PD1 and vegfr2 dual-binding agents

Abstract

To provide a combination with previously-proven cancer therapies such as anti-angiogenic agents in order to improve clinical response rates to anti-PD1 antibodies.SOLUTION: An antibody or an antigen-binding fragment thereof that binds specifically to both PD1 and VEGFR2, and a pharmaceutical composition comprising the same, are provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of UK Patent Application No. 2013180.1 filed August 24, 2020, and UK Patent Application No. 1914747.9 filed October 11, 2019, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Description of electronically submitted text files The contents of the text file submitted electronically herein are incorporated herein by reference in their entirety. A computer-readable copy of the Sequence Listing (Filename: UHEL_002_02WO_SeqList_ST25.txt, Date of Record: October 9, 2020, File size approximately 86,707 bytes).

[0003] The present invention relates to antibody molecules that specifically bind to both PD1 (also known as programmed cell death 1, PDCD1, CD279, PD-1, SLEB2, PD-1, SLE1) and VEGFR2 (also known as KDR, CD309, FLK1, kinase insert domain receptor) and their medical uses. [Background technology]

[0004] PD1 is a cell surface receptor that has been shown to be an immune "checkpoint" mediator. PD1 checkpoint activity minimizes autoimmunity risk by promoting apoptosis (programmed cell death) in lymph node-resident T cells reactive to self-antigens and facilitating the survival of regulatory (anti-inflammatory) T cells. Antagonism of PD1 activity with human or humanized monoclonal antibodies has proven to be a successful therapeutic approach for the treatment of multiple forms of cancer, potentially leading to T cell reactivation in tumors. This clinical success has led to a proliferation of anti-PD1 antibody molecules being investigated in clinical trials, although the majority of patients have yet to mount a sustained anti-cancer response when treated with anti-PD1 agents alone.

[0005] Increasing the number of patients who respond to anti-PD1 antibody therapy remains a major challenge. A potential strategy to improve clinical response rates to anti-PD1 antibodies is to combine them with previously proven cancer therapies, such as antiangiogenic agents. One such drug class is antibodies that block the VEGF signaling pathway, such as anti-VEGFR2 or anti-VEGF antibodies. However, such combination therapy requires the use of two separate, costly drugs, each with its own toxicity risks. There remains a need for a single drug that offers the benefits of combination therapy without the disadvantages of combining two separate drugs. Summary of the Invention

[0006] The present invention provides several anti-PD1 and anti-VEGFR2 dual antagonistic antibodies and their medical uses.

[0007] According to one aspect of the present invention, there is provided an antibody molecule that specifically binds to human PD1 and human VEGFR2, and optionally also specifically binds to cynomolgus PD1 and cynomolgus VEGFR2, or antigen-binding portions thereof.

[0008] In some aspects, the invention provides antibodies that specifically bind to both PD1 and VEGFR2, or antigen-binding portions of antibodies, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; (a) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (b) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (c) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 8) of LASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (d) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVYSIPWT; (e) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVYSIPWT; (f) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 8) of LASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVYSIPWT; (g) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 9) of LASQTIGTWLT, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG; (h) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 9) of LASQTIGTWLT, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (i) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG; (j) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG; (k) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 12) of LASQESGIWLG, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG; (l) The VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (m) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (n) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (o) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 40) of TASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (p) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 41) of AASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (q) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (r) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 40) of TASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (s) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 41) of AASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; or (t) The VH region amino acid sequence includes HCDR1 (sequence number 1) of GFTFSSYMMS, HCDR2 (sequence number 42) of TISGGGSNKYYVDSVKG, and HCDR3 (sequence number 64) of QVYYFDY, and the VL region amino acid sequence includes LCDR1 (sequence number 39) of RASQESGIWLS, LCDR2 (sequence number 43) of AASSLQS, and LCDR3 (sequence number 11) of QQVSVTPFT.

[0009] In some aspects, an anti-PD1 antibody, or antigen-binding portion thereof, is disclosed herein, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; The VH region amino acid sequence is (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) comprises an HCDR3 of SEQ ID NO: 3 or SEQ ID NO: 64; The VL region amino acid sequence is (a') 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; (b') LCDR2 of SEQ ID NO: 5, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 43, and (c') Contains LCDR3 of SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 11.

[0010] In some aspects, disclosed herein is an antibody or antigen-binding portion of an antibody that specifically binds to both PD1 and VEGFR2, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; (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: Including 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.

[0011] In some embodiments of the invention, HCDR1 of the antibody molecule or antigen-binding portion may exclude the sequence GFTFSSYMMS (SEQ ID NO: 1; MAb005 murine / humanized antibody HCDR1 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or HCDR2 of the antibody molecule or antigen-binding portion may exclude the sequence TISGGGANTYYPDSVKG (SEQ ID NO: 2; MAb005 murine / humanized antibody HCDR2 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or HCDR3 of the antibody molecule or antigen-binding portion may exclude the sequence QLYYFDY (SEQ ID NO: 3; MAb005 murine / humanized antibody HCDR3 disclosed in WO2015 / 085847A1, US2016 / 376367A1).

[0012] In some embodiments of the invention, LCDR1 of the antibody molecule or antigen-binding portion may exclude the sequence LASQTIGTWLT (SEQ ID NO: 9; MAb005 murine / humanized antibody LCDR1 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or LCDR2 of the antibody molecule or antigen-binding portion may exclude the sequence TATSLAD (SEQ ID NO: 5; MAb005 murine / humanized antibody LCDR2 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or LCDR3 of the antibody molecule or antigen-binding portion may exclude the sequence QQVYSIPWT (SEQ ID NO: 6; MAb005 murine / humanized antibody LCDR3 disclosed in WO2015 / 085847A1, US2016 / 376367A1).

[0013] Also provided in accordance with the present invention is an immunoconjugate comprising an antibody molecule, or antigen-binding portion thereof, as defined herein linked, fused or conjugated to a therapeutic agent.

[0014] In another aspect, the present invention provides a nucleic acid molecule encoding an antibody molecule, or an antigen-binding portion thereof, as defined herein. Further provided are vectors comprising the nucleic acid molecules of the invention.

[0015] Also provided is a host cell comprising a nucleic acid molecule or vector of the invention as defined herein.

[0016] In a further aspect, methods of producing an anti-PD1 / VEGFR2 antibody and / or antigen-binding portion thereof are provided, comprising culturing a host cell of the invention under conditions that result in expression and / or production of the antibody and / or antigen-binding portion thereof, and isolating the antibody and / or antigen-binding portion thereof from the host cell or culture.

[0017] In another aspect of the present invention there is provided a pharmaceutical composition comprising an antibody molecule of the invention as defined herein or an antigen-binding portion thereof, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein.

[0018] Furthermore, an effective amount of an antibody molecule of the invention as defined herein or an antigen-binding portion thereof, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a A method of enhancing an immune response in a subject is provided, comprising administering a pharmaceutical composition of the invention.

[0019] In a further aspect, there is provided a method for treating or preventing cancer in a subject comprising administering an effective amount of an antibody molecule of the invention as defined herein or an antigen-binding portion thereof, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0020] Further provided herein is an antibody molecule or antigen-binding portion thereof as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein, for use as a medicament. The present invention also provides an antibody molecule of the invention or an antigen-binding portion thereof as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, for use in the treatment of cancer.

[0021] In another aspect, the invention provides an antibody molecule or antigen-binding portion thereof or immunoconjugate or nucleic acid molecule or vector for use or a method of treatment of the invention as defined herein for separate, sequential or simultaneous use in combination with a second therapeutic agent, e.g., an anti-cancer agent.

[0022] In a further aspect, the present invention provides the use of an antibody molecule of the invention as defined herein or an antigen-binding portion thereof, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, in the manufacture of a medicament for use in the treatment of cancer.

[0023] The present invention also provides a method of treating or preventing an infectious disease in a subject comprising administering an effective amount of an antibody molecule or antigen-binding portion thereof as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein.

[0024] The infectious disease, in all aspects, can be selected from the group consisting of viral, bacterial, fungal or parasitic, hi one embodiment, the infectious disease is human immunodeficiency virus (HIV) infection.

[0025] Also provided is an antibody molecule or antigen-binding portion thereof as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein for use in the treatment of an infectious disease.

[0026] Also provided is an antibody molecule or antigen-binding portion thereof as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein in the manufacture of a medicament for use in the treatment of an infectious disease.

[0027] The invention comprises administering an effective amount of an antibody molecule or antigen-binding portion thereof as defined herein, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition as defined herein. Also provided are methods for treating or preventing infectious diseases in a subject.

[0028] The present invention also provides a method for producing an antibody molecule that specifically binds to human PD1 and human VEGFR2, and optionally also specifically binds to cynomolgus monkey PD1 and monkey VEGFR2, or an antigen-binding portion thereof, comprising: (1) grafting anti-PD1 CDRs from a non-human source onto a human v-domain framework to produce a humanized anti-PD1 antibody molecule, or antigen-binding portion thereof; (2) generating a library of clones of humanized anti-PD1 antibody molecules, or antigen-binding portions thereof, containing one or more mutations in the CDRs; (3) screening the library for antibodies that bind to human PD1 and human VEGFR2, and optionally also bind to cynomolgus monkey PD1 and rhesus monkey VEGFR2; (4) selecting clones from screening step (3) that have binding specificity for human PD1 and human VEGFR2, and optionally also have binding specificity for cynomolgus monkey PD1 and rhesus monkey VEGFR2; and (5) producing, from the clones selected from step (4), an antibody molecule or antigen-binding portion thereof that specifically binds to human PD1 and human VEGFR2, and optionally also specifically binds to cynomolgus monkey PD1 and rhesus monkey VEGFR2.

[0029] The method may include the further step of producing additional clones based on the clones selected in step (4), e.g., based on further exploratory mutagenesis at specific positions in the CDRs of the clones selected in step (4), to enhance humanization and / or minimize the amount of human T-cell epitopes and / or improve the manufacturing characteristics of the antibody molecule or antigen-binding portion thereof produced in step (5). [Brief explanation of the drawings]

