PD1 and VEGFR2 dual binders
Dual antagonistic antibodies targeting PD1 and VEGFR2 address the limitations of separate drug combinations by enhancing immune response and cancer treatment efficacy through simultaneous inhibition of both pathways.
Patent Information
- Application Number
- JP2022521074
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- Priority Date
- 2020-08-24
- Filing Date
- 2020-10-09
- Publication Date
- 2025-06-05
- Estimated Expiration
- 2040-10-09
AI Technical Summary
Current anti-PD1 antibody therapies for cancer treatment have limited clinical response rates, and combining them with anti-VEGFR2 agents requires two separate drugs, increasing cost and toxicity risks.
Development of dual antagonistic antibodies that specifically bind to both PD1 and VEGFR2, providing synergistic benefits without the need for multiple drugs.
The dual antagonistic antibodies enhance immune response and cancer treatment efficacy by simultaneously inhibiting both PD1 and VEGFR2 pathways, potentially improving clinical outcomes and reducing side effects.
Smart Images
Figure 0007689116000012 
Figure 0007689116000013 
Figure 0007689116000014
Abstract
Description
[Technical field]
[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of UK Patent Application No. 2013180.1 filed on August 24, 2020, and UK Patent Application No. 1914747.9 filed on October 11, 2019, the disclosures of each of which are incorporated herein by reference in their entireties.
[0002] Description of electronically submitted text files The contents of the text files 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 recording: 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 autoimmune risk by promoting apoptosis (programmed cell death) in lymph node-resident T cells that are reactive to self-antigens and by promoting 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, as it can lead to reactivation of T cells in tumors. This clinical success has led to a proliferation of anti-PD1 antibody molecules being explored in clinical trials, although the majority of patients have yet to mount a sustained anti-cancer response when treated with anti-PD1 drugs alone.
[0005] Increasing the number of patients who respond to anti-PD1 antibody therapy is a major challenge. A possible 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 provides 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 invention there is provided an antibody molecule which specifically binds to human PD1 and human VEGFR2, and optionally also specifically binds to cynomolgus PD1 and cynomolgus VEGFR2, or an antigen-binding portion thereof.
[0008] In some aspects, the invention provides an antibody that specifically binds to both PD1 and VEGFR2, or an antigen-binding portion of an antibody, the antibody comprising 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: 42) of TISGGGSNKYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes 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 includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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: 11) of QQVSVTPFT; (d) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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; (e) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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 QQVYSIPWT; (g) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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; (h) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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; (i) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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; (j) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of 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; (l) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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; (m) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGATYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGATYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGATYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, 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; (q) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGATYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes 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 includes 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 domain 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 domain 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) 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, the antibody comprising 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') comprises an 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, the antibody comprising 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: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 mouse / humanized antibody HCDR1 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or HCDR2 of the antibody molecule or antigen binding portion may exclude the sequence TISGGGATYYPDSVKG (SEQ ID NO:2; MAb005 mouse / 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 mouse / humanized antibody HCDR3 disclosed in WO2015 / 085847A1, US2016 / 376367A1).
[0012] In some aspects of the invention, the LCDR1 of the antibody molecule or antigen-binding portion may exclude the sequence LASQTIGTWLT (SEQ ID NO:9; MAb005 mouse / humanized antibody LCDR1 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or the LCDR2 of the antibody molecule or antigen-binding portion may exclude the sequence TATSLAD (SEQ ID NO:5; MAb005 mouse / humanized antibody LCDR2 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or the LCDR3 of the antibody molecule or antigen-binding portion may exclude the sequence QQVYSIPWT (SEQ ID NO:6; MAb005 mouse / humanized antibody LCDR3 disclosed in WO2015 / 085847A1, US2016 / 376367A1).
[0013] Also provided in accordance with the present invention is an immunoconjugate comprising an antibody molecule, or an 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 is a vector comprising the nucleic acid molecule 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, a method of producing an anti-PD1 / VEGFR2 antibody and / or antigen-binding portion thereof is provided, the method comprising culturing a host cell of the invention under conditions resulting 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 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] Further provided is a method of enhancing an immune response 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.
[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 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 an immunoconjugate or a nucleic acid molecule or a 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 a 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] Further provided is the antibody molecule or antigen-binding portion thereof as defined herein, or the immunoconjugate as defined herein, or the nucleic acid molecule as defined herein, or the vector as defined herein, or the pharmaceutical composition as defined herein in the manufacture of a medicament for use in the treatment of an infectious disease.
[0027] 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.
