 binding to PD1. Figure 2 In contrast, clones of the HCDR1, 2, and 3 libraries, which exhibited improved binding to VEGFR2, did not effectively compete with hMAb005, indicating that mutations in the VH domain that improve VEGFR2 affinity have led to epitope drift on PD1. Figure 2 Sequence analysis of all VEGFR2-improved clones maintaining PD1 binding in both LCDR1 (Table 3) and LCDR3 (Table 4) revealed a large number of mutations indicating positive selection.

[0160] Based on the above analysis, five representative clones with mutations in either LCDR1 or LCDR3 that exhibited high binding to two orthologs of PD1 and two orthologs of VEGFR2 were selected. Figure 1 ) and >75% reduction in the signal of hMAb005 / PD1 interaction ( Figure 2The IgG clones were used for expression and characterization of the IgG form (Table 5). Additionally, six experimental combinatorial IgG clones (clones MAB01-MAB06, Table 5) with potential beneficial mutations in both LCDR1 and LCDR3 were prepared. These 11 lead IgG clones were generated in a human IgG1 ineffective form.

[0161] Specificity and potency characteristics of lead IgG The binding of 11 lead IgG1 null clones outlined in Table 4, plus hMAb005 and allotype IgG1 null clones, to human and cyno PD1 was examined in titration ELISA. Figure 3A This analysis showed that all 11 lead clones exhibited similarly potent binding to human and cynoPD1 as hMAb005 IgG1, while the isotype control showed no binding to either protein. In contrast, when testing for binding to VEGFR2 protein, all 11 lead clones demonstrated significantly increased binding potency to both human and rhesus monkey orthologs compared to hMAb005. Figure 3B ).

[0162] To investigate whether this increased VEGFR2 responsiveness relative to hMab005 in the lead clone could alter the pharmacological relationship with the receptor, a human VEGFR2 reporter assay was used to examine the induction of luciferase expression under the control of the natural VEGF responsive element NFAT (Promega, performed according to the manufacturer's instructions). In this assay, soluble VEGF-165 protein was added to VEGFR2 reporter cells, and the ability of all IgG1 proteins to antagonize the VEGF-165-induced signal was examined. Figure 4 Both IgG1 hMAb005 and isotype IgG1 showed no antagonistic effect against VEGFR2 signaling. Surprisingly, however, all the lead IgG1 null antibodies exhibited potent antagonism against VEGF-VEGFR2 signaling, with many clones showing potency similar to that of the clinically approved anti-VEGFR2 cancer drug 'ramucirumab'. Figure 4 These were highly unexpected findings, as hMAb005 IgG1 has shown potent activation of VEGFR2. The above data indicate that PD1 epitope specificity was maintained, but the VEGFR2 pharmacology of all lead antibodies was completely reversed. To further examine these data, clones derived from the library ( Figures 5A-5E ) and clones with mutations in combined LCDR 1 and 3 ( Figure 5F-5K Separate analyses were generated. These analyses showed that antibodies MAB01-MAB06 are the most potent inhibitors of VEGFR2 signaling, demonstrating a synergistic effect in improving VEGFR2 binding mediated by each CDR.

[0163] The combinatorial analysis outlined in this article demonstrates, surprisingly, that in-depth sampling of the amino acid diversity in the CDRs of these antibodies allows for simultaneous optimization of PD1 and VEGFR2 target binding specificity, leading to the reversal of the VEGFR2 agonist phenotype of the progenitor antibody hMAb005 in multiple lead molecules. The resulting lead antibodies possess the beneficial characteristic of dual antagonism of clinically relevant PD1 and VEGFR2 signaling pathways.

[0164] Lead IgG variant generation and analysis Eight variants of clone MAB06 (MAB06.1–MAB06.8) were generated to experimentally interrogate multiple CDR residues by mutation into human germline sequences to reduce the risk of immunogenicity. These clones contained germline mutations in LCDR1 and 2 (Table 8) and in HCDR2 (Table 9). All eight clones were expressed and purified in IgG1-invalid form. All clones were readily expressed and purified via protein A affinity column, and the resulting proteins exhibited >94% monomeric IgG (as measured by SEC).

[0165] The ability of MAB06.1-MAB06.8 IgG to antagonize PD1 and VEGFR2 signaling was tested. All eight clones retained the ability to antagonize PD1 signaling in the PD1 / PD-L1 cell signaling bioassay, with potency similar to that of SHR-1210 IgG1-3M and nivolumab. Figure 6 Similarly, all eight clones retained the ability to antagonize VEGFR2 signaling in a concentration-dependent manner in the VEGF / VEGFR2 signaling bioassay, with potency similar to ramucirumab. Figure 7 Cloning MAB06.5 ( Figure 8A , 8B MAB06.6 Figure 8C , 8D MAB06.7 Figure 8E , 8F ) and MAB06.8 ( Figure 8G , 8H Comparative analysis of the PD1 and VEGFR2 antagonistic effects of clone MAB06 demonstrated that the sequence of clone MAB06 can accept humanization of multiple residues in LCDR1, LCDR2 (Table 8), and HCDR2 (Table 9) while retaining the ability to antagonize the signal transduction of both receptors. Importantly, it showed that the control SHR-1210 IgG13M (hMab005) could not inhibit VEGFR2 signaling in the presence of VEGF, as Figure 4 As shown in 5, 7 and 8.