[0030] [Figure 1]Figure 1. Direct binding ELISA of library-derived anti-PD1 Fabs against human and cynomolgus PD1 and human and rhesus VEGFR2 proteins. Clones were derived from multiple phage library selection branches, in which phage populations were selected on biotinylated target proteins in each round. After each round of selection, library-derived clones (black circles) were screened as periplasmically expressed Fab proteins against human (hu) and cynomolgus (cy) PD1 and human (hu) and rhesus (rh) VEGFR2. hMAb005 v-domain expressed as a human IgG1 Fab was used as a positive control on each plate (gray diamonds). [Figure 2] Figure 2A-2B. Epitope competition analysis of Fab proteins in AlphaScreen. Anti-PD1 clones were expressed as Fabs in E. coli, and peripreps were applied to epitope competition assays using AlphaScreen technology. In this assay, library-derived Fabs were analyzed for relative affinity and retention of the parent hMAb005 epitope by competing for hMAb005 IgG1 Null binding to human PD1 protein in solution. [Figure 3A] Figures 3A-3B. Direct titration ELISA for purified IgG1 null binding to human and cynomolgus PD1-Fc, human and rhesus VEGFR2 proteins. hMab005 in human IgG1 null format, isotype control IgG1, and library-derived clones were titrated (in nM) in direct binding ELISA against human and cynomolgus PD1 proteins (Figure 3A) and human and rhesus VEGFR2 proteins (Figure 3B). [Figure 3B] Same as above. [Figure 4]Figure 4. Cell-based VEGFR2 antagonism assay. Human IgG1 null format hMab005, isotype control IgG1, and library-derived clones were titrated (in nM) in a human VEGFR2 signaling assay, and human VEGF-165 protein was added to induce VEGFR2 signaling. Neither hMab005 nor the isotype IgG1 control protein demonstrated any concentration-dependent VEGFR2 antagonism. All 11 lead clones and the positive control anti-VEGFR2 IgG1 ramucirumab demonstrated potent antagonism in the nM range. [Figure 5A] Figures 5A-5K. Cell-based VEGFR2 antagonism assay—single clone analysis. VEGFR2 antagonism of the lead clones shown in Figure 4 was reanalyzed on a clone-by-clone basis. While all 11 lead clones exhibited potent antagonism, clones derived from the library with mutations found in only one CDR per clone (Figures 5A-5E) were found to be less potent than clones with combined mutations in both LCDR1 and LCDR3 (Figures 5F-5K). [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 5G] Same as above. [Figure 5H] Same as above. [Figure 5I] Same as above. [Figure 5J] Same as above. [Figure 5K] Same as above. [Figure 6]Figure 6. Cell-based PD1 antagonism assay. hMab005 (SHR-1210 IgG1-3M), isotype control IgG1, nivolumab IgG4, and clones MAB06.1-MAB06.8 (human IgG1 null format) were titrated in a human PD1 signaling assay where human PD1+ cells and human PD-L1+ cells were mixed, and antagonism of the PD1 / PD-L1 interaction resulted in increased signal. The isotype IgG1 control protein did not demonstrate any concentration-dependent PD1 antagonism. Clone MAB06.1-MAB06.8, hMab005, and positive control anti-PD1 IgG4 nivolumab demonstrated potent antagonism in the nM range. [Figure 7] Figure 7. Cell-based VEGFR2 antagonism assay. IgG1 hMab005 (SHR-1210 IgG1-3M), isotype control IgG1, nivolumab IgG4, and clones MAB06.1-MAB06.8 (in a human IgG1 null format) were titrated in a human VEGFR2 signaling assay, and human VEGF-165 protein was added to induce VEGFR2 signaling. Neither hMab005 nor the isotype IgG1 control protein exhibited any concentration-dependent VEGFR2 antagonism. Clone MAB06.1-MAB06.8 and the positive control anti-VEGFR2 IgG1 ramucirumab exhibited potent antagonism in the nM range. [Figure 8A] Figures 8A-8H. Cell-based PD1 and VEGFR2 antagonism assay—single clone analysis. PD1 and VEGFR2 antagonism for the lead clones shown in Figure 7 was reanalyzed on a clone-by-clone basis. Comparative analysis of PD1 and VEGFR2 antagonism for clones MAB06.5 (Figures 8A-8H), MAB06.6 (Figures 8C-8D), MAB06.7 (Figures 8E-8F), and MAB06.8 (Figures 8G-8H) demonstrated that the sequence of clone MAB06 can accommodate mutations of multiple residues in LCDR1, LCDR2, and HCDR2 while retaining the ability to antagonize signaling of both receptors. [Figure 8B] Same as above. [Figure 8C]Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above. [Figure 8F] Same as above. [Figure 8G] Same as above. [Figure 8H] Same as above. [Figure 9] 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.) in tumors can express PD-L1 and / or VEGF. These signals cooperate to suppress immune function in the tumor microenvironment. Therefore, dual antagonist antibodies (on the same or different cells) that can effectively block both PD1 and VEGFR2 signaling may have improved anti-tumor capabilities over PD1- or VEGFR2-blocking antibodies alone. [Figure 10] Figure 10. Cell-based VEGFR2 agonism assay. Ramucirumab, isotype control human IgG1, MAB06.5, MAB06.8, and VEGF-165 protein were titrated in a human VEGFR2 signaling assay. Neither ramucirumab, nor the isotype or clones MAB06.5 or MAB06.8, demonstrated any concentration-dependent VEGFR2 agonism. The positive control, VEGF-165, elicited potent antagonism in the nM range. [Figure 11A] Figures 11A-11F. Unidirectional human DC:T cell mixed lymphocyte reaction (MLR) assay. MAB06.5, MAB06.8, and nivolumab (anti-PD1) were titrated (nM) in a human MLR assay using three separate human donor pairs in duplicate runs. MAB6.5 and MAB6.8 demonstrated a concentration-dependent ability to drive PD1 blockade-driven IFN-γ signaling, comparable to nivolumab. This was found in both runs for donor pair 1 (Figures 11A and 11B), donor pair 2 (Figures 11C and 11D), and donor pair 3 (Figures 11E and 11F). [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 11F] Same as above. [Figure 12A] 12A-12D Direct titration ELISA for purified IgG1 null binding to human and cynomolgus PD1-Fc, human and cynomolgus VEGFR2 proteins. SHR-1210 IgG1-3M, isotype control IgG1, and seven third-generation clones in human IgG1 null format were titrated (in nM) in a direct binding ELISA against human and cynomolgus PD1 proteins (FIGS. 12A, 12B) and human and cynomolgus VEGFR2 proteins (FIGS. 12C, 12D). [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 13A] Figures 13A-B: Cell-based PD1 and VEGFR2 antagonism assay. Comparative analysis of PD1 (Figure 13A) and VEGFR2 (Figure 13B) antagonism for clone SHR-1210 IgG1-3M, nivolumab, isotype control IgG1, and seven third-generation clones in a human IgG1 null format. [Figure 13B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention is based on the discovery of an antibody that unexpectedly antagonizes 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 has been shown to not only promote angiogenesis but also enhance immunosuppression in the tumor microenvironment, and VEGFR2 is expressed on multiple immune cell types. However, the generation of a dual inhibitor antibody is challenging due to its dual targeting. The generation of a single antibody with potent regulation of proteins such as PD1 and VEGFR2, which differ significantly in amino acid sequence and structure, is a complex and unpredictable challenge. This dissimilarity in sequence and structure between the two targets precludes successful generation of such an antibody.

[0032] WO2015 / 085847A1 names an antagonistic murine anti-PD1 IgG molecule "MAb005" and also describes the preparation of a humanized form of MAb005. Humanized MAb005 (hMAb005) was shown to have a very rare side effect of inducing hemangiomas in clinical trials. In vitro studies successfully traced this issue back to off-target antibody reactivity, including binding to VEGFR2 (Finlay et al. (2019) mAbs, 11:1, 26-44). Importantly, binding of hMAb005 (also known as SHR-1210) to VEGFR2 was shown to lead to potent agonism (activation) of the receptor, which is likely the primary cause of the hemangiomas side effect. For the reasons discussed above, the humanized form of MAb005 described in WO2015 / 085847A1 is not ideal. Furthermore, it is not possible to predict a priori that changes in molecular properties can convert an antagonist / agonistic antibody, such as Mab005, into a dual antagonist of both the PD1 and VEGFR2 pathways. The present invention provides antibodies that maintain potent PD1 antagonism but also potently antagonize VEGFR2. Such antibodies may provide synergistic clinical benefits.

[0033] Provided herein are methods for generating antibodies derived from hMAb005 that specifically bind to PD1 and antagonize VEGF-VEGFR2 signaling. For example, such antibodies can be generated by introducing mutations into one or more CDR sequences of antibody hMAb005. In some embodiments, mutations are introduced into the LCDR1 and / or LCDR2 and / or HCDR2 sequences of antibody hMAb005.

[0034] In some aspects, an antibody that specifically binds to both human PD1 and human VEGFR2 or an antigen-binding portion thereof is provided. In some embodiments, the antibody or antigen-binding portion thereof can antagonize both the PD1-PDL1 signaling pathway and the VEGFR2-VEGF signaling pathway. In some embodiments, the antibody or antigen-binding portion thereof antagonizes the binding of human PD1 to human PD-L1 and antagonizes human VEGFR2 signaling in response to human VEGF.

[0035] In some embodiments, the anti-PD1 and anti-VEGFR2 antibodies or antigen-binding portions provided herein specifically bind to a PD1 protein comprising, or consisting of, SEQ ID NO: 32 or SEQ ID NO: 33. In some embodiments, the anti-PD1 and anti-VEGFR2 antibodies or antigen-binding portions provided herein specifically bind to a PD1 protein 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 SEQ ID NO: 32 or SEQ ID NO: 33.

[0036] In some embodiments, the anti-PD1 and anti-VEGFR2 antibodies or antigen-binding portions provided herein specifically bind to a VEGFR2 protein comprising or consisting of SEQ ID NO: 34 or SEQ ID NO: 35. In some embodiments, the anti-PD1 and anti-VEGFR2 antibodies or antigen-binding portions provided herein specifically bind to a VEGFR2 protein 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 SEQ ID NO: 34 or SEQ ID NO: 35.

[0037] In some embodiments of the invention, HCDR1 of the antibody molecule or antigen-binding portion may exclude the sequence GFTFSSYMMS (SEQ ID NO: 1; MAb005 murine / humanized antibody HCDR1 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or HCDR2 of the antibody molecule or antigen-binding portion may exclude the sequence TISGGGANTYYPDSVKG (SEQ ID NO: 2; MAb005 murine / humanized antibody HCDR2 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or HCDR3 of the antibody molecule or antigen-binding portion may exclude the sequence QLYYFDY (SEQ ID NO: 3; MAb005 murine / humanized antibody HCDR3 disclosed in WO2015 / 085847A1, US2016 / 376367A1).

[0038] In some embodiments of the invention, LCDR1 of the antibody molecule or antigen-binding portion may exclude the sequence LASQTIGTWLT (SEQ ID NO: 9; MAb005 murine / humanized antibody LCDR1 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or LCDR2 of the antibody molecule or antigen-binding portion may exclude the sequence TATSLAD (SEQ ID NO: 5; MAb005 murine / humanized antibody LCDR2 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or LCDR3 of the antibody molecule or antigen-binding portion may exclude the sequence QQVYSIPWT (SEQ ID NO: 6; MAb005 murine / humanized antibody LCDR3 disclosed in WO2015 / 085847A1, US2016 / 376367A1).

[0039] In some embodiments, the anti-PD1 antibodies of the present invention are selected to have equivalent binding specificity and affinity to both human PD1 and cynomolgus monkey PD1 (to facilitate maximal accuracy in primate toxicity and pharmacokinetic studies). Further refinement of the optimized antibody molecules described herein resulted in improved binding to the cynomolgus monkey orthologue of PD1 and / or maintained or improved potency in neutralizing PD1 / PD-L1 signaling. Crucially, these antibodies dramatically improved potential clinical activity and reduced risk of hemangioma induction compared to MAb005 (WO2015 / 085847A1; US2016 / 376367A1) by becoming potent antagonists of the human receptor VEGFR2.

[0040] In some aspects, the anti-PD1 and anti-VEGFR2 antibody molecules of the invention do not necessarily have the maximum number of human germline substitutions at the corresponding murine CDR or other (such as framework) amino acid positions. In some embodiments, a "maximally humanized" antibody molecule is not necessarily "maximally optimized" with respect to anti-PD1 or anti-VEGFR2 binding properties and / or other desirable characteristics.

[0041] The present invention encompasses modifications to the amino acid sequence of an antibody molecule or antigen-binding portion thereof as defined herein. For example, the present invention includes antibody molecules and their corresponding antigen-binding portions, including functionally equivalent variable regions and CDRs that do not significantly affect their properties, as well as variants with increased or decreased activity and / or affinity. For example, the amino acid sequence can be mutated to obtain an antibody with the desired binding affinity for PD1 and VEGFR2. Amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as insertions, including intrasequence insertions of single or multiple amino acid residues, are contemplated. Examples of terminal insertions include antibody molecules with an N-terminal methionyl residue or antibody molecules fused to an epitope tag. Other insertional variants of antibody molecules include fusions to the N- or C-terminus of the antibody of enzymes or polypeptides that increase the half-life of the antibody in the blood circulation.

[0042] Antibody molecules or antigen-binding portions of the invention may include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Antibody molecules or antigen-binding portions of the invention can be mutated to alter such post-translational modifications, for example, by adding, removing, or substituting one or more amino acid residues to create or remove glycosylation sites.

[0043] The antibody molecules or antigen-binding portions of the invention can be modified, for example, by amino acid substitutions to remove potential proteolytic sites in the antibody.