[0028] The 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, (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 a CDR; (3) screening the library for binding to human PD1 and human VEGFR2, and optionally also to cyno PD1 and rhesus 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 an antigen-binding portion thereof that specifically binds to human PD1 and human VEGFR2, and optionally also specifically binds to cynomolgus PD1 and rhesus 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 description 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, where phage populations were selected on biotinylated target proteins in each round. After each round of selection, library-derived clones (black circles) were screened against human (hu) and cynomolgus (cy) PD1 and human (hu) and rhesus (rh) VEGFR2 as periplasmically expressed Fab proteins. hMAb005 v-domain expressed as a human IgG1 Fab was used as a positive control on each plate (grey diamonds). [Diagram 2] Figure 2A-B Epitope competition analysis of Fab proteins in AlphaScreen Anti-PD1 clones were expressed as Fabs in E. coli and peripreps were applied in an epitope competition assay using AlphaScreen technology, in which Fabs from the library 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]3A-B 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 clones from the library were titrated (in nM) in a direct binding ELISA against human and cynomolgus PD1 proteins (FIG. 3A) and against human and rhesus VEGFR2 proteins (FIG. 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-K. Cell-based VEGFR2 antagonism assays - single clone analysis. VEGFR2 antagonism of the lead clones shown in Figure 4 was reanalyzed on a clone-by-clone basis. All 11 lead clones showed potent antagonism, however clones derived from the library with mutations found in only one CDR per clone (Figures 5A-E) proved to be less potent than clones combining mutations in both LCDR1 and LCDR3 (Figures 5F-K). [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 clone MAB06.1-MAB06.8 (human IgG1 null format) were titrated in a human PD1 signaling assay where human PD1+ and human PD-L1+ cells were mixed and antagonism of the PD1 / PD-L1 interaction results in increased signal. The isotype IgG1 control protein did not show any concentration-dependent PD1 antagonism. Clone MAB06.1-MAB06.8, hMab005 and positive control anti-PD1 IgG4 nivolumab showed 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 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 showed any concentration-dependent VEGFR2 antagonism. Clone MAB06.1-MAB06.8 and positive control anti-VEGFR2 IgG1 ramucirumab showed potent antagonism in the nM range. [Figure 8A]Figure 8A-8H Cell-based PD1 and VEGFR2 antagonism assays - 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 (Figure 8A-8B), MAB06.6 (Figure 8C-8D), MAB06.7 (Figure 8E-8F) and MAB06.8 (Figure 8G-8H) showed 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. Thus, dual antagonist antibodies (on the same or different cells) that can effectively block both PD1 and VEGFR2 signaling may therefore 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 and clones MAB06.5 and MAB06.8 demonstrated any concentration-dependent VEGFR2 agonism. The positive control VEGF-165 elicited potent antagonism in the nM range. [Figure 11A] Figure 11A-F 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 with three separate human donor pairs in duplicate replicate runs. MAB6.5 and MAB6.8 demonstrated a concentration-dependent ability to PD1 blockade-driven IFN-γ signaling that was comparable to nivolumab. This was found in both runs for donor pair 1 (Figure 11A, 11B), donor pair 2 (Figure 11C, 11D) and donor pair 3 (Figure 11E, 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-D 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 (FIG. 12A, B) and human and cynomolgus VEGFR2 proteins (FIG. 12C, D). [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 assays. Comparative analysis of PD1 (Figure 13A) and VEGFR2 (Figure 13B) antagonism for clones SHR-1210 IgG1-3M, nivolumab, isotype control IgG1 and seven third generation clones in human IgG1 null format. [Figure 13B] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[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 immune suppression in the tumor microenvironment, and VEGFR2 is expressed in multiple immune cell types. However, the generation of dual inhibitor antibodies is a complex and unpredictable challenge, as it involves the generation of a single antibody with strong regulation of two target proteins, e.g., PD1 and VEGFR2, which have quite different amino acid sequences and structures. This sequence and structural dissimilarity between the two targets hampers the successful generation of such antibodies.
[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 in clinical trials to have a very rare side effect of inducing hemangiomas. In vitro studies were successful in tracing this issue back to off-target antibody reactivity, including binding of 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 strong agonism (activation) of the receptor, which is likely the primary driver of the hemangiomas side effect. For the reasons discussed above, such a humanized form of MAb005 described in WO2015 / 085847A1 is not ideal. Furthermore, it cannot be predicted 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 is a method for generating an antibody derived from hMAb005 that specifically binds to PD1 and antagonizes VEGF-VEGFR2 signaling. For example, such an antibody can be generated by introducing mutations into one or more CDR sequences of antibody hMAb005. In some embodiments, the mutations are introduced into the LCDR1 and / or LCDR2 and / or HCDR2 sequences of antibody hMAb005.
[0034] In some aspects, an antibody is provided that specifically binds to both human PD1 and human VEGFR2 or the antigen-binding portion of the antibody. In some embodiments, the antibody or the antigen-binding portion thereof can antagonize both the PD1-PDL1 signaling pathway and the VEGFR2-VEGF signaling pathway. In some embodiments, the antibody or the antigen-binding portion thereof antagonizes the binding of human PD1 to human PD-L1 and antagonizes the signaling of human VEGFR2 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 mouse / humanized antibody HCDR1 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or HCDR2 of the antibody molecule or antigen binding portion may exclude the sequence TISGGGATYYPDSVKG (SEQ ID NO:2; MAb005 mouse / 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 mouse / humanized antibody HCDR3 disclosed in WO2015 / 085847A1, US2016 / 376367A1).
[0038] In some aspects of the invention, the LCDR1 of the antibody molecule or antigen-binding portion may exclude the sequence LASQTIGTWLT (SEQ ID NO:9; MAb005 mouse / humanized antibody LCDR1 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or the LCDR2 of the antibody molecule or antigen-binding portion may exclude the sequence TATSLAD (SEQ ID NO:5; MAb005 mouse / humanized antibody LCDR2 disclosed in WO2015 / 085847A1, US2016 / 376367A1), and / or the LCDR3 of the antibody molecule or antigen-binding portion may exclude the sequence QQVYSIPWT (SEQ ID NO:6; MAb005 mouse / humanized antibody LCDR3 disclosed in WO2015 / 085847A1, US2016 / 376367A1).
[0039] In some embodiments, the anti-PD1 antibodies of the present invention are selected to have equivalence in binding specificity and affinity to both human PD1 and cynomolgus PD1 (to facilitate primate toxicity and pharmacokinetic studies with maximum accuracy). Further refinement of the optimized antibody molecules described herein resulted in improved binding to cynomolgus orthologues 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 inducing hemangiomas 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 the 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 the properties and 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 to 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 envisioned. Examples of terminal insertions include antibody molecules with an N-terminal methionyl residue or antibody molecules fused to an epitope tag. Other insertion variants of antibody molecules include fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide that increases 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 that have other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. The 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, an antibody or antigen-binding portion of an antibody that binds to both PD1 and VEGFR2 is provided, the antibody comprising 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 TISGGGATYYPDSVKG, 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 TISGGGATYYPDSVKG, 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 TISGGGATYYPDSVKG, 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 TISGGGATYYPDSVKG, 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 TISGGGATYYPDSVKG, 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of 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 TISGGGATYYPDSVKG, 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 domain amino acid sequence comprises HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL domain 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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: 11) of QQVSVTPFT, [MAB06], or (l) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGATYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 TISGGGATYYVDSVKG, 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 TISGGGATYYVDSVKG, 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGATYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes 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 includes 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 domain 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 domain 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 VH region amino acid sequence includes 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 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 includes 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 includes 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 includes 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 includes 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, 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, [MAB06.8.6], 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 domain 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 domain 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 includes 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 includes 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 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, wherein the VH region amino acid sequence comprises or consists of EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSS (SEQ ID NO: 13).