[0166] The above findings demonstrate that the ability to antagonize both the PD1 and VEGFR2 pathways in a single molecule can provide clinical benefits superior to the antibody SHR-1210 (hMab005) by blocking two key immunosuppressive signals known to be highly active in the tumor microenvironment. In fact, the co-expression of both PD1 and VEGFR2 receptors on immune cells in human tumors has been demonstrated, suggesting that dual antagonists may act in a bifunctional manner on single cells, such as... Figure 9 As shown.

[0167] In the absence of receptor ligands, antibodies that bind to signal transduction receptors with high affinity risk becoming potent agonists of receptor activity. To examine this, the VEGFR2 bioassay was repeated in the absence of VEGF. In this assay, all proteins, including the high-affinity anti-VEGFR2 IgG1 control antibody ramucirumab, isotype control human IgG1, MAB06.5, MAB06.8, and VEGF-165, were titrated, with antibody protein concentrations starting at 1000 nM. Figure 10 Only VEGF-165 exhibited concentration-dependent receptor activation, confirming that MAB06.5 and MAB06.8 IgG1 are VEGFR2 antagonists in the presence of VEGF (Figure 8) and not VEGFR2 agonists in the absence of VEGF. Figure 10 ).

[0168] To further confirm the PD1 antagonistic efficacy of MAB06.5 and MAB06.8, both molecules were analyzed in a one-way MLR assay in human DC:T cells, with nivolumab (anti-PD1) as a positive control and IFN-γ measured as an indicator of activity (Figure 11). MLR assays were performed in two replicate runs using three separate human donor pairs. These analyses demonstrated that MAB6.5 and MAB6.8 exhibited concentration-dependent efficacy equivalent to nivolumab in PD1 blockade-driven IFN-γ signaling. For donor pair 1 ( Figure 11A , Figure 11B ), donor pair 2 ( Figure 11C , Figure 11D ) and donor pair 3 ( Figure 11E , Figure 11F This finding was observed in both runs, demonstrating high reproducibility in blocking PD1 function on T cells.

[0169] Generation and analysis of third-generation lead IgG variants WO2019170885A1 discloses numerous mutations that can be generated in the VH domain of hMab005 with the potential to enhance the molecule's affinity for PD1. To examine the potential utility of these previously defined mutations in the context of the novel clones described herein, nine cloned MAB06.8 variants were generated to experimentally inquire whether multiple VH CDR residues could be mutated to potentially further improve the ability to bind to PD1 and / or VEGFR2. All of these clones contained the MAB06.8 VL sequence (SEQ ID NO: 47, Table 8) expressed along with nine unique VH sequences (Table 10). All eight clones were expressed and purified in IgG1-ineffective form. All clones (except MAB06.8.3 and MAB06.8.5, both of which underwent aggregation) were readily expressed and purified via a protein A affinity column, and the resulting proteins exhibited >95% monomeric IgG % (as measured by SEC).

[0170] Seven well-expressing IgG1 inactive clones (MAB06.8.3 and MAB06.8.5 omitted) were examined in titration ELISA for binding to human and cyno PD1, along with hMAb005 and isotype IgG1 inactive clones. Figure 12A , Figure 12B The analysis showed that most of the seven clones retained binding to human and cyno PD1, while the isotype control showed no binding to either protein. MAB06.8.4 was a notable exception, as it lost the ability to bind to cyno PD1. Figure 12B When testing for binding of IgG to the VEGFR2 protein, all seven clones showed binding to both human and cynomolgus monkey orthologs. Figure 12C , Figure 12D ).

[0171] Although the epigenetic binding affinity for both PD1 and VEGFR2 was preserved in most clones, the beneficial qualities of MAB06.8 were neither preserved nor enhanced when examined in a PD1 and VEGFR2 antagonism bioassay across seven variants. Compared to the positive control ( Figure 13A , Figure 13B All seven clones showed reduced ability to antagonize PD1, VEGFR2, or both compared to MAB06 (Fig. 5), MAB06.5 (Fig. 8), and MAB06.8 (Fig. 8).

[0172] All documents or portions thereof cited herein (including, but not limited to, patents, patent applications, articles, books, and papers) are hereby expressly incorporated in their entirety by reference for any purpose. If any term defined in one or more of the incorporated documents or portions thereof conflicts with the definition of that term in this application, the definition appearing in this application shall prevail. However, any references, articles, publications, patents, patent publications, and patent applications cited herein are not and should not be construed as an admission or suggestion of any kind, but constitute valid prior art or are part of common general knowledge in any country of the world.

[0173] Although the invention has been described with reference to preferred or exemplary embodiments, those skilled in the art will recognize that various modifications and variations can be made to the invention without departing from its spirit and scope, and such modifications are expressly contemplated herein. The specific embodiments disclosed herein and set forth in the appended claims are not intended, nor should they be construed, as any limitation thereof.