[0044] In some aspects of the invention, there is provided an antibody or antigen-binding portion of an antibody that binds to both PD1 and VEGFR2, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; (a) The VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVYSIPWT. [MAB02A03] (b) The VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVYSIPWT [MAB02B03] (c) The VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes LCDR1 (SEQ ID NO: 8) of LASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVSVTPFT [MAB02D08] (d) The VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes LCDR1 (SEQ ID NO: 9) of LASQTIGTWLT, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG. [MAB05G03] (e) The VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes LCDR1 (SEQ ID NO: 9) of LASQTIGTWLT, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT [MAB05E08] (f) The VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG. [MAB01] (g) The VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG. [MAB02] (h) The amino acid sequence of the VH region is: HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and QLYYFDY and the VL region amino acid sequence includes LCDR1 (SEQ ID NO: 12) of LASQESGIWLG, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG. [MAB03] (i) The VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT. [MAB04] (j) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB05], or (k) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 8) of LASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06], or (l) The VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.1] or (m) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 40) of TASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.2], or (n) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 41) of AASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.3] or (o) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.4] or (p) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.5] or (q) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 40) of TASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.6] or (r) The amino acid sequence of the VH region is: HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and QLYYFD Y HCDR3 (SEQ ID NO: 3), and the VL region amino acid sequence includes LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 41) of AASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.7] or (s) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.8] or (t) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 61) of GFTFSSYLMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.8.1] or (u) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 61) of GFTFSSYLMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 64) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.8.2] or (v) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 61) of GFTFSSYLMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 65) of QLYGFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.8.3], or (w) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 61) of GFTFSSYLMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 66) of QLYYADY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.8.4] or (x) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 61) of GFTFSSYLMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 62) of QLYFFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.8.5], or (y) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 61) of GFTFSSYLMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 63) of QLYYYDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; [MAB06.8.6]; or (z) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 64) of QVYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.8.7] or (aa) The VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 66) of QLYYADY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, [MAB06.8.8] or (y) The VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 63) of QLYYYDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT. [MAB06.8.9] In some aspects, disclosed herein are anti-PD1 antibodies and anti-VEGFR2 antibodies, or antigen-binding portions thereof, wherein the antibodies comprise a heavy chain variable (VH) region and a light chain variable (VL) region, and the VH region amino acid sequence comprises or consists of EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSS (SEQ ID NO: 13).

[0045] In some aspects, anti-PD1 antibodies and anti-VEGFR2 antibodies, or antigen-binding portions thereof, disclosed herein, wherein the antibodies comprise a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises or consists of SEQ ID NO: 13, and the VL region comprises or consists of any one of the VL region amino acid sequences in Table 5 or Table 8.

[0046] In some aspects, disclosed herein is an antibody or antigen-binding portion of an antibody that specifically binds to both PD1 and VEGFR2, wherein the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; (a) the VH region amino acid sequence comprises SEQ ID NO: 13 and the VL region amino acid sequence comprises SEQ ID NO: 14; (b) the VH region amino acid sequence comprises SEQ ID NO: 13 and the VL region amino acid sequence comprises SEQ ID NO: 15; (c) the VH region amino acid sequence comprises SEQ ID NO: 13 and the VL region amino acid sequence comprises SEQ ID NO: 16; (d) the VH region amino acid sequence comprises SEQ ID NO: 13 and the VL region amino acid sequence comprises SEQ ID NO: 17; (e) the VH region amino acid sequence comprises SEQ ID NO: 13 and the VL region amino acid sequence comprises SEQ ID NO: 18; (f) the VH region amino acid sequence comprises SEQ ID NO: 13 and the VL region amino acid sequence comprises SEQ ID NO: 19; (g) the VH region amino acid sequence comprises SEQ ID NO: 13 and the VL region amino acid sequence comprises SEQ ID NO: 20; (h) the VH region amino acid sequence comprises SEQ ID NO: 13 and the VL region amino acid sequence comprises SEQ ID NO: 21; (i) the VH region amino acid sequence comprises SEQ ID NO: 13 and the VL region amino acid sequence comprises SEQ ID NO: 22; (j) the VH region amino acid sequence comprises SEQ ID NO: 13 and the VL region amino acid sequence comprises SEQ ID NO: 23; (k) the VH region amino acid sequence comprises SEQ ID NO: 13 and the VL region amino acid sequence comprises SEQ ID NO: 24; (l) the VH region amino acid sequence comprises SEQ ID NO: 48 and the VL region amino acid sequence comprises SEQ ID NO: 44; (m) the VH region amino acid sequence comprises SEQ ID NO: 48 and the VL region amino acid sequence comprises SEQ ID NO: 45; (n) the VH region amino acid sequence comprises SEQ ID NO: 48 and the VL region amino acid sequence comprises SEQ ID NO: 46; (o) the VH region amino acid sequence comprises SEQ ID NO: 48 and the VL region amino acid sequence comprises SEQ ID NO: 47; (p) the VH region amino acid sequence comprises SEQ ID NO: 49 and the VL region amino acid sequence comprises SEQ ID NO: 44; (q) the VH region amino acid sequence comprises SEQ ID NO: 49 and the VL region amino acid sequence comprises SEQ ID NO: 45; (r) the VH region amino acid sequence comprises SEQ ID NO: 49 and the VL region amino acid sequence comprises SEQ ID NO: 46; (s) the VH region amino acid sequence comprises SEQ ID NO: 49 and the VL region amino acid sequence comprises SEQ ID NO: 47; (t) the VH region amino acid sequence comprises SEQ ID NO: 67 and the VL region amino acid sequence comprises SEQ ID NO: 47; (u) the VH region amino acid sequence comprises SEQ ID NO: 68 and the VL region amino acid sequence comprises SEQ ID NO: 47; (v) the VH region amino acid sequence comprises SEQ ID NO: 69 and the VL region amino acid sequence comprises SEQ ID NO: 47; (w) the VH region amino acid sequence comprises SEQ ID NO: 70 and the VL region amino acid sequence comprises SEQ ID NO: 47; (x) the VH region amino acid sequence comprises SEQ ID NO: 71 and the VL region amino acid sequence comprises SEQ ID NO: 47; (y) the VH region amino acid sequence comprises SEQ ID NO: 72 and the VL region amino acid sequence comprises SEQ ID NO: 47; (z) the VH region amino acid sequence comprises SEQ ID NO: 73 and the VL region amino acid sequence comprises SEQ ID NO: 47; (aa) the VH region amino acid sequence comprises SEQ ID NO: 74 and the VL region amino acid sequence comprises SEQ ID NO: 47; or (bb) the VH region amino acid sequence comprises SEQ ID NO:75 and the VL region amino acid sequence comprises SEQ ID NO:47.

[0047] In some aspects, disclosed herein are anti-PD1 and anti-VEGFR2 antibodies, or antigen-binding portions thereof, wherein the antibodies comprise a heavy chain variable (VH) region and a light chain variable (VL) region; (a) the VH region amino acid sequence 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 VL region amino acid sequence 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 VH region amino acid sequence 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 VL region amino acid sequence 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: 15; (c) the VH region amino acid sequence is at least about 90% identical to SEQ ID NO: 13, 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, and the VL region amino acid sequence 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 VH region amino acid sequence 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 VL region amino acid sequence 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: 17; (e) the VH region amino acid sequence 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 VL region amino acid sequence 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 VH region amino acid sequence 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 VL region amino acid sequence 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 VH region amino acid sequence 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 VL region amino acid sequence 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: 20; (h) the VH region amino acid sequence 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 VL region amino acid sequence 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: 21; (i) the VH region amino acid sequence 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 VL region amino acid sequence 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 VH region amino acid sequence 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%, at least about 99%, at least about 100%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 11 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO: 23, and the VL region amino acid sequence 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: 23, (k) the VH region amino acid sequence 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 VL region amino acid sequence 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: 24; (l) the VH region amino acid sequence 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 VL region amino acid sequence 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 VH region amino acid sequence 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 VL region amino acid sequence 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; (n) the VH region amino acid sequence 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 VL region amino acid sequence 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; (o) the VH region amino acid sequence 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 VL region amino acid sequence 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:47; (p) the VH region amino acid sequence 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 VL region amino acid sequence 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; (q) the VH region amino acid sequence 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 VL region amino acid sequence 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 VH region amino acid sequence 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 VL region amino acid sequence 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; (s) the VH region amino acid sequence 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 VL region amino acid sequence 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: 47. In some embodiments, the CDR amino acid sequences of the anti-PD1 antibody and the anti-VEGFR2 antibody are 100% identical to the CDR amino acid sequences of the listed sequences, while the FR amino acid sequences are less than 100% identical to the FR amino acid sequences of the listed sequences.

[0048] In some aspects, an antibody or antigen-binding portion as defined herein may be isolated.

[0049] The antibody molecules or antigen-binding portions thereof defined herein can cross-compete for binding to PD1 and VEGFR2 with an antibody or antigen-binding portion thereof comprising a set of CDRs disclosed herein. In some embodiments, the invention provides anti-PD1 antibodies and anti-VEGFR2 antibodies or antigen-binding portions thereof, wherein the antibodies or antigen-binding portions cross-compete for binding to PD1 and VEGFR2 with an antibody or antigen-binding portion thereof comprising a set of CDRs disclosed herein, (a) comprise a complete germline human framework amino acid sequence, (b) specifically bind to human PD1, cynomolgus monkey PD1, human VEGFR2, and rhesus monkey VEGFR2, and (c) antagonize the binding of human PD1 to human PD-L1 and antagonize human VEGFR2 signaling in response to human VEGF.

[0050] In some embodiments, the anti-PD1 and anti-VEGFR2 antibodies or antigen-binding portions have minimal immunogenicity. In certain cases, the antibodies or antigen-binding portions exhibit reduced immunogenicity compared to an anti-PD1 antibody comprising an HCDR1 of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, an HCDR of SEQ ID NO: 3, an LCDR1 of SEQ ID NO: 9, an LCDR2 of SEQ ID NO: 5, and an LCDR3 of SEQ ID NO: 6. In some examples, the immunogenicity risk of an antibody or antigen-binding portion may be determined in silico by identifying T cell epitopes of the antibody or portion (e.g., in the variable regions of the antibody or portion).

[0051] For example, T cell epitopes of antibodies or antigen-binding moieties can be identified by using iTope™. iTope™ can be used to analyze the VL and VH region sequences of peptides with promiscuous, high-affinity binding to human MHC class II. Promiscuous, high-affinity MHC class II-binding peptides are thought to correlate with the presence of T cell epitopes, which are indicative of a high risk of clinical immunogenicity for drug proteins. iTope™ software identifies the amino acid side chains of peptides within the open-ended binding groove of 34 human MHC class II alleles and the specific binding pocket (particularly the pocket position). The study predicts favorable interactions between HLA-DR alleles (p1, p4, p6, p7, and p9). These alleles represent the most common HLA-DR alleles found worldwide, with no weighting attributed to their prevalence in any particular ethnic group. Twenty alleles contain the "open" p1 configuration and 14 alleles contain the "closed" configuration, in which glycine at position 83 is replaced with valine. Location of key binding residues is achieved by in silico generation of 9-mer peptides overlapping by eight amino acids across the test protein sequence. This process successfully distinguishes between peptides that do and do not bind to MHC class II molecules with high accuracy.

[0052] T cell epitopes of an antibody or antigen-binding portion can be identified by analyzing the VL and VH region sequences using TCED™ (T Cell Epitope Database™) to search for matches to T cell epitopes previously identified by in vitro human T cell epitope mapping analysis of other protein sequences. TCED™ is used to search any test sequence against a large (>10,000 peptides) database of peptides derived from unrelated protein and antibody sequences.

[0053] In some embodiments, the anti-PD1 and anti-VEGFR2 antibodies or antigen-binding portions may exhibit reduced immunogenicity because the antibodies or portions have a low number of one or more of the following peptides in their sequence: high affinity foreign ("HAF" - high immunogenic risk), low affinity foreign ("LAF" - low immunogenic risk), and / or TCED+ (epitopes previously identified in the TCED™ database).

[0054] In some embodiments, anti-PD1 antibodies and anti-VEGFR2 antibodies or antigen-binding portions thereof may have a high content of germline epitopes (GE) in their sequences. In some examples, anti-PD1 antibodies or antigen-binding portions thereof have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 (or more than 20) germline epitopes in their sequences (e.g., VL and / or VH region sequences). Germline epitopes may be defined as human germline peptide sequences with high MHC class II binding affinity. Germline epitope 9-mer peptides are unlikely to be immunogenic due to T cell tolerance, as has been verified in previous studies using a wide range of germline peptides. Importantly, such germline v-domain epitopes (further supported by similar sequences within human antibody constant regions) compete for MHC class II occupancy at the membrane of antigen-presenting cells, reducing the risk of exogenous peptide presentation sufficient to achieve the "activation threshold" required for T cell stimulation. High GE content is therefore a beneficial quality in the clinical development of antibody therapeutics, as it can provide low immunogenicity.

[0055] In certain embodiments, the anti-PD1 and anti-VEGFR2 antibodies or antigen-binding portions may have a reduced number of HAF, LAF, and / or TCED+ epitopes found in the frameworks of both the heavy and light chain variable regions compared to an anti-PD1 antibody comprising the variable domain sequence of antibody Mab005 (Tables 1 and 2). In some embodiments, the HAF, LAF, and / or TCED+ epitopes are absent from the VL and / or VH region sequences of the anti-PD1 antibody or antigen-binding portion.