[0045] In some aspects, anti-PD1 and anti-VEGFR2 antibodies, or antigen-binding portions thereof, are 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, the antibody comprising 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%, 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: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%, 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: 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 and anti-VEGFR2 antibodies 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 defined herein may be isolated.
[0049] The antibody molecules or antigen-binding portions thereof defined herein may 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 and anti-VEGFR2 antibodies or antigen-binding portions thereof, where 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 the signaling of human VEGFR2 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 that includes 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 the location of T cell epitopes of the antibody or portion (e.g., in the variable region of the antibody or portion).
[0051] For example, T cell epitopes of an antibody or antigen binding moiety can be identified by using iTope™. iTope™ can be used to analyze 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 believed to correlate with the presence of T cell epitopes that are high risk indicators of clinical immunogenicity of drug proteins. iTope™ software predicts favorable interactions between amino acid side chains of peptides in the open-ended binding groove of 34 human MHC class II alleles and specific binding pockets (particularly pocket positions; p1, p4, p6, p7 and p9). These alleles represent the most common HLA-DR alleles found worldwide, with no weighting due to those most widely found in any particular ethnic group. Twenty alleles include the "open" p1 configuration and 14 alleles include the "closed" configuration in which glycine at position 83 is replaced by valine. The 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 discriminated between peptides that did or did 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 analysis of 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 immunogenicity risk), low affinity foreign ("LAF" - low immunogenicity risk), and / or TCED+ (epitopes previously identified in the TCED™ database).
[0054] In some embodiments, anti-PD1 antibody and anti-VEGFR2 antibody or antigen-binding portion may have high content of germline epitopes (GE) in its sequence. In some examples, anti-PD1 antibody or antigen-binding portion has 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 its sequence (e.g., VL and / or VH region sequence). Germline epitopes may be defined as human germline peptide sequences with high MHC class II binding affinity. Germline epitope 9mer peptides are unlikely to be immunogenic due to T cell tolerance, as verified by 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 being 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, and 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-competition", "cross-block", "cross-blocked" and "cross-blocking" are used interchangeably to mean the ability of an antibody or portion thereof to directly or indirectly inhibit the binding of the anti-PD1 antibody and anti-VEGFR2 antibody of the present invention to targets PD1 and VEGFR2 (e.g., human PD1, human VEGFR2) via allosteric modulation. The extent to which an antibody or portion thereof can interfere with the binding of another antibody or portion thereof to a target can be determined using a competitive binding assay, regardless of whether it can be said to cross-block or cross-compete according to the present invention. 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 the 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 one that binds to a target in a cross-competition assay such that, for example, during the assay, in the presence of a second antibody or portion thereof, the recorded displacement of an immunoglobulin single variable domain according to the 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 needs to be strength blocked) by a potentially cross-competing antibody or fragment thereof present in a given amount. It is preferred that a cross-competing antibody or portion thereof has a recorded displacement that is between 10% and 100%, or between 50% and 100%.
[0057] Antibody molecules or antigen-binding portions defined herein may contain 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. The 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 such 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, may 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 may 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 with the corresponding HCDR sequences inserted. The antibody molecule or antigen-binding portion thereof may comprise a VH region comprising an IGHV3-7 human germline scaffold amino acid sequence with the corresponding set of HCDR1, HCDR2 and HCDR3 amino acid sequences 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 with the corresponding HCDR sequences inserted and an IGKV1-39 human germline scaffold with the corresponding LCDR sequences inserted. The antibody molecule or antigen binding portion thereof may comprise a VH region comprising the IGHV3-7 human germline scaffold amino acid sequence with the corresponding set of HCDR1, HCDR2 and HCDR3 amino acid sequences inserted and a VL region comprising the amino acid sequence of the IGKV1-39 human germline scaffold with the corresponding set of LCDR1, LCDR2 and LCDR3 amino acid sequences inserted. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences were determined 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, MAB06.9, MAB06.10, MAB06.11, MAB06.12, MAB06.13, MAB06.14, MAB06.15, MAB06.16, MAB06.17, MAB06.18, MAB06.19, MAB06.20, MAB06.21, MAB06.22, MAB06.23, MAB06.24, MAB06.25, MAB06.26, MAB06.27, MAB06.28, MAB06.29, MAB06.30, MAB06.31, MAB06.32, MAB06.33, MAB06.34, MAB06.35, MAB06.36, MAB06.37, MAB06.38, MAB06.39, MAB06.40, MAB06.41, MAB06.42, MAB06.43, MAB06.44, MAB06.45, MAB06.46, MAB06.47, MAB06.48, MAB06.49, MAB06.49, MAB06.49, MAB06.40, MAB0 The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acid sequences of any one of the six CDR sequences (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 the 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 the 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, the anti-PD1 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, the anti-PD1 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, 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 (Ward et al., 1995 Therap. Immunol.2:77-94).In some embodiments, the immunoglobulin constant region may comprise an RDELT (SEQ ID NO: 36) motif or a 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 and anti-VEGFR2 antibodies 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 and anti-VEGFR2 antibodies may comprise an immunoglobulin heavy chain constant region comprising any one of the Fc region amino acid sequences in Table 6, and an immunoglobulin light chain constant region that is a kappa light chain constant region or 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 TISGGGATYYPDSVKG, 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 TISGGGATYYPDSVKG, 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 TISGGGATYYPDSVKG, 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 TISGGGATYYPDSVKG, 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 TISGGGATYYPDSVKG, 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 TISGGGATYYPDSVKG, 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 TISGGGATYYPDSVKG, 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 an HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, an HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and an HCDR3 (SEQ ID NO: 3) of QLYYFDY; 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; 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 TISGGGATYYPDSVKG, 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 TISGGGATYYPDSVKG, 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 TISGGGATYYPDSVKG, 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 TISGGGATYYVDSVKG, 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 TISGGGATYYVDSVKG, 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 TISGGGATYYVDSVKG, 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 an HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, an HCDR2 (SEQ ID NO: 38) of TISGGGATYYVDSVKG, and an HCDR3 (SEQ ID NO: 3) of QLYYFDY; 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; 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 any one of SEQ ID NOs: 25 to 31; (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 (SEQ ID NO: 1) of GFTSSYMMS, an HCDR2 (SEQ ID NO: 42) of TISGGGSNKYYVDSVKG, and an HCDR3 (SEQ ID NO: 3) of QLYYFDY, 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, 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; 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 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. (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 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: 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 can 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 the 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, and cell killing by CDC, ADCC, and / or ADCP can be measured. In some embodiments, anti-PD1 and anti-VEGFR2 antibodies (see Table 6) that comprise the amino acid sequence of the Fc region of human IgG1-3M are effector null. In some embodiments, anti-PD1 and anti-VEGFR2 antibodies (see Table 6) that comprise the amino acid sequence of the Fc region of human IgG1-3M are not effector null.