[0174] Implementation plan with numbering Despite the appended claims, this disclosure sets forth embodiments numbered as follows: 1. An antibody or an antigen-binding moiety of said antibody that specifically binds to both PD1 and VEGFR2, wherein said antibody or antigen-binding moiety comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein (a) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (b) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (c) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQESGIWLS (SEQ ID NO:8), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (d) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVYSIPWT (SEQ ID NO:6); (e) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVYSIPWT (SEQ ID NO:6); (f) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of LASQESGIWLS (SEQ ID NO:8), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVYSIPWT (SEQ ID NO:6); (g) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQTIGTWLT (SEQ ID NO:9), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVAELPFG (SEQ ID NO:10); (h) The amino acid sequence of the VH region includes HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region includes LCDR1 of LASQTIGTWLT (SEQ ID NO:9), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (i) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVAELPFG (SEQ ID NO:10); (j) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVAELPFG (SEQ ID NO:10); (k) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of LASQESGIWLG (SEQ ID NO:12), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVAELPFG (SEQ ID NO:10); (l) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of LASQGIGPWLS (SEQ ID NO:4), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (m) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO:2) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of LASQPLGIWLS (SEQ ID NO:7), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (n) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TATSLAD (SEQ ID NO:5) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (o) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TASSLAD (SEQ ID NO:40) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (p) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLAD (SEQ ID NO:41) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (q) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO:38), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (r) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42) and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of TASSLAD (SEQ ID NO:40) and LCDR3 of QQVSVTPFT (SEQ ID NO:11); (s) The amino acid sequence of the VH region comprises HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42), and HCDR3 of QLYYFDY (SEQ ID NO:3); and the amino acid sequence of the VL region comprises LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLAD (SEQ ID NO:41), and LCDR3 of QQVSVTPFT (SEQ ID NO:11); or (t) The amino acid sequence of the VH region contains HCDR1 of GFTFSSYMMS (SEQ ID NO:1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO:42) and HCDR3 of QVYYFDY (SEQ ID NO:64); and the amino acid sequence of the VL region contains LCDR1 of RASQESGIWLS (SEQ ID NO:39), LCDR2 of AASSLQS (SEQ ID NO:43) and LCDR3 of QQVSVTPFT (SEQ ID NO:11).

[0175] 2. The antibody or antigen-binding portion as described in Embodiment 1, wherein (a) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:44; (b) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:47; (c) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:24; (d) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:14; (e) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:15; (f) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:16; (g) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:17; (h) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:18; (i) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:19; (j) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:20; (k) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:21; (l) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:22; (m) The amino acid sequence of the VH region contains SEQ ID NO:13, and the amino acid sequence of the VL region contains SEQ ID NO:23; (n) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:44; (o) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:45; (p) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:46; (q) The amino acid sequence of the VH region contains SEQ ID NO:48, and the amino acid sequence of the VL region contains SEQ ID NO:47; (r) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:45; (s) The amino acid sequence of the VH region contains SEQ ID NO:49, and the amino acid sequence of the VL region contains SEQ ID NO:46; or (t) The amino acid sequence of the VH region contains SEQ ID NO:73, and the amino acid sequence of the VL region contains SEQ ID NO:47.

[0176] 3. The antibody or antigen-binding portion as described in embodiment 1 or 2, wherein the antibody or antigen-binding portion is capable of antagonizing both the PD1-PDL1 and VEGFR2-VEGF signaling pathways.

[0177] 4. An antibody or an antigen-binding portion of said antibody that specifically binds to PD1 and VEGFR2, wherein said antibody or antigen-binding portion cross-competes with the antibody or antigen-binding portion of any one of embodiments 1-3 for binding to both PD1 and VEGFR2; and (a) Contains a complete human germline framework amino acid sequence; (b) Specifically binds to human PD1, cynomolgus monkey PD1, human VEGFR2, and rhesus monkey VEGFR2; and (c) It antagonizes the binding of human PD1 to human PD-L1 and antagonizes human VEGFR2 signaling in response to human VEGF.

[0178] 5. An antibody or antigen-binding portion as described in any one of embodiments 1-4, wherein the antibody is human, humanized, or chimeric.

[0179] 6. The antibody or antigen-binding portion as described in any one of embodiments 1-5, wherein the VH region, the VL region, or both the VH region and the VL region comprise one or more human framework region amino acid sequences.

[0180] 7. The antibody or antigen-binding portion as described in any one of embodiments 1-6, wherein the VH region, the VL region, or both the VH region and the VL region comprise a human variable region framework amino acid sequence that has been inserted into the CDR.

[0181] 8. The antibody or antigen binding portion as described in embodiment 1 or 3, wherein the VH region comprises an IGHV3-7 human germline scaffold amino acid sequence into which the amino acid sequences of HCDR1, HCDR2 and HCDR3 have been inserted.

[0182] 9. An antibody or antigen-binding portion as described in any one of embodiments 1, 3, and 8, wherein the VL region comprises an IGKV1-39 human germline scaffold amino acid sequence into which the amino acid sequences of LCDR1, LCDR2, and LCDR3 have been inserted.

[0183] 10. An antibody or antigen-binding portion as described in any one of embodiments 1-9, wherein the antibody comprises an immunoglobulin constant region.

[0184] 11. The antibody or antigen-binding portion as described in embodiment 10, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

[0185] 12. The antibody or antigen-binding portion as described in embodiment 11, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0186] 13. The antibody or antigen binding portion as described in embodiment 10, wherein the immunoglobulin constant region is immune inert.

[0187] 14. The antibody or antigen binding region as described in embodiment 10, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region containing amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region containing amino acid substitutions L234A, L235A and G237A, or a wild-type human IgG2 constant region.

[0188] 15. The antibody or antigen-binding portion as described in embodiment 13, wherein the immunoglobulin constant region comprises any one of SEQ ID NO:25-31.

[0189] 16. The antigen-binding portion as described in any one of embodiments 1-15, wherein the antibody or antigen-binding portion is Fab, Fab', F(ab')2, Fd, Fv, scFv, macroantibody, microantibody, intracellular antibody, biantibody, triantibody, tetraantibody, or biscFv.