[0056] As used herein, the terms "cross-compete," "cross-compete," "cross-block," "cross-blocked," and "cross-blocking" are used interchangeably to refer to the ability of an antibody or portion thereof to directly or indirectly inhibit the binding of the anti-PD1 and anti-VEGFR2 antibodies of the invention to their targets PD1 and VEGFR2 (e.g., human PD1, human VEGFR2) through allosteric modulation. The extent to which an antibody or portion thereof can interfere with the binding of another antibody or portion thereof to its target is therefore within the scope of the present invention. Whether or not cross-blocking or cross-competition can be described as specifically described can be determined using competitive binding assays. One example of a binding competition assay is Homogeneous Time-Resolved Fluorescence (HTRF). One particularly suitable quantitative cross-competition assay uses a FACS or AlphaScreen-based approach to measure competition between a labeled (e.g., His-tagged, biotinylated, or radiolabeled) antibody or portion thereof and another antibody or portion thereof for binding to a target. Generally, a cross-competing antibody or portion thereof is a cross-competing antibody or portion thereof that binds to a target in a cross-competition assay such that, for example, in the presence of a second antibody or portion thereof during the assay, the recorded displacement of an immunoglobulin single variable domain described in the present invention is up to 100% (e.g., in a FACS-based competition assay) of the theoretical maximum displacement (e.g., of a cold (e.g., unlabeled) antibody or fragment thereof that must be blocked) by a potentially cross-competing antibody or fragment thereof present in a given amount. Preferably, a cross-competing antibody or portion thereof has a recorded displacement that is between 10% and 100%, or between 50% and 100%.

[0057] The antibody molecules or antigen-binding portions defined herein may comprise one or more substitutions, deletions and / or insertions that remove post-translational modification (PTM) sites, such as glycosylation sites (N-linked or O-linked), deamination sites, phosphorylation sites or isomerization / fragmentation sites.

[0058] More than 350 types of PTMs are known. Major forms of PTMs include phosphorylation, glycosylation (N-linked and O-linked), sumoylation, palmitoylation, acetylation, sulfation, myristoylation, prenylation, and methylation (of K and R residues). Statistical methods for identifying putative amino acid sites involved in specific PTMs are well known in the art (see Zhou et al., 2016, Nature Protocols 1:1318-1321). It is contemplated to remove these sites by substitution, deletion, and / or insertion and optionally test (experimentally and / or theoretically) for (a) binding activity and / or (b) loss of PTM.

[0059] The antibody molecule or antigen-binding portion thereof may be human, humanized or chimeric.

[0060] An antibody molecule, or antigen-binding portion thereof, can comprise one or more human variable domain framework scaffolds into which the CDRs are inserted. For example, the VH region, the VL region, or both the VH and VL regions can comprise one or more human framework region amino acid sequences.

[0061] The antibody molecule, or antigen-binding portion thereof, may comprise an IGHV3-7 human germline scaffold into which the corresponding HCDR sequences have been inserted. The antibody molecule, or antigen-binding portion thereof, may comprise a VH region comprising an IGHV3-7 human germline scaffold amino acid sequence into which the corresponding set of HCDR1, HCDR2 and HCDR3 amino acid sequences have been inserted.

[0062] The antibody molecule, or antigen-binding portion thereof, may comprise an IGKV1-39 human germline scaffold into which the corresponding LCDR sequences have been inserted. The antibody molecule, or antigen-binding portion thereof, may comprise a VL region comprising an IGKV1-39 human germline scaffold amino acid sequence into which the corresponding set of LCDR1, LCDR2 and LCDR3 amino acid sequences have been inserted.

[0063] The antibody molecule or antigen-binding portion thereof may comprise an IGHV3-7 human germline scaffold into which the corresponding HCDR sequences have been inserted and an IGKV1-39 human germline scaffold into which the corresponding LCDR sequences have been inserted. The antibody molecule or antigen-binding portion thereof may comprise a VH region comprising an IGHV3-7 human germline scaffold amino acid sequence into which the corresponding sets of HCDR1, HCDR2, and HCDR3 amino acid sequences have been inserted, and an IGKV1-39 human germline scaffold into which the corresponding sets of LCDR1, LCDR2, and LCDR3 amino acid sequences have been inserted. The VL region may comprise the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences are as follows: MAB02A03, MAB02B03, MAB02D08, MAB05G03, MAB05E08, MAB01, MAB02, MAB03, MAB04, MAB05, MAB06, MAB06.1, MAB06.2, MAB06.3, MAB06.4, MAB06.5, MAB06.6, MAB06 The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of any one of 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 (all six CDR sequences are derived from the same clone).

[0064] In some aspects, the antibody molecule, or antigen-binding portion thereof, may comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In additional embodiments, the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. The antibody molecule, or antigen-binding portion thereof, may comprise an immunologically inert constant region. In some aspects, the anti-PD1 antibody and anti-VEGFR2 antibody, or antigen-binding portion thereof, may comprise an immunoglobulin constant region comprising a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, and G237A, or a human IgG1 constant region comprising amino acid substitutions L234A, L235A, G237A, and P331S. In some aspects, the anti-PD1 antibody and anti-VEGFR2 antibody, or antigen-binding portion thereof, may comprise an immunoglobulin constant region comprising a wild-type human IgG2 constant region or a wild-type human IgG4 constant region. In some embodiments, anti-PD1 antibodies and anti-VEGFR2 antibodies may comprise an immunoglobulin constant region comprising any one of the amino acid sequences in Table 6. The Fc region sequences in Table 6 begin with the CH1 domain. In some embodiments, anti-PD1 antibodies and anti-VEGFR2 antibodies may comprise an immunoglobulin constant region 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, the human IgG4(S228P) Fc region comprises the following substitution compared to the wild-type human IgG4 Fc region: S228P. For example, a human IgG1-3M Fc region contains the following substitutions compared to the wild-type human IgG1 Fc region: L234A, L235A, and G237A, and a human IgG1-4M Fc region contains the following substitutions compared to the wild-type human IgG1 Fc region: L234A, L235A, G237A, and P331S. In some embodiments, the amino acid residue positions of the constant regions of immunoglobulin molecules are numbered according to the EU nomenclature system (Ward et al., 1995 Therap. Immunol. 2:77-94).In some embodiments, the immunoglobulin constant region may comprise 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 proportion of the human population than the RDELT (SEQ ID NO: 36) allotype. In some embodiments, the anti-PD1 antibody and the anti-VEGFR2 antibody may comprise an immunoglobulin constant region comprising any one of SEQ ID NOs: 25-31. In some embodiments, the anti-PD1 antibody and the anti-VEGFR2 antibody are selected from 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, MAB0 and any one of the Fc region amino acid sequences in Table 6. In some embodiments, the anti-PD1 antibody and the anti-VEGFR2 antibody comprise an immunoglobulin heavy chain constant region and a kappa light chain comprising any one of the Fc region amino acid sequences in Table 6. The constant region may comprise an immunoglobulin light chain constant region that is a lambda light chain constant region.

[0065] In some aspects, disclosed herein are anti-PD1 and anti-VEGFR2 antibodies, or antigen-binding portions thereof, wherein the antibodies comprise a heavy chain variable (VH) region and a light chain variable (VL) region and a heavy chain constant region; (a) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVYSIPWT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (b) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVYSIPWT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (c) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 8) of LASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVYSIPWT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (d) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 9) of LASQTIGTWLT, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (e) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 9) of LASQTIGTWLT, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (f) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (g) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (h) The VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LASQESGIWLG the LCDR1 (SEQ ID NO: 12) of QQVAELPFG, the LCDR2 (SEQ ID NO: 5) of TATSLAD, and the LCDR3 (SEQ ID NO: 10) of QQVAELPFG, and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (i) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (j) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (k) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 8) of LASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (l) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (m) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 40) of TASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (n) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 41) of AASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (o) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (p) The VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; and the heavy chain constant region comprises SEQ ID NO: 25 Contains one of the following: (q) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY; the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 40) of TASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (q) the VH region amino acid sequence comprises an HCDR1 of GFTSSYMMS (SEQ ID NO: 1), an HCDR2 of TISGGGSNKYYVDSVKG (SEQ ID NO: 42), and an HCDR3 of QLYYFDY (SEQ ID NO: 3); the VL region amino acid sequence comprises an LCDR1 of RASQESGIWLS (SEQ ID NO: 39), an LCDR2 of AASSLAD (SEQ ID NO: 41), and an LCDR3 of QQVSVTPFT (SEQ ID NO: 11); and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; or (s) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31. In some embodiments, the antibody further comprises an immunoglobulin light chain constant region that is a kappa light chain constant region or a lambda light chain constant region.

[0066] In some aspects, disclosed herein are anti-PD1 and anti-VEGFR2 antibodies, or antigen-binding portions thereof, wherein the antibodies comprise a heavy chain variable (VH) region and a light chain variable (VL) region and a heavy chain constant region; 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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (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 a wild-type human IgG4 constant region. a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (l) 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: 44; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (m) 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: 45; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region containing the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; and the amino acid substitutions L234A, L235A, and G a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, (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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (o) 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: 47; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (p) 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: 44; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (q) 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: 45; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, (r) 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: 46; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S; 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; the heavy chain constant region comprises a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgG2 constant region, a wild-type human IgG1 constant region; a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, and G237A; or a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S.

[0067] In some aspects, disclosed herein are anti-PD1 and anti-VEGFR2 antibodies, or antigen-binding portions thereof, wherein the antibodies comprise a heavy chain variable (VH) region and a light chain variable (VL) region and a heavy chain constant region; 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 NOs: 25 to 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 NOs: 25 to 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 of SEQ ID NOs: 25 to 31; Including any one of (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: 17, and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 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 NOs: 25 to 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 NOs: 25 to 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 NOs: 25 to 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 NOs: 25 to 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 NOs: 25 to 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 NOs: 25 to 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 NOs: 25 to 31; (l) 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: 44, and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (m) 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: 45, and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 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 NOs: 25 to 31; (o) 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: 47, and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (p) 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: 44, and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (q) 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: 45, and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 31; (r) 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: 46, and the heavy chain constant region comprises any one of SEQ ID NOs: 25 to 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 NOs: 25 to 31.

[0068] In some embodiments, the anti-PD1 and anti-VEGFR2 antibodies may be immune effector null. In some embodiments, the anti-PD1 and anti-VEGFR2 antibodies, or antigen-binding portions thereof, do not induce immune effector function, and optionally suppress immune effector function. In some embodiments, the anti-PD1 and anti-VEGFR2 antibodies lack measurable binding to human FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb receptors, but maintain binding to human FcγRIIb receptors, and optionally maintain binding to human FcRn receptors. FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb are examples of activating receptors. FcγRIIb is an example of an inhibitory receptor. FcRn is an example of a recycling receptor. In some embodiments, the binding affinity of the anti-PD1 and anti-VEGFR2 antibodies, or antigen-binding portions thereof, for human Fc receptors can be measured by BIACORE® analysis. In some embodiments, homogeneous time-resolved fluorescence (HTRF) can be used to test the binding of anti-PD1 and anti-VEGFR2 antibodies to human Fc receptors. In one example of HTRF, human IgG1 (wild-type) is labeled with a full set of Fc gamma receptors, and then antibodies with engineered Fc fragments are used in a titration competition. In some embodiments, PD1-positive cells and / or VEGFR2-positive cells can be mixed with human white blood cells and anti-PD1 and anti-VEGFR2 antibodies to measure cell killing by CDC, ADCC, and / or ADCP. In some embodiments, anti-PD1 and anti-VEGFR2 antibodies comprising the amino acid sequence of the Fc region of human IgG1-3M (see Table 6) are effector-null. In some embodiments, anti-PD1 and anti-VEGFR2 antibodies comprising the amino acid sequence of the Fc region of human IgG1-3M (see Table 6) are not effector-null.

[0069] The antibody molecule, or antigen-binding portion thereof, may be a Fab fragment, F(ab)2 fragment, Fv fragment, tetrameric antibody, tetravalent antibody, multispecific antibody (e.g., bivalent antibody), domain-specific antibody, single-domain antibody, monoclonal antibody, or fusion protein. In one embodiment, the antibody may 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 other than PD1 or VEGFR2. Antibody molecules and methods for their construction and use are described, for example, in Holliger & Hudson (2005, Nature Biotechnol. 23(9):1126-1136).