[0069] An antibody molecule or an antigen-binding portion thereof may be a Fab fragment, F(ab) 2The antibody may be a fragment, an Fv fragment, a tetrameric antibody, a tetravalent antibody, a multispecific antibody (e.g., a bivalent antibody), a domain-specific antibody, a single domain antibody, a monoclonal antibody, or a 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 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, antiangiogenic agents, antiproliferative agents, proapoptotic agents, and cytostatic and cytolytic enzymes (e.g., ribonucleases). Additional therapeutic agents include therapeutic nucleic acids, such as genes encoding immunomodulatory agents, antiangiogenic agents, antiproliferative agents, or proapoptotic 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, maytansines, 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, photosensitizers may also be used in immunoconjugates. In addition, conjugates can be made using secondary carriers as cytotoxic agents, such as liposomes or polymers, and suitable cytotoxins include agents that inhibit or prevent the function of cells and / or cause the destruction of cells. Representative cytotoxins include antibiotics, inhibitors of tubulin polymerization, alkylating agents that bind to and destroy DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins, such as protein kinases, phosphatases, topoisomerases, enzymes and cyclins.
[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, gugerotin, puromycin, tegafur, thiazofuridine, adhamycin, cisplatin, carboplatin, cyclophosphamide ... Famide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine, methotrexate, fluorouracil, etoposide, taxol, taxol analogues, 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 anti-hormonal agents that block hormone action on the tumor and immunosuppressants that suppress cytokine production, downregulate self-antigen expression, or mask MHC antigens.
[0075] Also provided is a nucleic acid molecule encoding an antibody molecule of the present invention or an antigen-binding portion thereof as defined herein. The nucleic acid molecule 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 an anti-PD1 antibody and an anti-VEGFR2 antibody or an antigen-binding portion thereof as described herein. In some embodiments, the nucleic acid molecule as defined herein may be isolated.
[0076] There is further provided a vector comprising a nucleic acid molecule of the invention as defined herein. The vector may be an expression vector.
[0077] Also provided is a host cell comprising the nucleic acid molecule or vector of the present invention as defined herein. The host cell may be a recombinant host cell. In some embodiments, the host cell may comprise a vector comprising a nucleic acid molecule encoding both the VH region amino acid sequence and the VL region amino acid sequence of the anti-PD1 antibody and the anti-VEGFR2 antibody or antigen-binding portion thereof as described herein. In some embodiments, the host cell 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 antibody and the anti-VEGFR2 antibody or antigen-binding portion thereof as defined herein.
[0078] In a further aspect, a method of producing anti-PD1 and anti-VEGFR2 antibodies and / or antigen-binding portions thereof is provided, the method comprising culturing a host cell of the invention under conditions resulting 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 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.
[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 of treating or preventing cancer in a subject comprising administering to the subject an effective amount of 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. 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, 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.
[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 an immunoconjugate or a nucleic acid molecule or a 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 invention provides anti-PD1 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 may comprise a pharmaceutically acceptable excipient, carrier or diluent.The pharmaceutically acceptable excipient may be a compound or a combination of compounds that does not induce secondary reactions and allows, for example, the smooth administration of anti-PD1 and anti-VEGFR2 antibody molecule, increase its life span and / or increase its effectiveness in the body or increase its solubility in solution.These pharmaceutically acceptable vehicles are well known and will be adapted by those skilled in the art as a function of the mode of administration of anti-PD1 and anti-VEGFR2 antibody molecule.
[0089] In some embodiments, the anti-PD1 and anti-VEGFR2 antibody molecules may be provided in lyophilized form for reconstitution prior to administration. For example, the lyophilized antibody molecules may be reconstituted in sterile water and mixed with saline prior to administration to an individual.
[0090] Anti-PD1 and anti-VEGFR2 antibody molecules are usually administered in the form of a pharmaceutical composition, which may contain at least one component in addition to the antibody molecule. Thus, the pharmaceutical composition may contain, in addition to the anti-PD1 and anti-VEGFR2 antibody molecules, pharma- ceutically 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 depends on the route of administration, which may be by bolus administration, infusion, injection or any other suitable route, as discussed below.
[0091] Parenterally, for example, by subcutaneous injection or intravenous administration, for example, by injection, the pharmaceutical composition comprising the anti-PD1 antibody molecule and the anti-VEGFR2 antibody molecule may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions using isotonic vehicles, such as, for example, sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants and / or other additives 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 molecules and the anti-VEGFR2 antibody molecules can be administered alone or in combination with other therapeutic agents, either simultaneously or sequentially, depending on the condition to be 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., treatment prior to 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 usually in a "therapeutically effective amount" that is 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 practitioners. Prescription of treatment, such as determining dosage, is within the responsibility of general practitioners and other physicians and may depend on the severity of symptoms and / or progression of the disease being treated. Appropriate doses of antibody molecules are well known in the art (Ledermann JA et al., 1991, Int. J. Cancer 47:659-664; Bagshawe KD et al., 1991, Antibody, Immunoconjugates and Radiopharmaceutics 4:915-922). Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) so that the type of agent administered is appropriately used. The therapeutically effective amount or appropriate dosage of an antibody molecule may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods are known for extrapolating effective dosages in mice and other test animals to humans. 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 exact 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 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., 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, and also for other antibody formats proportional to molecular weight. Treatment may be repeated at daily, twice-weekly, weekly, or monthly intervals, at the discretion of the physician. 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-operatively and / or post-operatively, and / or may be 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, e.g., using an auto-injector 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 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more in an individual.