[0190] 17. An antibody or antigen-binding portion as described in any one of embodiments 1-16, wherein the antibody is monoclonal.

[0191] 18. The antibody or antigen-binding portion as described in any one of embodiments 1-17, wherein the antibody is a tetrameric antibody, a tetravalent antibody, or a multispecific antibody.

[0192] 19. An antibody or antigen-binding portion as described in any one of embodiments 1-18, wherein the antibody or antigen-binding portion specifically binds (a) human PD1 or (b) human PD1 and cynomolgus monkey PD1 or (c) human PD1 and rhesus monkey PD1 or (d) human PD1, cynomolgus monkey PD1 and rhesus monkey PD1.

[0193] 20. An antibody or antigen-binding portion as described in any one of embodiments 1-19, wherein the antibody or antigen-binding portion specifically binds to (a) human VEGFR2 or (b) human VEGFR2 and cynomolgus monkey VEGFR2 or (c) human VEGFR2 and rhesus monkey VEGFR2 or (d) human VEGFR2, cynomolgus monkey VEGFR2 and rhesus monkey VEGFR2.

[0194] 21. An immunoconjugate comprising an antibody or antigen-binding portion of any one of embodiments 1-20 linked to a therapeutic agent.

[0195] 22. The immunoconjugate as described in embodiment 21, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, an anti-angiogenic agent, an antiproliferative agent, an apoptosis-promoting agent, a cell inhibitory enzyme, a cell lysing enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an antiproliferative agent, or an apoptosis-promoting agent.

[0196] 23. A pharmaceutical composition comprising an antibody or antigen-binding portion as described in any one of embodiments 1-20 or an immunoconjugate as described in embodiments 21 or 22, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0197] 24. A nucleic acid molecule encoding the following portion of the antibody or antigen-binding portion according to any one of embodiments 1-20: (a) The amino acid sequence of the VH region; (b) the amino acid sequence of the VL region; or (c) Both the amino acid sequences of the VH region and the VL region.

[0198] 25. An expression vector comprising the nucleic acid molecule described in embodiment 24.

[0199] 26. A recombinant host cell comprising the nucleic acid molecule described in embodiment 24 or the expression vector described in embodiment 25.

[0200] 27. A method for generating an anti-PD1 antibody or its antigen-binding portion, the method comprising: Recombinant host cells containing the expression vector described in Embodiment 25 are cultured under conditions expressing the nucleic acid molecule to generate the antibody or antigen-binding moiety; and Isolate the antibody or antigen-binding portion from the host cells or culture.

[0201] 28. A method for enhancing the immune response of a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion of any one of embodiments 1-20, an immunoconjugate of embodiment 21 or 22, or a pharmaceutical composition of embodiment 23.

[0202] 29. A method for treating or preventing a subject from cancer, an infectious disease, or an immune disease, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding moiety of any one of embodiments 1-20, an immunoconjugate of embodiment 21 or 22, or a pharmaceutical composition of embodiment 23.

[0203] 30. The method as described in embodiment 29, wherein the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or hematologic malignancy.

[0204] 31. The method of embodiment 29, wherein the infectious disease is a viral, bacterial, fungal, or parasitic infection.

[0205] 32. The method of embodiment 29, wherein the infectious disease is human immunodeficiency virus (HIV) infection.

[0206] 33. The antibody or antigen-binding moiety as described in any one of embodiments 1-20, the immunoconjugate as described in embodiment 21 or 22, or the pharmaceutical composition as described in embodiment 23, for use in the treatment of cancer, infectious diseases, or immune diseases.

[0207] 34. The antibody or antigen-binding moiety, immunoconjugate, or pharmaceutical composition used as described in embodiment 33, wherein the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or hematologic malignancy.

[0208] 35. The antibody or antigen-binding moiety, immunoconjugate, or pharmaceutical composition used as described in embodiment 33, wherein the infectious disease is a virus, bacteria, fungus, or parasite.

[0209] 36. The antibody or antigen-binding moiety, immune conjugate, or pharmaceutical composition used as described in embodiment 33, wherein the infectious disease is human immunodeficiency virus (HIV) infection.

[0210] 37. An antibody or antigen-binding moiety as described in any one of embodiments 1-20, an immunoconjugate as described in embodiment 21 or 22, or a pharmaceutical composition as described in embodiment 23, used as a medicine.

[0211] Table 1. Amino acid sequences of the hMAb005 VL mutagen library.

[0212]

[0213] As stated above, the "unified" CDR definition used in this manuscript is an extended definition compared to the classic Kabat definition. The light chain frame region (LFR) and CDR for each of the above sequences are shown in bold, with the location where mutagenesis was applied underlined.

[0214] Table 2. Amino acid sequences of the hMAb005 VH mutagen library.

[0215]

[0216] As stated above, the "unified" CDR definition used in this manuscript is an extended definition compared to the classic Kabat definition. Each sequence above shows the light chain frame region (HFR) and CDR in bold, with the location where mutagenesis was applied underlined.

[0217] Table 3. Position-specific mutations found in LCDR1 clones that exhibit improved binding with VEGFR2.

[0218]

[0219] The original LCDR1 sequence LASQTIGTWLT (SEQ ID NO:9) is displayed at the top. LASQX1X2X3X4WLX5 are variant sequences, where X1 is T, E, G, K, P, or S, X2 is I, A, L, S, or V, X3 is G, R, or S, X4 is T, G, I, L, or P, and X5 is T or S (SEQ ID NO:59).