[0070] In another aspect of the present invention, there is provided an immunoconjugate comprising an antibody molecule of the invention, or an antigen-binding portion thereof, as defined herein, linked to a therapeutic agent.

[0071] Examples of suitable therapeutic agents include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulatory agents, anti-angiogenic agents, anti-proliferative agents, pro-apoptotic agents, and cytostatic and cytolytic enzymes (e.g., ribonucleases). Additional therapeutic agents include therapeutic nucleic acids, such as genes encoding immunomodulatory agents, anti-angiogenic agents, anti-proliferative agents, or pro-apoptotic agents. These drug descriptors are not mutually exclusive, and thus, a therapeutic agent may be described using one or more of the above terms.

[0072] Examples of suitable therapeutic agents for use in immunoconjugates include taxanes, maytansine, CC-1065 and duocarmycins, calicheamicins and other enediynes and auristatins. Other examples include antifolates, vinca alkaloids, and anthracyclines. Plant toxins, other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes, and photosensitizers may also be used in immunoconjugates. Additionally, conjugates can be made using secondary carriers as cytotoxic agents, such as liposomes or polymers, and suitable cytotoxins inhibit cell function. Cytotoxins include agents that disrupt or interfere with, and / or result in the destruction of, cells. Exemplary cytotoxins include antibiotics, inhibitors of tubulin polymerization, alkylating agents that bind to and damage DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins such as protein kinases, phosphatases, topoisomerases, enzymes, and cyclins.

[0073] Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclavicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluridine, pentostatin, broxafuridine, capecitabine, cladobine, decitabine, floxafuridine, fludarabine, gugelotin, puromycin, tegafur, thiazolidine, adhamycin, cisplatin, carboplatin, and cyclophosphamide. These include phenidin, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine, methotrexate, fluorouracil, etoposide, taxol, taxol analogs, platins such as cisplatin and carboplatin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, autramycin, azaserine, bleomycin, tamoxifen, idarubicin, dolastatins / auristatins, hemiasterin, esperamycin, and maytansinoids.

[0074] Suitable immunomodulatory agents include antihormonal agents that block hormone action on the tumor and immunosuppressants that suppress cytokine production, downregulate self-antigen expression, or mask MHC antigens.

[0075] Nucleic acid molecules encoding the antibody molecules of the present invention, or antigen-binding portions thereof, defined herein are also provided. The nucleic acid molecules may encode (a) a VH region amino acid sequence, (b) a VL region amino acid sequence, or (c) both the VH and VL region amino acid sequences of the anti-PD1 and anti-VEGFR2 antibodies, or antigen-binding portions thereof, described herein. In some embodiments, the nucleic acid molecules defined herein may be isolated.

[0076] Further provided is a vector comprising a nucleic acid molecule of the invention as defined herein. The vector may be an expression vector.

[0077] Also provided are host cells comprising the nucleic acid molecules or vectors of the invention defined herein. The host cells may be recombinant host cells. In some embodiments, the host cells may comprise vectors comprising nucleic acid molecules encoding both the VH region amino acid sequence and the VL region amino acid sequence of the anti-PD1 and anti-VEGFR2 antibodies, or antigen-binding portions thereof, described herein. In some embodiments, the host cells may comprise a first vector comprising a nucleic acid molecule encoding the VH region amino acid sequence and a second vector comprising a nucleic acid molecule encoding the VL region amino acid sequence of the anti-PD1 and anti-VEGFR2 antibodies, or antigen-binding portions thereof, defined herein.

[0078] In a further aspect, methods of producing anti-PD1 and anti-VEGFR2 antibodies and / or antigen-binding portions thereof are provided, comprising culturing a host cell of the invention under conditions that result in expression and / or production of the antibody and / or antigen-binding portion thereof, and isolating the antibody and / or antigen-binding portion thereof from the host cell or culture.

[0079] In another aspect of the invention, an antibody molecule or antigen-binding portion thereof of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, is provided. Pharmaceutical compositions comprising the vectors are provided.

[0080] Further provided is a method of enhancing an immune response in a subject, comprising administering to the subject an effective amount of an antibody molecule of the invention as defined herein or an antigen-binding portion thereof, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0081] In a further aspect, there is provided a method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of an antibody molecule of the invention or antigen-binding portion thereof as defined herein, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein. In some embodiments, the cancer is associated with expression or overexpression of PD1 and / or VEGFR2.

[0082] For example, the cancer may be pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial 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 or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0083] The present invention also provides an antibody molecule of the invention as defined herein or an antigen-binding portion thereof, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, for use in the treatment of cancer.

[0084] In another aspect, the invention provides an antibody molecule or antigen-binding portion thereof or immunoconjugate or nucleic acid molecule or vector for use or a method of treatment of the invention as defined herein for separate, sequential or simultaneous use in combination with a second therapeutic agent, e.g., an anti-cancer agent.

[0085] In a further aspect, the present invention provides the use of an antibody molecule of the invention as defined herein or an antigen-binding portion thereof, or an immunoconjugate of the invention as defined herein, or a nucleic acid molecule of the invention as defined herein, or a vector of the invention as defined herein, or a pharmaceutical composition of the invention as defined herein, in the manufacture of a medicament for use in the treatment of cancer.

[0086] The present invention also provides a method for treating or preventing cancer or an immune disease in a subject, comprising administering to the subject an effective amount of an antibody molecule of the invention as defined herein or an antigen-binding portion thereof, or an immunoconjugate as defined herein, or a nucleic acid molecule as defined herein, or a vector as defined herein, or a pharmaceutical composition of the invention as defined herein.

[0087] In one embodiment, the present invention provides anti-PD1 antibodies and anti-VEGFR2 antibodies, or antigen-binding portions thereof, comprising the amino acid sequences disclosed herein for use in therapy.

[0088] The pharmaceutical composition of the present invention can comprise a pharmaceutically acceptable excipient, carrier or diluent.The pharmaceutically acceptable excipient can be a compound or a combination of compounds that does not induce secondary reactions and can, for example, facilitate the administration of anti-PD1 and anti-VEGFR2 antibody molecules, increase their life span and / or increase their effectiveness in the body or increase their solubility in solution.These pharmaceutically acceptable vehicles are well known and can be used for the anti-PD1 and anti-VEGFR2 antibody molecules. It will be adapted by the skilled artisan as a function of the mode of administration of the VEGFR2 antibody molecule.

[0089] In some embodiments, the anti-PD1 antibody molecules and anti-VEGFR2 antibody molecules may be provided in lyophilized form for reconstitution prior to administration. For example, lyophilized antibody molecules may be reconstituted in sterile water and mixed with saline prior to administration to an individual.

[0090] The anti-PD1 and anti-VEGFR2 antibody molecules are typically administered in the form of a pharmaceutical composition, which may contain at least one component in addition to the antibody molecule. Thus, in addition to the anti-PD1 and anti-VEGFR2 antibody molecules, the pharmaceutical composition may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances 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 exact nature of the carrier or other material will depend on the route of administration, which may be by bolus administration, infusion, injection, or any other suitable route, as discussed below.

[0091] For parenteral administration, e.g., by subcutaneous injection or intravenous administration, e.g., by injection, pharmaceutical compositions containing anti-PD1 antibody molecules and anti-VEGFR2 antibody molecules may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity, and stability. Those skilled in the art can prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, and lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may include buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; 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 non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0092] Pharmaceutical compositions comprising the anti-PD1 antibody molecule and the anti-VEGFR2 antibody molecule can be administered alone or in combination with other therapeutic agents, either simultaneously or sequentially, depending on the condition being treated.

[0093] The anti-PD1 and anti-VEGFR2 antibody molecules described herein may be used in methods of treatment of the human or animal body, including prophylactic or preventative treatment (e.g., treating before the onset of a condition in an individual to reduce the risk of developing the condition in the individual; delaying its onset; or reducing its severity after onset). The treatment method may include administering the anti-PD1 and anti-VEGFR2 antibody molecules to an individual in need thereof.

[0094] Administration is typically in a "therapeutically effective amount," sufficient to show benefit to the patient. Such benefit may be at least an improvement in at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration, and other factors known to medical professionals. Prescribing treatment, e.g., determining dosage, etc., is within the responsibility of general practitioners and other physicians, and will depend on the symptoms and / or duration of the disease being treated. The dosage may depend on the severity of the disease or progression. Appropriate dosages 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 Radiopharmaceutics 4:915-922). Specific dosages may be provided herein or in the Physician's Desk Reference (2003) to ensure that the type of agent being administered is used appropriately. A therapeutically effective amount or appropriate dosage of an antibody molecule can be determined by comparing its in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other test animals to humans are known. The exact dosage depends on several factors, including whether the antibody is for prophylaxis or therapy, the size and location of the area to be treated, the precise nature of the antibody (e.g., whole antibody, fragment), and the nature of any detectable label or other molecule attached to the antibody.

[0095] Typical antibody doses range from 100 μg to 1 g for systemic applications and from 1 μg to 1 mg for topical applications. A higher initial loading dose may be administered, followed by one or more lower doses. Typically, the antibody is a whole antibody, e.g., an IgG1 or IgG4 isotype. This is the dose for a single treatment of an adult patient and may be adjusted proportionally for children and infants, as well as for other antibody formats based on molecular weight. Treatment may be repeated at daily, twice-weekly, weekly, or monthly intervals, at the physician's discretion. 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.

[0096] Treatment may be cyclical, with the period between administrations being about 2 weeks or more, e.g., about 3 weeks or more, about 4 weeks or more, about 1 month or more, about 5 weeks or more, or about 6 weeks or more. For example, treatment may be every 2-4 weeks or every 4-8 weeks. Treatment may be administered pre- and / or post-operatively and / or administered or applied directly to the anatomical site of surgery or invasive procedure. Suitable formulations and routes of administration are described above.

[0097] In some embodiments, the anti-PD1 and anti-VEGF antibody molecules described herein may be administered as a subcutaneous injection, which may be administered using an auto-injector, for example, for long-term prophylaxis / treatment.

[0098] In some embodiments, the therapeutic effect of the anti-PD1 and anti-VEGF antibody molecules may last for several half-lives, depending on the dose. For example, the therapeutic effect of a single administration of the anti-PD1 and anti-VEGF antibody molecules may last for one or more months, two or more months, three or more months, four or more months, five or more months, or six or more months in an individual.

[0099] The present invention also provides a method for producing an antibody molecule that specifically binds to human PD1 and human VEGFR2, and optionally also specifically binds to cynomolgus monkey PD1 and monkey VEGFR2, or an antigen-binding portion thereof, comprising: (1) grafting anti-PD1 CDRs from a non-human source onto a human v-domain framework to produce a humanized anti-PD1 antibody molecule, or antigen-binding portion thereof; (2) generating a library of clones of humanized anti-PD1 antibody molecules, or antigen-binding portions thereof, containing one or more mutations in the CDRs; (3) screening the library for binding to human PD1, and optionally cynomolgus PD1, and also for binding to human VEGFR2 and rhesus VEGFR2; (4) Screening for clones that have binding specificity for human PD1, and optionally also for cynomolgus monkey PD1 and human VEGFR2 and rhesus monkey VEGFR2. selecting from step (3); (5) producing antibody molecules that specifically bind to human PD1 and, optionally, antibody molecules that specifically bind to cynomolgus PD1, human VEGFR2, and rhesus VEGFR2, or antigen-binding portions thereof, from the clones selected from step (4); (6) conducting germline mutagenesis studies in the CDRs to identify possibilities for further improving the molecular quality of the antibody.

[0100] The method may include the further step of producing additional clones based on the clones selected in step (4), e.g., based on further exploratory mutagenesis at specific positions in the CDRs of the clones selected in step (4), to enhance humanization and / or minimize the amount of human T-cell epitopes and / or improve the manufacturing characteristics of the antibody molecule or antigen-binding portion thereof produced in step (5).

[0101] As used herein, the term "PD1" refers to programmed cell death protein 1 and variants thereof that retain at least some of the biological activity of PD1. As used herein, PD1 can include all species of native sequence PD1, including human, rat, mouse, and chicken. The term "PD1" can be used to include variants, isoforms, and species homologs of human PD1.