[0099] The 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, (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 a CDR; (3) screening the library for binding to human PD1, and optionally cynomolgus PD1, and also for binding to human VEGFR2 and rhesus VEGFR2; (4) selecting clones from screening step (3) that have binding specificity for human PD1, and optionally also binding specificity for cynomolgus PD1 and human VEGFR2 and rhesus VEGFR2; (5) producing, from the clones selected from step (4), 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; (6) performing 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. The antibodies of the invention may cross-react with PD1 from species other than human, in particular PD1 from cynomolgus monkeys (Macaca fascicularis). Examples of human and cynomolgus monkey PD1 amino acid sequences are provided in Table 7. In certain embodiments, the 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 a portion of the biological activity of VEGFR2. As used herein, VEGFR2 may include all species of native sequence VEGFR2, including human, rat, mouse and chicken. The term "VEGFR2" may 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. The antibodies of the present invention may cross-react with VEGFR2 of species other than human, particularly VEGFR2 from rhesus monkeys (Macaca mulatta). Examples of human and rhesus VEGFR2 amino acid sequences are provided in Table 7. In certain embodiments, the 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 antibodies" (synonymous terms "anti-PD1 and anti-VEGFR2 antibodies") used in the context of the antibodies of the invention refer to antibodies 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 a cellular response to PD1 and VEGFR2. For purposes of the present invention, the term "anti-PD1 and anti-VEGFR2 antagonist antibody" is expressly understood to include 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 is substantially abolished, reduced or neutralized to any meaningful extent.
[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 other receptors.
[0106] An "antibody molecule" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., 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 chains thereof, fusion proteins comprising antibodies, and any other modified configurations of immunoglobulin molecules that contain 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 an antibody of any class, such as IgG, IgA or IgM (or a subclass thereof), and 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., lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2. 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 may 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, Fd fragment consisting of the VH and CH1 domains, 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 an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined to extend from an 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 may 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 known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions, which contribute to the formation of the antigen-binding site of the antibody. When selecting the FRs adjacent to the CDRs, for example, when humanizing or optimizing an antibody, FRs from an antibody 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 competing, schemes created in the field, and is based on a combination of immunoglobulin repertoire analysis and structural analysis of isolated antibodies and co-crystals 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 the 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 the functional activity. A preferred example of a "conservative substitution" is the substitution of one amino acid with another amino acid having a value of 0 or greater in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89:10915-10919): [Table 1]
[0114] 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, not the method by which it is produced. The monoclonal antibodies of the invention preferably exist in a homogenous or substantially homogenous population.
[0115] "Humanized" antibody molecules refer to forms of non-human (e.g., murine) antibody molecules or antigen-binding portions thereof, such as chimeric immunoglobulins, immunoglobulin chains or fragments thereof that contain minimal sequence derived from non-human immunoglobulin (Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies). Humanized antibodies may be human immunoglobulins (recipient antibody) in which residues from the CDRs of the recipient are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
[0116] 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 carrying human antibody genes.
[0117] 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.
[0118] "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.
[0119] 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.
[0120] The term "isolated molecule" (where the molecule is, for example, 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 associated in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) 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 originates 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 well known in the art for purification.
[0121] The term "epitope" refers to a portion of a molecule or antigen-binding portion thereof that can be 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 a combination of secondary structural units or structural domains of a target that are recognized by the antigen-binding region or antigen-binding portion of an antibody. Similarly, an epitope can consist of a defined chemically active surface grouping of a molecule, such as amino acids or sugar side chains, and has 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, such as, for example, conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structure determination methods (e.g., NMR).
[0122] 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 (kD)" to the association rate. off ) or "On Rate (k on )" is also called "K D is k off / k on and is expressed as molar concentration (M). Then, K D The smaller the K, the stronger the binding affinity. Therefore, a K of 1 μM 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.
[0123] 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.
[0124] 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 required to provide a therapeutic benefit to a subject, but less than a toxic amount.
[0125] The term "inhibit" or "neutralize" as used herein with respect to a biological activity of an antibody molecule of the invention refers to the ability of the antibody to substantially antagonize, prohibit, prevent, limit, slow down, eliminate, stop, reduce or reverse the progression or severity that is inhibited, including, but not limited to, the biological activity or binding interaction of the antibody molecule to PD1 or VEGFR2.
[0126] 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 mutations. A host cell includes cells transfected in vivo with a polynucleotide of the invention.
[0127] 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.
[0128] As used herein, unless otherwise indicated, the term "treating" refers to reversing, alleviating, inhibiting progression, slowing progression, delaying onset, 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 treatment 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 also include references to curative, palliative, and prophylactic treatment.
[0129] Where an embodiment is described herein with the term "comprising," it is understood that similar embodiments otherwise described with the terms "consisting of" and / or "consisting essentially of" are also provided.
[0130] When aspects or embodiments of the invention are described in terms of a Markush group or other alternative groupings, 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 not present. The invention also contemplates the explicit exclusion of one or more group members in the claimed invention.
[0131] 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 otherwise required by context, the singular includes the plural and the plural includes the singular. Any examples following the term "for example" are not meant to be exhaustive or limiting.
[0132] 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.
[0133] Specific non-limiting embodiments of the present invention will now be described with reference to the accompanying drawings. EXAMPLES
[0134] Example 1 Generation of Optimized Anti-PD1 Therapeutic Antibodies Introduction In this example, we have successfully generated a panel of antagonistic, optimized anti-PD1 antibodies that are simultaneously VEGFR2 antagonists, which are well expressed, biophysically stable, highly soluble, and have maximized identity to the preferred human germline.
[0135] 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 E. coli TG-1 cells, and the phage repertoire was essentially rescued as previously described in detail (Finlay et al., 2011, Methods Mol Biol 681: 383-401).
[0136] Phage selection was performed by coating streptavidin magnetic microbeads with biotinylated PD1 or VEGFR2 target protein (either human or monkey), washing the beads three times with PBS, and resuspending them in PBS pH 7.4 and 5% skim milk protein. These beads were coated with 100 nM of target protein in round 1 of selection, followed by three successive rounds of decreasing antigen concentrations. At each round, phages were eluted using trypsin and then re-infected into TG1 cells.