[0220] Table 4. Position-specific mutations found in LCDR3 clones that exhibit improved binding with VEGFR2.

[0221]

[0222] The original LCDR3 sequence QQVYSIPWT (SEQ ID NO:6) is displayed at the top. QQVX1X2X3PX4X5 are variant sequences, where X1 is Y, A, F, N, or S, X2 is S, A, E, M, N, Q, or V, X3 is I, L, T, or V, X4 is W or F, and X5 is T, A, G, N, R, or S (SEQ ID NO:60).

[0223] Table 5. Light chain variable region sequences of IgGs exhibiting improved binding with VEGFR2.

[0224]

[0225] Table 6. Examples of amino acid sequences in the Fc region of antibodies.

[0226] Human IgG4 wild type ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 25) Human IgG4 (S228P) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK (SEQ ID NO: 26) Human IgG1 wild type ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27) Human IgG1-3M ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28) Human IgG2 wild type ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 29) Human IgG1 wild-type "REEM" allotype ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 30) Human IgG1-3M "REEM" allotype ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEM KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31) Table 7. Examples of amino acid sequences of membrane proteins.

[0227] Human PD1 sequence MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ IDNO: 32) cynomolgus monkey PD1 sequence MQIPQAPWPVVWAVLQLGWRPGWFLESPDRPWNAPTFSPALLLVTEGDNATFTSNSNASESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTRLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSSPRPAGQFQALVVGVVGGLLGSLVLLVWVLAVICSRAAQGTIEARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPAPCVPEQTEYATIVFPSGLGTSSPARRGSADGPRSPRPLRPEDGHCSWPL(SEQ ID NO: 33) Human KDR (VEGFR2) sequence Rhesus monkey KDR (VEGFR2) sequence Table 8. Amino acid sequences of the light chain variable region of IgG that exhibits improved binding with VEGFR2.

[0228]

[0229] Table 9. Amino acid sequences of the heavy chain variable region of IgG that exhibit improved binding with VEGFR2.

[0230]

[0231] Table 10. Combining the amino acid sequence of the heavy chain variable region with the VL amino acid sequence of SEQ ID NO: 47 to prepare third-generation IgG.

[0232]

Claims

1. An antibody or antigen-binding portion of the antibody that specifically binds to both PD1 and VEGFR2, wherein the antibody or antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein (a) the VH region amino acid sequence comprises a HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), a HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO: 42), and a HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises a LCDR1 of RASQESGIWLS (SEQ ID NO: 39), a LCDR2 of TATSLAD (SEQ ID NO: 5), and a LCDR3 of QQVSVTPFT (SEQ ID NO: 11); (b) the VH region amino acid sequence comprises a HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), a HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO: 42), and a HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises a LCDR1 of RASQESGIWLS (SEQ ID NO: 39), a LCDR2 of AASSLQS (SEQ ID NO: 43), and a LCDR3 of QQVSVTPFT (SEQ ID NO: 11); (c) the VH region amino acid sequence comprises a HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), a HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO: 2), and a HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises a LCDR1 of LASQESGIWLS (SEQ ID NO: 8), a LCDR2 of TATSLAD (SEQ ID NO: 5), and a LCDR3 of QQVSVTPFT (SEQ ID NO: 11); (d) the VH region amino acid sequence comprises a HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), a HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO: 2), and a HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises a LCDR1 of LASQGIGPWLS (SEQ ID NO: 4), a LCDR2 of TATSLAD (SEQ ID NO: 5), and a LCDR3 of QQVYSIPWT (SEQ ID NO: 6). ​ ​ ​ ​ (e) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO: 2), and HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises LCDR1 of LASQPLGIWLS (SEQ ID NO: 7), LCDR2 of TATSLAD (SEQ ID NO: 5), and LCDR3 of QQVYSIPWT (SEQ ID NO: 6); (f) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO: 2), and HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises LCDR1 of LASQESGIWLS (SEQ ID NO: 8), LCDR2 of TATSLAD (SEQ ID NO: 5), and LCDR3 of QQVYSIPWT (SEQ ID NO: 6); (g) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO: 2), and HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises LCDR1 of LASQTIGTWLT (SEQ ID NO: 9), LCDR2 of TATSLAD (SEQ ID NO: 5), and LCDR3 of QQVAELPFG (SEQ ID NO: 10); (h) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO: 2), and HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises LCDR1 of LASQTIGTWLT (SEQ ID NO: 9), LCDR2 of TATSLAD (SEQ ID NO: 5), and LCDR3 of QQVSVTPFT (SEQ ID NO: 11); (i) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO: 2), and HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises LCDR1 of LASQGIGPWLS (SEQ ID NO: 4), LCDR2 of TATSLAD (SEQ ID NO: 5), and LCDR3 of QQVAELPFG (SEQ ID NO: 10); (j) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO: 2), and HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises LCDR1 of LASQPLGIWLS (SEQ ID NO: 7), LCDR2 of TATSLAD (SEQ ID NO: 5), and LCDR3 of QQVAELPFG (SEQ ID NO: 10); (k) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO: 2), and HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises LCDR1 of LASQESGIWLG (SEQ ID NO: 12), LCDR2 of TATSLAD (SEQ ID NO: 5), and LCDR3 of QQVAELPFG (SEQ ID NO: 10); (l) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO: 2), and HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises LCDR1 of LASQGIGPWLS (SEQ ID NO: 4), LCDR2 of TATSLAD (SEQ ID NO: 5), and LCDR3 of QQVSVTPFT (SEQ ID NO: 11); (m) the VH region amino acid sequence comprises a HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), a HCDR2 of TISGGGANTYYPDSVKG (SEQ ID NO: 2), and a HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises a LCDR1 of LASQPLGIWLS (SEQ ID NO: 7), a LCDR2 of TATSLAD (SEQ ID NO: 5), and a LCDR3 of QQVSVTPFT (SEQ ID NO: 11); (n) the VH region amino acid sequence comprises a HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), a HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO: 38), and a HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises a LCDR1 of RASQESGIWLS (SEQ ID NO: 39), a LCDR2 of TATSLAD (SEQ ID NO: 5), and a LCDR3 of QQVSVTPFT (SEQ ID NO: 11); (o) the VH region amino acid sequence comprises a HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), a HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO: 38), and a HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises a LCDR1 of RASQESGIWLS (SEQ ID NO: 39), a LCDR2 of TASSLAD (SEQ ID NO: 40), and a LCDR3 of QQVSVTPFT (SEQ ID NO: 11); (p) the VH region amino acid sequence comprises a HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), a HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO: 38), and a HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises a LCDR1 of RASQESGIWLS (SEQ ID NO: 39), a LCDR2 of AASSLAD (SEQ ID NO: 41), and a LCDR3 of QQVSVTPFT (SEQ ID NO: 11); (q) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGANTYYVDSVKG (SEQ ID NO: 38), and HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises LCDR1 of RASQESGIWLS (SEQ ID NO: 39), LCDR2 of AASSLQS (SEQ ID NO: 43), and LCDR3 of QQVSVTPFT (SEQ ID NO: 11); (r) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO: 42), and HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises LCDR1 of RASQESGIWLS (SEQ ID NO: 39), LCDR2 of TASSLAD (SEQ ID NO: 40), and LCDR3 of QQVSVTPFT (SEQ ID NO: 11); (s) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO: 42), and HCDR3 of QLYYFDY (SEQ ID NO: 3); and the VL region amino acid sequence comprises LCDR1 of RASQESGIWLS (SEQ ID NO: 39), LCDR2 of AASSLAD (SEQ ID NO: 41), and LCDR3 of QQVSVTPFT (SEQ ID NO: 11); or (t) the VH region amino acid sequence comprises HCDR1 of GFTFSSYMMS (SEQ ID NO: 1), HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO: 42), and HCDR3 of QVYYFDY (SEQ ID NO: 64); and the VL region amino acid sequence comprises LCDR1 of RASQESGIWLS (SEQ ID NO: 39), LCDR2 of AASSLQS (SEQ ID NO: 43), and LCDR3 of QQVSVTPFT (SEQ ID NO: 11).