[0102] In some embodiments, "PD1" refers to the wild-type human form of PD1. Antibodies of the invention may cross-react with PD1 from species other than human, particularly PD1 from cynomolgus monkeys (Macaca fascicularis). Examples of human and cynomolgus monkey PD1 amino acid sequences are provided in Table 7. In certain embodiments, antibodies may be completely specific for human PD1 and may not exhibit non-human cross-reactivity.

[0103] As used herein, the term "VEGFR2" refers to vascular endothelial growth factor receptor 2 (also known as KDR or FLK1) and variants thereof that retain at least some of the biological activity of VEGFR2. As used herein, VEGFR2 can include all species of native-sequence VEGFR2, including human, rat, mouse, and chicken. The term "VEGFR2" can be used to include variants, isoforms, and species homologs of human VEGFR2. In some embodiments, "VEGFR2" refers to the wild-type human form of VEGFR2. Antibodies of the present invention may cross-react with VEGFR2 from species other than human, particularly VEGFR2 from rhesus monkeys (Macaca mulatta). Examples of human and rhesus monkey VEGFR2 amino acid sequences are provided in Table 7. In certain embodiments, antibodies may be fully specific for human VEGFR2 and may not exhibit non-human cross-reactivity.

[0104] As used herein, "antagonist" or "anti-PD1 and anti-VEGFR2 antagonist antibody" (synonymously "anti-PD1 and anti-VEGFR2 antibody") used in the context of the antibodies of the invention refers to an antibody capable of binding to PD1 and VEGFR2 and inhibiting PD1 and VEGFR2 biological activity and / or downstream pathways mediated by PD1 and / or VEGFR2 signaling. Anti-PD1 and anti-VEGFR2 antagonist antibodies include antibodies that can block, antagonize, inhibit, or reduce (including significantly) PD1 and VEGFR2 biological activity, including downstream pathways mediated by PD1 and / or VEGFR2, such as receptor binding and / or eliciting cellular responses to PD1 and VEGFR2. For purposes of the present invention, the term "anti-PD1 and anti-VEGFR2 antagonist antibody" includes all terms, titles, and functional states and characteristics whereby PD1 and VEGFR2 itself and PD1 and VEGFR2 biological activity (including, but not limited to, its ability to inhibit activation of T cell anti-tumor cell activity) or the result of the activity or biological activity are realized to any meaningful extent. Qualitatively nullified, diminished or neutralized is expressly understood.

[0105] An antibody "specifically binds," "specifically interacts," "preferentially binds," "binds," or "interacts with" PD1 or VEGFR2 if it binds with greater affinity, avidity, more readily, and / or for longer than it binds to other receptors.

[0106] An "antibody molecule" is an immunoglobulin molecule that can specifically bind to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody molecule" includes not only intact polyclonal or monoclonal antibodies, but also any antigen-binding fragment (e.g., "antigen-binding portion") or single chain thereof, fusion proteins comprising an antibody, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site, including, for example, but not limited to, scFv, single domain antibodies (e.g., shark and camel antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv.

[0107] An "antibody molecule" encompasses antibodies of any class, such as IgG, IgA, or IgM (or subclass thereof); the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG1, and IgG2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0108] As used herein, the term "antigen-binding portion" of an antibody molecule refers to one or more fragments of an intact antibody that retain the ability to specifically bind to PD1. The antigen-binding function of an antibody molecule can be performed by fragments of an intact antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody molecule include Fab, Fab', F(ab')2, an Fd fragment consisting of the VH and CH1 domains, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, single-domain antibody (dAb) fragments, and isolated complementarity-determining regions (CDRs).

[0109] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native-sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The numbering of residues in the Fc region is that of the EU index as in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. As is known in the art, the Fc region can exist in a dimeric or monomeric form.

[0110] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist 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 selecting FRs adjacent to CDRs, for example, when humanizing or optimizing an antibody, FRs derived from antibodies containing the same standard class CDR sequence are preferred.

[0111] The definition of CDR used in this application combines domains used in many different, often conflicting, schemes developed in the field and is based on a combination of immunoglobulin repertoire analysis and structural analysis of isolated antibodies and cocrystals with antigens (Swindells et al., 2016, abYsis: Integrated Antibody Sequence and Structure-Management, Analysis, and Prediction. J Mol Biol. [PMID: 27561707; Epub 22 August 2016]). The CDR definition used herein (the "integrated" definition) incorporates the lessons of all such previous findings and includes all appropriate loop positions necessary to sample the complete residue landscape that may mediate target binding complementarity.

[0112] Table 1 shows the amino acid sequence of the VL domain of MAb005 humanized anti-PD1 antibody with CDRs defined herein. Table 2 shows the amino acid sequence of the VH domain of MAb005 humanized anti-PD1 antibody with CDRs defined herein.

[0113] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid that does not significantly adversely alter functional activity. A preferred example of a "conservative substitution" is the substitution of one amino acid with another amino acid that has a value of 0 or greater in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89:10915-10919):

[0114] [Table 1]

[0115] The term "monoclonal antibody" (Mab) refers to an antibody or antigen-binding portion thereof that is derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Preferably, the monoclonal antibodies of the invention exist in a homogeneous or substantially homogeneous population.

[0116] A "humanized" antibody molecule refers to a form of a non-human (e.g., murine) antibody molecule or antigen-binding portion thereof, derived from a chimeric immunoglobulin, immunoglobulin chain, or non-human immunoglobulin. These fragments (Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) contain the minimal sequence required for humanization. Humanized antibodies can be human immunoglobulins (recipient antibody) in which residues from the recipient CDRs are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.

[0117] A "human antibody or fully human antibody" refers to an antibody molecule or antigen-binding portion thereof derived from a transgenic mouse or a human cell harboring human antibody genes.

[0118] The term "chimeric antibody" refers to an antibody molecule or antigen-binding portion thereof in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody molecule in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0119] "Antibody drug conjugate" and "immunoconjugate" refer to an antibody molecule or antigen-binding portion thereof, including antibody derivatives that bind to PD1 and VEGFR2 and are conjugated to a cytotoxic agent, a cytostatic agent, and / or a therapeutic agent.

[0120] The antibody molecules or antigen-binding portions thereof of the present invention can be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques or a combination of such techniques, or other techniques readily known in the art.

[0121] The term "isolated molecule" (e.g., a polypeptide, polynucleotide, or antibody) refers to a molecule that, by its origin or source of derivation, (1) is not associated with naturally associated components with which it is naturally associated; (2) is substantially free of other molecules from the same species; (3) is expressed by cells from a different species; or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally occurs is "isolated" from its naturally associated components. A molecule may also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule may be analyzed by several means well known in the art. For example, the purity of a polypeptide sample may be analyzed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means for purification well known in the art.

[0122] The term "epitope" refers to a portion of a molecule, or antigen-binding portion thereof, capable of being recognized and bound by an antibody molecule at the antigen-binding region of one or more antibody molecules. An epitope can consist of a defined region of primary, secondary, or tertiary protein structure, including any combination of secondary structural units or structural domains of a target recognized by the antigen-binding region of an antibody or antigen-binding portion thereof. Similarly, an epitope can consist of a defined chemically active surface grouping of molecules, such as amino acids or sugar side chains, and possess specific three-dimensional structural characteristics as well as specific charge characteristics. As used herein, the term "antigenic epitope" is defined as a portion of a polypeptide to which an antibody molecule can specifically bind, as determined by any method known in the art, for example, conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structure determination methods (e.g., NMR).

[0123] The term "binding affinity" or "KD" refers to the dissociation rate of a particular antigen-antibody interaction. KD is the ratio of the rate of dissociation, which is expressed as the "off rate (k)" to the associated rate. off ) or "On Rate (k on )" is also called "K D is k off / k on and expressed as molar concentration (M). Then, K D The smaller is, the more Therefore, the affinity of the binding is strong. D has a K of 1 nM D The KD value of an antibody can be determined using methods established in the art. One method for determining the KD of an antibody is to use surface plasmon resonance (SPR), typically using a biosensor system such as a Biacore® system.

[0124] The term "titer" refers to IC 50 The effective concentration of an antibody or antibody drug conjugate against the antigen PD1 or VEGFR2 that inhibits 50% of the activity measured in the PD1 or VEGFR2 activity assay described herein can be expressed as the effective concentration of an antibody or antibody drug conjugate against the antigen PD1 or VEGFR2 that inhibits 50% of the activity measured in the PD1 or VEGFR2 activity assay described herein.

[0125] As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to the amount (dosage and duration and means of administration) necessary to achieve the desired therapeutic result. 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.

[0126] The terms "inhibit" or "neutralize" as used herein with respect to a biological activity of an antibody molecule of the invention refer to the ability of the antibody to substantially antagonize, prohibit, prevent, limit, slow down, eliminate, stop, reduce or reverse the progression or severity of the inhibited disease, including, but not limited to, the biological activity or binding interaction of the antibody molecule to PD1 or VEGFR2.

[0127] A "host cell" includes an individual cell or cell culture that can be or has been the recipient of a vector for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, and the progeny may not be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide of the invention.

[0128] As used herein, "vector" refers to a construct capable of delivering, and preferably expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0129] As used herein, unless otherwise indicated, the term "treating" means reversing, alleviating, inhibiting progression, slowing progression, delaying the onset of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. As used herein, unless otherwise indicated, the term "treatment" refers to the act of treating as defined above. The term "treating" also includes adjuvant and neoadjuvant treatment of a subject. For the avoidance of doubt, references to "treatment" herein include references to curative, palliative, and prophylactic treatment. For the avoidance of doubt, references to "treatment" herein, in plural, also include references to curative, palliative, and prophylactic treatment.

[0130] Where embodiments are described herein using the term "comprising," it is understood that similar embodiments otherwise described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0131] Aspects or embodiments of the present invention may be incorporated by reference in their entirety. When written in terms, the invention encompasses not only the entire group recited as a whole, but also each member of the group individually and all possible subgroups of the main group, as well as the main group in which one or more group members are absent. The invention also contemplates the explicit exclusion of one or more group members in the claimed invention.

[0132] 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 belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the term "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a specified integer or group of integers but not the exclusion of any other integer or group of integers. Unless the context dictates otherwise, the singular includes the plural and the plural includes the singular. The term "for example" followed by any examples is not meant to be exhaustive or limiting.

[0133] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology.

[0134] Specific non-limiting embodiments of the present invention will now be described with reference to the accompanying drawings. [Example]

[0135] Example 1 Generation of an optimized anti-PD1 therapeutic antibody Introduction In this example, we successfully generated a panel of optimized antagonistic anti-PD1 antibodies that are simultaneously VEGFR2 antagonists, and that are well expressed, biophysically stable, highly soluble, and have maximized identity to the preferred human germline. material and method PD1 library generation and selection The PD1 Fab repertoire was constructed by mass oligo synthesis and PCR. Mutagenesis was applied to the CDR and CDR-proximal regions of the VL (Table 1) and VH (Table 2) domains. The amplified Fab repertoire was then cloned into a phagemid vector via restriction-ligation and transformed into Escherichia coli (E. coli) TG-1 cells. The phage repertoire was essentially rescued as previously described in detail (Finlay et al., 2011, Methods Mol Biol 681: 383-401).