[0137] 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 resuspended in PBS pH 7.4. The supernatants containing soluble Fab were collected after shaking and centrifugation at room temperature.
[0138] 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, G237A mutations in the lower hinge that suppress normal immunoglobulin ADCC, ADCP and CDC functions, respectively) and human Cκ domains. 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.
[0139] Direct binding ELISA for Fab and IgG Binding and cross-reactivity to the lead panel recombinant proteins was first assessed by binding ELISA. Human PD1 human Fc-tagged recombinant protein and cynomolgus monkey PD1 human Fc-tagged recombinant protein were coated on the surface of MaxiSorp™ flat-bottom 96-well plates at 1 μg / ml. Purified Fab or IgG samples were titrated in two-fold serial dilutions 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.
[0140] AlphaScreen epitope competition assay for Fab peripreps AlphaScreen assays (Perkin Elmer) were performed in 384-well white microtiter plates (Greiner) in a final volume of 25 μl. Reaction buffer contained 1x PBS pH 7.3 (Oxoid, Cat. No. BR0014G) and 0.05% (v / v) Tween® 20 (Sigma, Cat. No. 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 min 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 h at room temperature, followed by the addition of streptavidin donor beads at 20 μg / ml (final concentration) and incubation for 30 min at room temperature. Light emission was measured in an EnVision multilabel plate reader (Perkin Elmer) and analyzed using the EnVision management software. Values were reported as Counts Per Second (CPS) and corrected for crosstalk. Percent signal reduction was calculated relative to unrelated samples.
[0141] PD1 / PD-L1 cell-based antagonism assay The ability of antibodies in blocking 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 at 100μl per well into each of the inner 60 wells of two 96-well white flat-bottom assay plates. Cell collection medium was added to each of the outer wells and the assay plate and incubated overnight at 37℃ / 5% CO2. On the day of the assay, sample IgG was diluted 4-fold from 300nM to 0,04nM in assay buffer (99% RPMI 1640 / 1% FBS) and 40μL per dilution was added to the assay plate containing PD-L1 aAPC / CHO-K1 cells. Positive inhibition controls included mAb005, IgG1 null and nivolumab IgG4. An irrelevant IgG was included as a negative inhibition control. PD1 effector cells were then thawed in assay buffer (99% RPMI 1640 / 1% FBS) and cell suspensions were added to wells of the assay plate containing 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, equilibrated 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 minutes, after which luminescence signals were measured at 10, 20, and 30 minutes.
[0142] 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.
[0143] 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 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 (Table 2). These libraries were transformed into E. coli, rescued, and phage populations from each library were subjected to four rounds of selection on human and cynomolgus PD1 and human and rhesus VEGFR2 proteins.
[0144] We then performed ELISA screening of the Fabs in peri-prep format to examine 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 showed binding to both the positive control hMAb005 Fab and to PD1 orthologues with similar signals. Importantly, binding analyses also demonstrated that in some libraries, enriched populations of clones not only retained PD1 activity but also showed significantly improved binding to human and / or cynomolgus VEGFR2. We picked 168 clones from these selected populations and further characterized both by 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 compete effectively with hMAb005, suggesting that VEGFR2 affinity-improving mutations in the VH domain had 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).
[0145] Based on the above analysis, five representative clones with mutations in LCDR1 or LCDR3 that showed high binding to both the orthologues of PD1 and VEGFR2 (Figure 1) and a signal reduction rate of hMAb005 / PD1 interaction of more than 75% (Figure 2) were selected for expression and characterization in IgG format (Table 5). In addition, 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 human IgG1 null format.
[0146] Characterization of lead IgG specificity and potency The 11 derived IgG1 null clones outlined in Table 4 as well as hMAb005 and isotype IgG1 null were examined in a titration ELISA binding to human and cynomolgus PD1 (Figure 3A). This analysis showed that all 11 lead clones had similar binding capacity 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 a significant increase in binding potency to both the human and rhesus orthologues compared to hMAb005 (Figure 3B).
[0147] To investigate whether this increased VEGFR2 reactivity to hMab005 in the lead clones could alter the pharmacological relationship with the receptor, a human VEGFR2 reporter assay was used to investigate the induction of luciferase expression under the control of the natural VEGF response element NFAT (Promega, performed according to the manufacturer's instructions). In this assay, soluble VEGF-165 protein was added to VEGFR2 reporter cells and all IgG1 proteins were examined for their ability to antagonize the signal induced by VEGF-165 (Figure 4). Both IgG1 hMAb005 and isotype IgG1 showed no antagonism of VEGFR2 signaling. However, to our surprise, all lead IgG1 null antibodies showed potent antagonism of VEGF-VEGFR2 signaling induction, with some clones showing a similar range of potency to 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 suggested a complete reversal of VEGFR2 pharmacology for all lead antibodies, while maintaining PD1 epitope specificity. To further explore this data, individual analyses were generated for library-derived clones (Figures 5A-5E) and clones combining mutations in LCDR1 and 3 (Figures 5F-5K). These analyses suggested that antibodies MAB01-MAB06 were the most potent inhibitors of VEGFR2 signaling, demonstrating that improvements in VEGFR2 binding mediated by each CDR could be combined synergistically.
[0148] 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 precursor antibody hMAb005 in multiple lead molecules. The resulting lead antibodies possess the beneficial features of dual antagonism of clinically relevant PD1 and VEGFR2 signaling pathways.
[0149] Generation and analysis of lead IgG variants Eight variants of clone MAB06 (MAB06.1-MAB06.8) were generated and mutated to human germline sequences of multiple CDR residues to experimentally test their ability to reduce immunogenicity risk. These clones contained germline mutations in LCDR1 and 2 (Table 8), as well as HCDR2 (Table 9). All eight clones were expressed and purified in an IgG1 null format. All clones were readily expressed and purified via a Protein A affinity column, and the resulting proteins exhibited >94% monomeric IgG% (as measured by SEC).