2. The antibody or antigen binding portion of claim 1, wherein (a) the VH region amino acid sequence comprises SEQ ID NO: 49, and the VL region amino acid sequence comprises SEQ ID NO: 44; (b) the VH region amino acid sequence comprises SEQ ID NO: 49, and the VL region amino acid sequence comprises SEQ ID NO: 47; (c) the VH region amino acid sequence comprises SEQ ID NO: 13, and the VL region amino acid sequence comprises SEQ ID NO: 24; (d) the VH region amino acid sequence comprises SEQ ID NO: 13, and the VL region amino acid sequence comprises SEQ ID NO: 14; (e) the VH region amino acid sequence comprises SEQ ID NO: 13, and the VL region amino acid sequence comprises SEQ ID NO: 15; (f) the VH region amino acid sequence comprises SEQ ID NO: 13, and the VL region amino acid sequence comprises SEQ ID NO: 16; (g) the VH region amino acid sequence comprises SEQ ID NO: 13, and the VL region amino acid sequence comprises SEQ ID NO: 17; (h) the VH region amino acid sequence comprises SEQ ID NO: 13, and the VL region amino acid sequence comprises SEQ ID NO: 18; (i) the VH region amino acid sequence comprises SEQ ID NO: 13, and the VL region amino acid sequence comprises SEQ ID NO: 19; (j) the VH region amino acid sequence comprises SEQ ID NO: 13, and the VL region amino acid sequence comprises SEQ ID NO: 20; (k) the VH region amino acid sequence comprises SEQ ID NO: 13, and the VL region amino acid sequence comprises SEQ ID NO: 21; (l) the VH region amino acid sequence comprises SEQ ID NO: 13, and the VL region amino acid sequence comprises SEQ ID NO: 22; (m) the VH region amino acid sequence comprises SEQ ID NO: 13, and the VL region amino acid sequence comprises SEQ ID NO: 23; (n) the VH region amino acid sequence comprises SEQ ID NO: 48, and the VL region amino acid sequence comprises SEQ ID NO: 44; (o) the VH region amino acid sequence comprises SEQ ID NO: 48, and the VL region amino acid sequence comprises SEQ ID NO: 45; (p) the VH region amino acid sequence comprises SEQ ID NO: 48, and the VL region amino acid sequence comprises SEQ ID NO: 46; (q) the VH region amino acid sequence comprises SEQ ID NO: 48, and the VL region amino acid sequence comprises SEQ ID NO: 47; (r) the VH region amino acid sequence comprises SEQ ID NO: 49, and the VL region amino acid sequence comprises SEQ ID NO: 45; (s) the VH region amino acid sequence comprises SEQ ID NO: 49, and the VL region amino acid sequence comprises SEQ ID NO: 46; or (t) the VH region amino acid sequence comprises SEQ ID NO: 73, and the VL region amino acid sequence comprises SEQ ID NO:

47.