[0136] Streptavidin magnetic microbeads were coated with biotinylated PD1 or VEGFR2 target proteins (either human or monkey), and the beads were washed three times with PBS. Phage selection was performed by resuspending the beads in pH 7.4 and 5% skim milk protein. The beads were coated with 100 nM of target protein in round 1 of selection, followed by three successive rounds of decreasing antigen concentrations. After each round, phage were eluted using trypsin and then reinfected into TG1 cells. Production of extraperiplasmic extracts (small scale) Production of soluble Fab in individual E. coli clones was performed. E. coli TG1 cells in logarithmic growth phase were induced with isopropyl 1-thio-D-β-galactopyranoside. Periplasmic extracts containing soluble Fab were generated by freeze / thaw cycles: bacterial cell pellets were frozen overnight at -20°C, then thawed at room temperature and diluted with PBS pH 7. The supernatant containing the soluble Fab was collected after shaking at room temperature and centrifugation. IgG expression and purification Mammalian codon-optimized synthetic genes encoding the heavy and light chain variable domains of the lead panel anti-PD1 antibodies and Mab005 were cloned into mammalian expression vectors containing effector function-null human IgG1 ("IgG1null"; a human IgG1 containing L234A, L235A, and G237A mutations in the lower hinge that suppress normal immunoglobulin ADCC, ADCP, and CDC functions, respectively) and a human Cκ domain. Co-transfection of the heavy and light chain-containing vectors into a mammalian expression system was followed by Protein A-based IgG purification, quantification, and QC on denaturing and non-denaturing SDS-PAGE. Direct binding ELISA for Fab and IgG Binding and cross-reactivity to the lead panel's recombinant proteins were first assessed by binding ELISA. Human PD1 human Fc-tagged recombinant protein and cynomolgus monkey PD1 human Fc-tagged 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 two-fold serial dilutions starting from 500 nM to 0.98 nM and allowed to bind to the coated antigen. Fabs were detected using a mouse anti-c-myc antibody followed by donkey anti-mouse IgG conjugated to horseradish peroxidase. IgG was detected using mouse anti-human IgG conjugated to horseradish peroxidase. Binding signals were visualized with 3,3',5,5'-tetramethylbenzidine substrate solution (TMB) and absorbance measured at 450 nm. AlphaScreen epitope competition assay for Fab periprep AlphaScreen assays (Perkin Elmer) were performed in 384-well white microtiter plates (Greiner) in a final volume of 25 μl. The reaction buffer contained 1x PBS pH 7.3 (Oxoid, catalog number BR0014G) and 0.05% (v / v) Tween® 20 (Sigma, catalog number P9416). Periprep 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 hMAb005 IgG and anti-human IgG1 acceptor beads were added at 20 μg / ml (final concentration), and the mixture was incubated for 1 hour at room temperature. Subsequently, streptavidin donor beads were added at 20 μg / ml (final concentration) and incubated for 30 minutes at room temperature. Light emission was measured with an EnVision multilabel plate reader (Perkin Elmer) and analyzed using EnVision management software. Values ​​were reported as counts per second (CPS) and corrected for crosstalk. Percent signal reduction was calculated relative to an unrelated sample. PD1 / PD-L1 cell-based antagonism assay The ability of antibodies to block PD1 / PD-L1 interaction was measured using a PD1 / PD-L1 blocking cell-based bioassay (Promega). The day before the assay, PD-L1 aAPC / CHO-K1 cells were thawed and transferred to cell collection medium (90% Ham's F12 / 10% FBS). The cell suspension was dispensed into each of the inner 60 wells of two 96-well white flat-bottom assay plates at 100 μl per well. Cell collection medium was added to each of the outer wells and the assay plate, and the plate was incubated overnight at 37°C / 5% CO2. On the day of the assay, sample IgG was added to assay buffer (99 The PD-L1 effector cells were then thawed in assay buffer (99% RPMI 1640 / 1% FBS) and the cell suspension was added to wells of the assay plate containing the PD-L1 aAPC / CHO-K1 cells and IgG titration samples. The assay plate was incubated in a 37°C / 5% CO2 incubator for 6 hours, allowed to equilibrate to ambient temperature for 5-10 minutes, and then 80 μl of Bio-Glo™ Reagent (Promega) was added. The assay plate was incubated at ambient temperature for an additional 5-30 min, after which the luminescent signal was measured at 10, 20, and 30 min. 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). The mo-DCs were further matured in culture by the addition of TNFα. Pan-T cells were isolated from allogeneic PBMC donors. One-way MLRs were set up by co-culturing mature DCs with freshly isolated pan-T cells at a T cell:DC ratio of 10:1 in the presence of titrated test substances for approximately 5 days. Activity was measured by quantifying IFN-γ production on day 5 of the MLR via ELISA. Results and Discussion Library generation and screening The variable domains of the antagonistic anti-PD1 IgG hMAb005 were cloned into a phage display vector in a human IgG1-kappa Fab format. Oligonucleotide mutagenesis using NNK randomization was applied to 5-6 consecutive residues per sublibrary, resulting in the generation of 5 VL sublibraries (Table 1) and 6 VH sublibraries (Table 2). These libraries were transformed into E. coli, rescued, and phage populations from each library were subjected to four rounds of selection against human and cynomolgus monkey PD1 and human and rhesus monkey VEGFR2 proteins.

[0137] Next, we performed ELISA screening of the Fabs in a peri-prep format to examine the binding of individual clones from each library selection to human PD1, cynomolgus PD1, human VEGFR2, and cynomolgus VEGFR2 (Figure 1). These analyses demonstrated that individual clones in all selection rounds exhibited binding to both the positive control hMAb005 Fab and PD1 orthologs with similar signals. Importantly, binding analyses also demonstrated that in some libraries, enriched populations of clones not only retained PD1 activity but also exhibited significantly improved binding to human and / or cynomolgus VEGFR2. We selected 168 clones from these selected populations and further characterized them by both Alphascreen PD1 epitope competition with hMAb005 IgG and DNA sequencing of the VL and VH domains. Alphascreen analysis shows that unique sequences from both the LCDR1 and LCDR3 libraries with improved binding to VEGFR2 (Figure 1) maintained epitope competition for hMAb005 binding to PD1 (Figure 2). In contrast, clones from the HCDR1, 2, and 3 libraries that showed improved binding to VEGFR2 did not effectively compete with hMAb005, suggesting that VEGFR2 affinity-improving mutations in the VH domain resulted in epitope drift on PD1 (Figure 2). Sequence analysis of all VEGFR2-improved clones that maintained PD1 binding demonstrated that a significant number of mutations were positively selected in both LCDR1 (Table 3) and LCDR3 (Table 4).

[0138] Based on the above analysis, five representative clones with mutations in LCDR1 or LCDR3 that showed high binding to both PD1 and VEGFR2 orthologs (Figure 1) and a signal reduction rate of >75% for the hMAb005 / PD1 interaction (Figure 2) were selected for expression and characterization in IgG format (Table 5). Additionally, six experimental combination IgG clones were generated that combined potentially beneficial mutations in both LCDR1 and LCDR3 (clones MAB01-MAB06, Table 5). These 11 lead IgG clones were generated in a human IgG1 null format. Characterization of lead IgG specificity and potency The 11 lead IgG1 null clones outlined in Table 4, as well as hMAb005 and isotype IgG1 null, were tested in a titration ELISA binding to human and cynomolgus PD1 (Figure 3A). This analysis demonstrated that all 11 lead clones had similar binding capabilities to human and cynomolgus PD1 as hMAb005 IgG1, while the isotype control showed no binding to either protein. In contrast, when the IgGs were tested for binding to VEGFR2 protein, all 11 lead clones showed significantly increased binding potency to both the human and rhesus orthologs compared to hMAb005 (Figure 3B).

[0139] To investigate whether this increased VEGFR2 reactivity to hMAb005 in the lead clones could alter its pharmacological relationship with the receptor, we used a human VEGFR2 reporter assay (Promega, performed according to the manufacturer's instructions) to examine the induction of luciferase expression under the control of the natural VEGF response element NFAT. In this assay, soluble VEGF-165 protein was added to VEGFR2 reporter cells, and all IgG1 proteins were tested for their ability to antagonize VEGF-165-induced signaling (Figure 4). Both IgG1 hMAb005 and isotype IgG1 showed no antagonism of VEGFR2 signaling. Surprisingly, however, all lead IgG1 null antibodies showed potent antagonism of VEGF-VEGFR2 signaling induction, with several clones exhibiting a range of potency similar to that of the clinically approved anti-VEGFR2 cancer drug ramucirumab (Figure 4). This was a highly unexpected finding, as hMAb005 IgG1 has been shown to potently stimulate VEGFR2. The above data suggest a complete reversal of VEGFR2 pharmacology for all lead antibodies while maintaining PD1 epitope specificity. To further explore this data, we generated individual analyses for library-derived clones (Figures 5A-5E) and clones combining mutations in LCDRs 1 and 3 (Figures 5F-5K). These analyses suggested that antibodies MAB01-MAB06 were the most potent inhibitors of VEGFR2 signaling, demonstrating that the improvements in VEGFR2 binding mediated by each CDR can be combined synergistically.

[0140] The combinatorial analysis outlined herein surprisingly demonstrated that deep sampling of amino acid diversity in the CDRs of these antibodies enabled simultaneous optimization of PD1 and VEGFR2 target binding specificity, resulting in reversal of the VEGFR2 agonistic phenotype of the precursor antibody hMAb005 in multiple lead molecules. The resulting lead antibodies possess the beneficial signature of dual antagonism of clinically relevant PD1 and VEGFR2 signaling pathways. Generation and analysis of lead IgG variants Eight variants of clone MAB06 (MAB06.1-MAB06.8) were generated and mutated to human germline sequences at multiple CDR residues to experimentally test their ability to reduce immunogenicity risk. These clones contained germline mutations in LCDR1 and 2 (Table 8) and HCDR2 (Table 9). All eight clones were expressed and purified in an IgG1 null format. All clones were purified via a protein A affinity column. It was easily expressed and purified, and the resulting protein exhibited >94% monomeric IgG% (as determined by SEC).

[0141] MAB06.1-MAB06.8 IgGs were tested for their ability to antagonize both PD1 and VEGFR2 signaling. All eight clones retained the ability to antagonize PD1 signaling in a PD1 / PD-L1 cell signaling bioassay, with potency similar to that of both SHR-1210 IgG1-3M and nivolumab (Figure 6). Similarly, all eight clones retained the concentration-dependent ability to antagonize VEGFR2 signaling in a VEGF / VEGFR2 signaling bioassay, with potency similar to that of ramucirumab (Figure 7). Comparative analysis of PD1 and VEGFR2 antagonism for clones MAB06.5 (Figures 8A and 8B), MAB06.6 (Figures 8C and 8D), MAB06.7 (Figures 8E and 8F), and MAB06.8 (Figures 8G and 8H) demonstrated that the sequence of clone MAB06 can accommodate humanization of multiple residues in LCDR1, LCDR2 (Table 8), and HCDR2 (Table 9) while retaining the ability to antagonize signaling of both receptors. Importantly, the control SHR-1210 IgG13M (hMab005) was shown to be unable to inhibit VEGFR2 signaling in the presence of VEGF, as shown in Figures 4, 5, 7, and 8.

[0142] These findings suggest that the ability to antagonize both the PD1 and VEGFR2 pathways in a single molecule may provide clinical benefit to the antibody SHR-1210 (hMab005) by blocking two important immunosuppressive signals known to be highly active in the tumor microenvironment. Indeed, both PD1 and VEGFR2 receptors have been demonstrated to be coexpressed on immune cells in human tumors, suggesting the potential for dual antagonists to act in a bifunctional manner on a single cell, as shown in Figure 9.

[0143] Antibodies that bind to signaling receptors with high affinity run the risk of becoming potent agonists of receptor activity in the absence of receptor ligand. To investigate this, we repeated the VEGFR2 bioassay in the absence of VEGF. Ramucirumab, a high-affinity anti-VEGFR2 IgG1 control antibody, isotype control human IgG1, MAB06.5, MAB06.8, and VEGF-165 protein were all titrated in this assay, with the antibody protein starting at a concentration of 1000 nM (Figure 10). Only VEGF-165 demonstrated concentration-dependent receptor activation, confirming that MAB06.5 IgG1 and MAB06.8 IgG1 are VEGFR2 antagonists in the presence of VEGF (Figure 8) but not agonists in the absence of VEGF (Figure 10).

[0144] To further confirm the PD1 antagonistic potency of MAB06.5 and MAB06.8, both molecules were analyzed in a human DC:T cell unidirectional MLR assay, measuring nivolumab (anti-PD1) as a positive control and IFN-γ as an indicator of activity (Figure 11). MLR assays were performed in duplicate using three separate human donor pairs. These analyses demonstrated that both MAB6.5 and MAB6.8 exhibited a concentration-dependent ability to block PD1 and drive IFN-γ signaling comparable to nivolumab. This was found in both donor pair 1 (Figures 11A and 11B), donor pair 2 (Figures 11C and 11D), and donor pair 3 (Figures 11E and 11F) runs, demonstrating a high reproducibility of blocking PD1 function on T cells. Generation and analysis of third-generation lead IgG variants WO2019170885A1 discloses several mutations that can be made in the VH domain of hMab005 that have the potential to increase the affinity of the molecule for PD1. To examine the possible utility of these previously defined mutations in the context of the novel clones described herein, nine clonal MAB06.8 variants were generated and analyzed. The ability to mutate VH CDR residues of MAB06.8.8 was experimentally investigated to potentially further improve binding to PD1 and / or VEGFR2. All eight clones contained the MAB06.8 VL sequence (SEQ ID NO: 47, Table 8) expressed in nine unique VH sequences (Table 10). All eight clones were expressed and purified in an IgG1 null format. All clones (except MAB06.8.3 and MAB06.8.5, both of which suffer from aggregation) were readily expressed and purified via a Protein A affinity column, and the resulting proteins exhibited greater than 95% monomeric IgG% (as measured by SEC).