[0150] MAB06.1-MAB06.8 IgG were tested for their ability to antagonize both PD1 and VEGFR2 signaling. All eight clones retained the ability to antagonize PD1 signaling in the PD1 / PD-L1 cell signaling bioassay, with similar potency to both SHR-1210 IgG1-3M and nivolumab (Figure 6). Similarly, all eight clones retained the concentration-dependent ability to antagonize VEGFR2 signaling in the VEGF / VEGFR2 signaling bioassay, with similar potency to ramucirumab (Figure 7). Comparative analysis of PD1 and VEGFR2 antagonism for clones MAB06.5 (Figure 8A, Figure 8B), MAB06.6 (Figure 8C, Figure 8D), MAB06.7 (Figure 8E, Figure 8F) and MAB06.8 (Figure 8G, Figure 8H) showed 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.
[0151] The above findings indicate 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 co-expressed 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.
[0152] Antibodies that bind to high affinity signaling receptors run the risk of being potent agonists of receptor activity in the absence of receptor ligand. To examine this, the VEGFR2 bioassay was repeated 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 antibody protein starting at a concentration of 1000 nM (Figure 10). Only VEGF-165 showed concentration-dependent receptor activation, confirming that MAB06.5 IgG1 and MAB06.8 IgG1 are antagonists of VEGFR2 in the presence of VEGF (Figure 8) and not agonists of VEGFR2 in the absence of VEGF (Figure 10).
[0153] 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-gamma as an indicator of activity (Figure 11). MLR assays were performed in duplicate replicate runs using three separate human donor pairs. These analyses demonstrated that both MAB6.5 and MAB6.8 exhibited a concentration-dependent ability to block PD1 driving IFN-γ signaling comparable to nivolumab. This was found in both donor pair 1 (Figure 11A, 11B), donor pair 2 (Figure 11C, 11D) and donor pair 3 (Figure 11E, 11F) runs, demonstrating high reproducibility in blocking the function of PD1 on T cells.
[0154] 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 investigate 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 experimentally investigated for the ability to mutate multiple VH CDR residues to potentially further improve binding to PD1 and / or VEGFR2. All of these clones contained the MAB06.8VL sequence (SEQ ID NO: 47, Table 8) expressed in nine unique VH sequences (Table 10). All eight clones were expressed and purified in IgG1 null format. All clones were easily expressed and purified via a Protein A affinity column (except for MAB06.8.3 and MAB06.8.5, both of which suffer from aggregation), and the resulting proteins showed >95% monomeric IgG% (measured by SEC).
[0155] Seven well-expressing IgG1 null clones (MAB06.8.3 and MAB06.8.5 omitted) as well as hMAb005 and isotype IgG1 null were examined in titration ELISA binding to human and cynomolgus PD1 (Figure 12A, Figure 12B). This analysis showed that the majority 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 (Figure 12C, Figure 12D).
[0156] Despite the apparent retention of binding affinity for both PD1 and VEGFR2 in most clones, the benefit of MAB06.8 was neither retained 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 control, MAB06 (Figure 5), MAB06.5 (Figure 8), and MAB06.8 (Figure 8) (Figure 13A,B).
[0157] 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 of the incorporated documents or portions of documents defines a term that contradicts the definition in the application of that term, the definition set forth in the application of the present invention shall control. However, the 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.
[0158] While the invention has been described with reference to preferred or exemplary embodiments, those skilled in the art will recognize that various modifications and variations thereto can be effected 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, and no should be inferred.
[0159] Numbered embodiments Without limiting the scope of the appended claims, the present disclosure describes the following numbered embodiments.
[0160] 1. An antibody or antigen-binding portion of an antibody that specifically binds to both PD1 and VEGFR2, wherein the antibody or antigen-binding portion comprises a heavy chain variable (VH) region and a light chain variable (VL) region; (a) the VH region amino acid sequence includes 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 includes 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 includes 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 includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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: 11) of QQVSVTPFT; (d) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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; (e) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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 QQVYSIPWT; (g) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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; (h) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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; (i) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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; (j) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of 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; (l) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, 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; (m) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 2) of TISGGGATYYPDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGATYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGATYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGATYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, 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; (q) the VH region amino acid sequence includes HCDR1 (SEQ ID NO: 1) of GFTFSSYMMS, HCDR2 (SEQ ID NO: 38) of TISGGGATYYVDSVKG, and HCDR3 (SEQ ID NO: 3) of QLYYFDY, and the VL region amino acid sequence includes 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 includes 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 includes 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 domain 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 domain 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.
[0161] 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.
[0162] 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.
[0163] 4. An antibody or antigen-binding portion of an antibody 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) 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 thereof that antagonizes the binding of human PD1 to human PD-L1 and antagonizes human VEGFR2 signaling in response to human VEGF.
[0164] 5. The antibody or antigen-binding portion of any one of embodiments 1 to 4, wherein the antibody is human, humanized or chimeric.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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 are inserted.
[0169] 10. The antibody or antigen-binding portion of any one of embodiments 1 to 9, wherein the antibody comprises an immunoglobulin constant region.
[0170] 11. The antibody or antigen-binding portion of embodiment 10, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.
[0171] 12. The antibody or antigen-binding portion of embodiment 11, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.
[0172] 13. The antibody or antigen-binding portion of embodiment 10, wherein the immunoglobulin constant region is immunologically inert.
[0173] 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.
[0174] 15. The antibody or antigen-binding portion of embodiment 13, wherein the immunoglobulin constant region comprises any one of SEQ ID NOs: 25-31.
[0175] 16. The antibody or antigen-binding portion is a 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.
[0176] 17. The antibody or antigen-binding portion of any one of embodiments 1 to 16, wherein the antibody is monoclonal.
[0177] 18. The antibody or antigen-binding portion of any one of embodiments 1 to 17, wherein the antibody is a tetrameric antibody, a tetravalent antibody or a multispecific antibody.
[0178] 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.
[0179] 20. The antibody or antigen-binding portion of any one of embodiments 1 to 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.
[0180] 21. An immunoconjugate comprising the antibody or antigen-binding portion of any one of embodiments 1 to 20 linked to a therapeutic agent.
[0181] 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.
[0182] 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 pharma- ceutically acceptable carrier, diluent or excipient.