3. The antibody or antigen-binding portion of claim 1 or 2, wherein the antibody or antigen-binding portion is capable of antagonizing both the PD1-PDL1 and VEGFR2-VEGF signaling pathways.

4. An antibody or antigen-binding portion of the antibody that specifically binds to PD1 and VEGFR2, wherein the antibody or antigen-binding portion cross-competes for binding to both PD1 and VEGFR2 with the antibody or antigen-binding portion of any one of claims 1-3; and (a) comprises a fully human germline framework amino acid sequence; (b) specifically binds to human PD1, cynomolgus monkey PD1, human VEGFR2, and rhesus monkey VEGFR2; and (c) antagonizes the binding of human PD1 to human PD-L1 and antagonizes human VEGFR2 signaling in response to human VEGF.

5. The antibody or antigen-binding portion of any one of claims 1-4, wherein the antibody is human, humanized, or chimeric.

6. The antibody or antigen-binding portion of any one of claims 1-5, wherein the VH region, the VL region, or both the VH region and the VL region comprise one or more human framework region amino acid sequences.

7. The antibody or antigen-binding portion of any one of claims 1-6, wherein the VH region, the VL region, or both the VH region and the VL region comprise a human variable region framework scaffold amino acid sequence that has been inserted into the CDRs.

8. The antibody or antigen-binding portion of claim 1 or 3, wherein the VH region comprises an IGHV3-7 human germline scaffold amino acid sequence that has been inserted into the HCDR1, HCDR2, and HCDR3 amino acid sequences.

9. The antibody or antigen-binding portion of any one of claims 1, 3, and 8, wherein the VL region comprises an IGKV1-39 human germline scaffold amino acid sequence that has been inserted into the LCDR1, LCDR2, and LCDR3 amino acid sequences.

10. The antibody or antigen-binding portion of any one of claims 1-9, wherein the antibody comprises an immunoglobulin constant region.

11. The antibody or antigen-binding portion of claim 10, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

12. The antibody or antigen-binding portion of claim 11, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

13. The antibody or antigen-binding portion of claim 10, wherein the immunoglobulin constant region is immunologically inert.

14. The antibody or antigen-binding portion of claim 10, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A, or a wild-type human IgG2 constant region. ​ ​ ​ 15. The antibody or antigen-binding portion of claim 13, wherein the immunoglobulin constant region comprises any one of SEQ ID NOs: 25-31.

16. The antigen-binding portion of any one of claims 1-15, wherein the antibody or antigen-binding portion is a Fab, Fab', F(ab')2, Fd, Fv, scFv, maxibody, minibody, intrabody, diabody, triabody, tetrabody, or bis-scFv.

17. The antibody or antigen-binding portion of any one of claims 1-16, wherein the antibody is monoclonal.

18. The antibody or antigen-binding portion of any one of claims 1-17, wherein the antibody is a tetrameric antibody, tetravalent antibody, or multispecific antibody.

19. The antibody or antigen-binding portion of any one of claims 1-18, wherein the antibody or antigen-binding portion specifically binds (a) human PD1 or (b) human PD1 and cynomolgus monkey PD1 or (c) human PD1 and rhesus monkey PD1 or (d) human PD1, cynomolgus monkey PD1, and rhesus monkey PD1.

20. The antibody or antigen-binding portion of any one of claims 1-19, wherein the antibody or antigen-binding portion specifically binds (a) human VEGFR2 or (b) human VEGFR2 and cynomolgus monkey VEGFR2 or (c) human VEGFR2 and rhesus monkey VEGFR2 or (d) human VEGFR2, cynomolgus monkey VEGFR2, and rhesus monkey VEGFR2.

21. An immunoconjugate comprising the antibody or antigen-binding portion of any one of claims 1-20 linked to a therapeutic agent.

22. The immunoconjugate of claim 21, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, an anti-proliferative agent, a pro-apoptotic agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

23. A pharmaceutical composition comprising the antibody or antigen-binding portion of any one of claims 1-20 or the immunoconjugate of claim 21 or 22, and a pharmaceutically acceptable carrier, diluent, or excipient.

24. A nucleic acid molecule encoding the following portion of the antibody or antigen-binding portion of any one of claims 1-20: (a) the VH region amino acid sequence; (b) the VL region amino acid sequence; or (c) both the VH region and the VL region amino acid sequences.

25. An expression vector comprising the nucleic acid molecule of claim 24.

26. A recombinant host cell comprising the nucleic acid molecule of claim 24 or the expression vector of claim 25.

27. A method of producing an anti-PD1 antibody or antigen-binding portion thereof, the method comprising: culturing a recombinant host cell comprising the expression vector of claim 25 under conditions wherein the nucleic acid molecule is expressed, thereby producing the antibody or antigen-binding portion; and isolating the antibody or antigen-binding portion from the host cell or culture.

28. A method for enhancing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding portion of any one of claims 1-20, the immunoconjugate of claim 21 or 22, or the pharmaceutical composition of claim 23.