[0145] Seven well-expressing IgG1 null clones (MAB06.8.3 and MAB06.8.5 omitted), as well as hMAb005 and isotype IgG1 null, were tested in titration ELISA binding to human and cynomolgus PD1 (Figures 12A and 12B). This analysis showed that many of these seven clones retained binding to human and cynomolgus PD1, while the isotype controls showed no binding to either protein. One notable exception was MAB06.8.4, which lost the ability to bind cynomolgus PD1 (Figure 12B). When the IgGs were tested for binding to VEGFR2 protein, all seven clones showed binding to both the human and cynomolgus orthologues (Figures 12C and 12D).

[0146] Despite the apparent retention of binding affinity for both PD1 and VEGFR2 in most clones, the benefit of MAB06.8 was neither maintained nor enhanced when seven variants were tested in PD1 and VEGFR2 antagonism bioassays. All seven clones showed a reduced ability to antagonize PD1, VEGFR2, or both compared to the positive controls, MAB06 (Figure 5), MAB06.5 (Figure 8), and MAB06.8 (Figure 8) (Figures 13A, 13B).

[0147] All documents or portions of documents cited herein, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more incorporated documents or portions of documents define a term that contradicts the definition in the application of that term, the definition set forth in the application of this invention shall control. However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be, made as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0148] While the present 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 thereto without departing from the spirit and scope of the invention, and such modifications are expressly contemplated herein. No limitations with respect to the specific embodiments disclosed herein and set forth in the appended claims are intended, nor should any be inferred. Numbered Embodiments Without limiting the scope of any appended claims, the present disclosure describes the following numbered embodiments.

[0149] 1. An antibody or antigen-binding portion thereof 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; (a) The VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises RASQESGIWL LCDR1 (SEQ ID NO: 39) of S, LCDR2 (SEQ ID NO: 5) of TATSLAD and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT, (b) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (c) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 8) of LASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (d) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVYSIPWT; (e) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVYSIPWT; (f) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 8) of LASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 6) of QQVYSIPWT; (g) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 9) of LASQTIGTWLT, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG; (h) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 9) of LASQTIGTWLT, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (i) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG; (j) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG; (k) The VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LASQESGIWLG LCDR1 (SEQ ID NO: 12) of QQVAELPFG, LCDR2 (SEQ ID NO: 5) of QQVAELPFG, and LCDR3 (SEQ ID NO: 10) of QQVAELPFG. (l) The VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 4) of LASQGIGPWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (m) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGANTYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 7) of LASQPLGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (n) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 5) of TATSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (o) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 40) of TASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (p) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 41) of AASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (q) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGANTYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (r) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 40) of TASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; (s) the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 41) of AASSLAD, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT; or (t) An antibody or antigen-binding portion thereof, wherein the VH region amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 64) of QVYYFDY, and the VL region amino acid sequence comprises LCDR1 (SEQ ID NO: 39) of RASQESGIWLS, LCDR2 (SEQ ID NO: 43) of AASSLQS, and LCDR3 (SEQ ID NO: 11) of QQVSVTPFT.

[0150] 2. (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 antibody or antigen-binding portion of embodiment 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 73 and the VL region amino acid sequence comprises SEQ ID NO: 47.

[0151] 3. The antibody or antigen-binding portion of embodiment 1 or 2, wherein the antibody or antigen-binding portion is capable of antagonizing both the PD1-PDL1 and VEGFR2-VEGF signaling pathways.

[0152] 4. An antibody or antigen-binding portion thereof that binds to both PPD1 and VEGFR2, wherein the 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; (a) comprising a complete germline human framework amino acid sequence; (b) specifically binds to human PD1, cynomolgus monkey PD1, human VEGFR2, and rhesus monkey VEGFR2; (c) Antagonizing the binding of human PD1 to human PD-L1 and inhibiting human V in response to human VEGF. An antibody or antigen-binding portion that antagonizes EGFR2 signaling.

[0153] 5. The antibody or antigen-binding portion of any one of embodiments 1 to 4, wherein the antibody is human, humanized, or chimeric.

[0154] 6. The antibody or antigen-binding portion of any one of embodiments 1 to 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.

[0155] 7. The antibody or antigen-binding portion of any one of embodiments 1 to 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 into which the CDRs are inserted.

[0156] 8. The antibody or antigen-binding portion of embodiment 1 or 3, wherein the VH region comprises an IGHV3-7 human germline scaffold amino acid sequence into which the HCDR1, HCDR2 and HCDR3 amino acid sequences are inserted.

[0157] 9. The antibody or antigen-binding portion of 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 LCDR1, LCDR2 and LCDR3 amino acid sequences have been inserted.

[0158] 10. The antibody or antigen-binding portion of any one of embodiments 1 to 9, wherein the antibody comprises an immunoglobulin constant region.

[0159] 11. The antibody or antigen-binding portion of embodiment 10, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA, or IgY.

[0160] 12. The antibody or antigen-binding portion of embodiment 11, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0161] 13. The antibody or antigen-binding portion of embodiment 10, wherein the immunoglobulin constant region is immunologically inert.

[0162] 14. The antibody or antigen-binding portion of embodiment 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.

[0163] 15. The antibody or antigen-binding portion of embodiment 13, wherein the immunoglobulin constant region comprises any one of SEQ ID NOs: 25-31.

[0164] 16. The antibody or antigen-binding portion is Fab, Fab', F(ab') 2、 16. The antigen-binding portion of any one of embodiments 1 to 15, which is an Fd, Fv, scFv, maxibody, minibody, intrabody, diabody, triabody, tetrabody or bis-scFv.

[0165] 17. The antibody or antigen-binding portion of any one of embodiments 1 to 16, wherein the antibody is monoclonal.

[0166] 18. Any of embodiments 1 to 17, wherein the antibody is a tetrameric antibody, a tetravalent antibody, or a multispecific antibody. 1. The antibody or antigen-binding portion thereof according to any one of claims 1 to 9.

[0167] 19. The antibody or antigen-binding portion of any one of embodiments 1-18, wherein the antibody or antigen-binding portion specifically binds to (a) human PD1, or (b) human PD1 and cynomolgus PD1, or (c) human PD1 and rhesus PD1, or (d) human PD1, cynomolgus PD1, and rhesus PD1.

[0168] 20. The antibody or antigen-binding portion of 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 VEGFR2, or (c) human VEGFR2 and rhesus VEGFR2, or (d) human VEGFR2, cynomolgus VEGFR2, and rhesus VEGFR2.

[0169] 21. An immunoconjugate comprising the antibody or antigen-binding portion of any one of embodiments 1 to 20 linked to a therapeutic agent.

[0170] 22. The immunoconjugate of embodiment 21, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, an antiangiogenic agent, an antiproliferative agent, a proapoptotic agent, a mitogenic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an antiangiogenic agent, an antiproliferative agent, or a proapoptotic agent.

[0171] 23. A pharmaceutical composition comprising the antibody or antigen-binding portion of any one of embodiments 1 to 20, or the immunoconjugate of embodiment 21 or 22, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0172] 24. The antibody or antigen-binding portion according to any one of embodiments 1 to 20. (a) VH region amino acid sequence; (b) a VL region amino acid sequence or (c) both the VH and VL region amino acid sequences A nucleic acid molecule encoding

[0173] 25. An expression vector comprising the nucleic acid molecule of embodiment 24.

[0174] 26. A recombinant host cell comprising the nucleic acid molecule of embodiment 24 or the expression vector of embodiment 25.

[0175] 27. A method for making an anti-PD1 antibody or antigen-binding portion thereof, comprising: Culturing a recombinant host cell comprising the expression vector of embodiment 25 under conditions wherein the nucleic acid molecule is expressed, thereby producing the antibody or antigen-binding portion; A method comprising isolating the antibody or antigen-binding portion from the host cell or culture.

[0176] 28. A method for enhancing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding portion of any one of embodiments 1 to 20, the immunoconjugate of embodiment 21 or 22, or the pharmaceutical composition of embodiment 23.

[0177] 29. A method for treating or preventing cancer, an infectious disease, or an immune disease in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding portion of any one of embodiments 1 to 20, the immunoconjugate of embodiment 21 or 22, or the pharmaceutical composition of embodiment 23.

[0178] 30. The method of embodiment 29, wherein the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain 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, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0179] 31. The method of embodiment 29, wherein the infectious disease is viral, bacterial, fungal or parasitic.

[0180] 32. The method of embodiment 29, wherein the infectious disease is human immunodeficiency virus (HIV) infection.

[0181] 33. The antibody or antigen-binding portion of any one of embodiments 1 to 20, the immunoconjugate of embodiment 21 or 22, or the pharmaceutical composition of embodiment 23 for use in treating cancer, an infectious disease, or an immune disorder.

[0182] 34. The antibody or antigen-binding portion, immunoconjugate, or pharmaceutical composition for use according to 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 or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0183] 35. The antibody or antigen-binding portion, immunoconjugate or pharmaceutical composition for use according to embodiment 33, wherein the infectious disease is caused by a virus, bacteria, fungus or parasite.

[0184] 36. The antibody or antigen-binding portion, immunoconjugate or pharmaceutical composition for use according to embodiment 33, wherein the infectious disease is human immunodeficiency virus (HIV) infection.

[0185] 37. An antibody or antigen-binding portion according to any one of embodiments 1 to 20, an immunoconjugate according to embodiment 21 or 22, or a pharmaceutical composition according to embodiment 23 for use as a medicament.

[0186] [Table 2]

[0187] [Table 3]

[0188] [Table 4]

[0189] [Table 5]

[0190] [Table 6]

[0191] [Table 7]

[0192] [Table 8]

[0193]

Table 9

[0194]

Table 10

[0195]

Table 11

Claims

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

2. (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 antibody or antigen-binding portion of claim 1, wherein 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. joining part.

4. An antibody or antigen-binding portion thereof that specifically binds to both PD1 and VEGFR2, wherein the antibody or antigen-binding portion cross-competes with the antibody or antigen-binding portion of any one of claims 1 to 3 for binding to both PD1 and VEGFR2; (a) comprising a complete germline human framework amino acid sequence; (b) specifically binds to human PD1, cynomolgus monkey PD1, human VEGFR2, and rhesus monkey VEGFR2; (c) An antibody or antigen-binding portion that 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 to 4, wherein the antibody is human, humanized or chimeric.

6. The antibody or antigen-binding portion of any one of claims 1 to 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. 7. The antibody or antigen-binding portion of claim 1, 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 into which the CDRs are inserted.

8. 4. 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 into which the HCDR1, HCDR2 and HCDR3 amino acid sequences are inserted.

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 into which the LCDR1, LCDR2 and LCDR3 amino acid sequences are inserted.

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

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

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

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

14. 11. 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. 14. The antibody or antigen-binding portion of claim 13, wherein the immunoglobulin constant region comprises any one of SEQ ID NOs: 25 to 31.

16. The antibody or antigen-binding portion thereof may be Fab, Fab', F(ab') ２、 16. The antigen-binding portion of any one of claims 1 to 15, which is an 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 to 16, wherein the antibody is monoclonal.

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

19. The antibody or antigen-binding portion of any one of claims 1 to 18, wherein the antibody or antigen-binding portion specifically binds to (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. 20. The antibody or antigen-binding portion of any one of claims 1 to 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.

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

22. 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 mitogenic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

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

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

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

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

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

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

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

30. 30. The method of claim 29, wherein the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric 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 pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

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

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

33. 24. The antibody or antigen-binding portion of any one of claims 1 to 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. 34. The antibody or antigen-binding portion, immunoconjugate or pharmaceutical composition for use according to claim 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 or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma or cancer of the blood tissue.

35. 34. The antibody or antigen-binding portion, immunoconjugate or pharmaceutical composition for use according to claim 33, wherein the infectious disease is caused by a virus, bacteria, fungus or parasite.

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

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

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