[0183] 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 domain amino acid sequence, or (c) both the VH and VL domain amino acid sequences A nucleic acid molecule encoding
[0184] 25. An expression vector comprising the nucleic acid molecule of embodiment 24.
[0185] 26. A recombinant host cell comprising the nucleic acid molecule of embodiment 24 or the expression vector of embodiment 25.
[0186] 27. A method for making an anti-PD1 antibody or an antigen-binding portion thereof, comprising: Culturing a recombinant host cell comprising the expression vector of embodiment 25 under conditions in which the nucleic acid molecule is expressed, thereby producing the antibody or antigen-binding portion; and isolating the antibody or antigen-binding portion from the host cell or culture.
[0187] 28. A method for enhancing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion thereof 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.
[0188] 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 an antibody or antigen-binding portion thereof 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.
[0189] 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.
[0190] 31. The method of embodiment 29, wherein the infectious disease is viral, bacterial, fungal or parasitic.
[0191] 32. The method of embodiment 29, wherein the infectious disease is human immunodeficiency virus (HIV) infection.
[0192] 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 the treatment of cancer, an infectious disease or an immune disease.
[0193] 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 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 a cancer of the blood tissue.
[0194] 35. The antibody or antigen-binding portion, immunoconjugate or pharmaceutical composition for use according to embodiment 33, wherein the infectious disease is viral, bacterial, fungal or parasitic.
[0195] 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.
[0196] 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. [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9]
Table 10
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 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 domain 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 domain 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; or (d) 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: 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.
2. The antibody or antigen-binding portion of claim 1, 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: 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.
3. The antibody or antigen-binding portion of claim 1, 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: 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.
4. The antibody or antigen-binding portion of claim 1, 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: 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.
5. The antibody or antigen-binding portion of claim 1, 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: 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.
6. (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:49 and the VL region amino acid sequence comprises SEQ ID NO:45; or (d) The antibody or antigen-binding portion of claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO:49 and the VL region amino acid sequence comprises SEQ ID NO:
46.
7. The antibody or antigen-binding portion of claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO:49 and the VL region amino acid sequence comprises SEQ ID NO:
44.
8. The antibody or antigen-binding portion of claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO:49 and the VL region amino acid sequence comprises SEQ ID NO:
47.
9. The antibody or antigen-binding portion of claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO:49 and the VL region amino acid sequence comprises SEQ ID NO:
45.
10. The antibody or antigen-binding portion of claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO:49 and the VL region amino acid sequence comprises SEQ ID NO:
46.
11. The antibody or antigen-binding portion of any one of claims 1 to 5, wherein the antibody is humanized or chimeric.
12. 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.
13. 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 a human variable region framework scaffold amino acid sequence into which the CDRs are inserted.
14. The antibody or antigen-binding portion of any one of claims 1 to 5, 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.
15. The antibody or antigen-binding portion of any one of claims 1 to 5 and 14, 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.
16. The antibody or antigen-binding portion of any one of claims 1 to 15, wherein the antibody or antigen-binding portion comprises an immunoglobulin constant region.
17. 17. The antibody or antigen-binding portion of claim 16, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.
18. 18. The antibody or antigen-binding portion of claim 17, wherein the immunoglobulin constant region is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.
19. 17. The antibody or antigen-binding portion of claim 16, wherein the immunoglobulin constant region is immunologically inert.
20. 17. The antibody or antigen-binding portion of claim 16, 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.
21. 17. The antibody or antigen-binding portion of claim 16, wherein the immunoglobulin constant region comprises an amino acid sequence represented by SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:
31.
22. The antibody or antigen-binding portion is selected from the group consisting of Fab, Fab', F(ab') 2 22. The antibody or antigen-binding portion of any one of claims 1 to 21, which is an Fv, scFv, maxibody, minibody, diabody, triabody, tetrabody or bis-scFv. (a) an antibody or antigen-binding portion thereof according to any one of claims 1 to 22, and (b) Therapeutic Agent Including, An immunoconjugate, wherein the antibody or antigen-binding portion is linked to the therapeutic agent.
24. 24. The immunoconjugate of claim 23, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, a mitogenic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.
25. A pharmaceutical composition comprising the antibody or antigen-binding portion of any one of claims 1 to 22 or the immunoconjugate of claim 23 or 24 and a pharma- ceutically acceptable carrier, diluent or excipient.
26. Both the VH and VL domain amino acid sequences of the antibody or antigen-binding portion of any one of claims 1 to 22 A nucleic acid molecule encoding the
27. 27. An expression vector comprising the nucleic acid molecule of claim 26.
28. 28. A recombinant host cell comprising the nucleic acid molecule of claim 26 or the expression vector of claim 27.
29. 1. A method of making an anti-PD1 and anti-VEGFR2 antibody or antigen-binding portion thereof, comprising: Culturing the recombinant host cell of claim 28 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.
30. An antibody or antigen-binding portion according to any one of claims 1 to 22, an immunoconjugate according to claim 23 or 24 or a pharmaceutical composition according to claim 25 for use in enhancing an immune response in a subject.
31. An antibody or antigen-binding portion of any one of claims 1 to 22, an immunoconjugate of claim 23 or 24, or a pharmaceutical composition of claim 25, for use in the treatment of cancer, an infectious disease or an immune disease.
32. 32. The antibody or antigen-binding portion, immunoconjugate or pharmaceutical composition for use according to claim 31 , 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 a cancer of the blood tissue.
33. 32. The antibody or antigen-binding portion, immunoconjugate or pharmaceutical composition for use according to claim 31 , wherein the infectious disease is viral, bacterial, fungal or parasitic.
34. 32. The antibody or antigen-binding portion, immunoconjugate or pharmaceutical composition for use according to claim 31 , wherein the infectious disease is human immunodeficiency virus (HIV) infection.
35. An antibody or antigen-binding portion according to any one of claims 1 to 22, an immunoconjugate according to claim 23 or 24 or a pharmaceutical composition according to claim 25 for use as a medicament.
Citation Information
Patent Citations
PD-1 antibody, antigen-binding fragment thereof, and medical application thereof
WO2015085847A1
PD1 binding agents
WO2019170885A1