29. A method of treating or preventing cancer, an infectious disease, or an immune disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding portion of any one of claims 1-20, the immunoconjugate of claim 21 or 22, or the pharmaceutical composition of claim 23.

30. The method of claim 29, wherein the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cavity or pharynx cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.

31. The method of claim 29, wherein the infectious disease is viral, bacterial, fungal, or parasitic.

32. The method of claim 29, wherein the infectious disease is human immunodeficiency virus (HIV) infection.

33. The antibody or antigen binding portion of any one of claims 1-20, the immunoconjugate of claim 21 or 22, or the pharmaceutical composition of claim 23 for use in the treatment of cancer, an infectious disease, or an immune disease.

34. The antibody or antigen binding portion, immunoconjugate, or pharmaceutical composition for use of claim 33, wherein the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cavity or pharynx cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.

35. The antibody or antigen binding portion, immunoconjugate, or pharmaceutical composition for use of claim 33, wherein the infectious disease is viral, bacterial, fungal, or parasitic.

36. The antibody or antigen binding portion, immunoconjugate, or pharmaceutical composition for use of claim 33, wherein the infectious disease is human immunodeficiency virus (HIV) infection.

37. The antibody or antigen binding portion of any one of claims 1-20, the immunoconjugate of claim 21 or 22, or the pharmaceutical composition of claim 23 for use as a medicament.

38. A method of determining direct binding ELISA of Fabs and IgGs, the steps of the method being as follows: First, assess the binding and cross-reactivity of the lead panel to recombinant proteins by binding ELISA; Human PD1 human Fc tagged recombinant protein and cynomolgus PD1 human Fc tagged recombinant protein were coated onto the surface of MaxiSorp™ flat-bottom 96-well plates at 1 µg / ml; Purified Fab or IgG samples were titrated in two-fold serial dilutions from 500 nM to 0.98 nM and allowed to bind to the coated antigen; Fabs were detected using mouse anti-c-myc antibody followed by donkey anti-mouse IgG conjugated to horseradish peroxidase; IgGs were detected using mouse anti-human IgG conjugated to horseradish peroxidase; Bound signal was visualized with 3,3',5,5'-tetramethylbenzidine substrate solution (TMB) and absorbance at 450 nm was measured.

39. A method of performing an AlphaScreen epitope competition assay on a Fab periplasmic preparation, the steps of the method being as follows: AlphaScreen assay was performed in a 25 µl final volume in a 384-well white microtiter plate; The reaction buffer contained lx PBS pH 7.3 and 0.05% (v / v) Tween® 20; Periplasmic preparation samples diluted in reaction buffer were incubated with biotinylated human PD1-His / AviTag at a final concentration of 0.6 nM for 20 minutes at room temperature; 0.3 nM of hMAb005 IgG and 20 µg / ml (final concentration) of anti-human IgG1 acceptor beads were added and the mixture was incubated for 1 hour at room temperature, followed by the addition of 20 µg / ml (final concentration) of streptavidin donor beads and incubation for 30 minutes at room temperature; Light emission was measured in an EnVision multilabel plate reader (Perkin Elmer) and analyzed using EnVision Manager software; Values were reported as counts per second and corrected for cross-talk; signal reduction percentage was calculated relative to an irrelevant sample.

40. A method of determining based on PD1 / PD-L1 cell-based antagonism, the steps of the method being as follows: A PD1 / PD-L1 blockade cell-based bioassay (Promega) was used to measure the potency of antibodies blocking PD1 / PD-L1 interaction; PD-L1 aAPC / CHO-K1 cells were thawed and transferred into cell recovery medium (90% Ham’s F12 / 10% FBS) one day before the assay; Cell suspension was dispensed at 100 µl / well into each of the inner 60 wells of two 96-well white flat-bottom assay plates; Cell recovery medium was added to each of the outer wells of the assay plates and incubated overnight at 37°C / 5% CO2; On the day of the assay, sample IgG was diluted 4-fold from 300 nM to 0.04 nM in assay buffer (99% RPMI 1640 / 1% FBS) and 40 μΐ of each dilution was added to the assay plate containing PD-L1 aAPC / CHO-K1 cells; positive inhibition controls included mAb005 in an IgG1 null format and nivolumab IgG4; as a negative inhibition control, an irrelevant IgG was included; PD1 effector cells were then thawed in assay buffer (99% RPMI 1640 / 1% FBS) and the cell suspension was added to the wells of the assay plate containing PD-L1 aAPC / CHO-K1 cells and IgG titration samples; The assay plate was incubated for six hours at 37°C / 5% C02 incubator, allowed to equilibrate to ambient temperature for 5-10 minutes, and then 80 μΐ of Bio-Glo™ reagent was added; The assay plate was incubated for a further 5-30 minutes at ambient temperature and luminescent signal was subsequently measured at 10, 20 and 30 minutes.

41. A method for a one-way human DC:T cell mixed lymphocyte reaction (MLR) assay, the steps of the method being as follows: CD14+ monocytes were isolated from PBMC of three donors and cultured in vitro in the presence of GM-CSF and IL-4 to generate immature monocyte-derived dendritic cells (mo-DCs); mo-DCs were further matured in culture by the addition of TNFa; Pan T cells were isolated from allogeneic PBMC donors; A one-way MLR was established by co-culturing mature DCs with freshly isolated pan T cells at a 10:1 T cell:DC ratio in the presence of titrated test article for approximately 5 days; Activity was measured by quantifying IFN-g production at day 5 of the MLR by ELISA.

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