[go: up one dir, main page]

WO2025016011A1 - Anticorps anti-pd-1/ctla-4/vegf et leur utilisation - Google Patents

Anticorps anti-pd-1/ctla-4/vegf et leur utilisation Download PDF

Info

Publication number
WO2025016011A1
WO2025016011A1 PCT/CN2024/089982 CN2024089982W WO2025016011A1 WO 2025016011 A1 WO2025016011 A1 WO 2025016011A1 CN 2024089982 W CN2024089982 W CN 2024089982W WO 2025016011 A1 WO2025016011 A1 WO 2025016011A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
cancer
antigen
binding fragment
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/089982
Other languages
English (en)
Inventor
Yaxuan LV
Fei Peng
Juehua XU
Zhen Qian
Huanhuan Cao
Xueyan YANG
Qian Ding
Yongcong TAN
Shuhua Han
Qinglin Du
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GENOR BIOPHARMA CO Ltd
Original Assignee
GENOR BIOPHARMA CO Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by GENOR BIOPHARMA CO Ltd filed Critical GENOR BIOPHARMA CO Ltd
Publication of WO2025016011A1 publication Critical patent/WO2025016011A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to anti-PD-1 antibodies, trispecific antibodies specifically binding to PD-1, CTLA-4 and VEGF, or antigen-binding fragments thereof, polynucleotides encoding the antibodies or antigen-binding fragments thereof, and methods of making and using the foregoing.
  • PD-1 Programmed cell death protein 1
  • PD-1 regulates T cell function by inhibiting T cell activation and proliferation, as well as promoting T cell exhaustion (Liu et al., 2021; Sharpe & Pauken, 2018) .
  • PD-1 is activated by its ligands, PD-L1 and PD-L2, which are expressed on certain tumor cells and other antigen-presenting cells.
  • PD-1 inhibition has been shown to enhance T cell activity and promote anti-tumor immune responses in preclinical and clinical studies (Chen et al., 2021; B. Zhao, Zhao, & Zhao, 2020) .
  • PD-1 inhibitors such as Nivolumab and Pembrolizumab have been approved by the FDA for the treatment of several cancer types, including lung cancer, melanoma, and bladder cancer.
  • Keytruda is used to treat several types of cancer, including melanoma, non-small cell lung cancer, and head and neck cancer.
  • Nivolumab is used to treat cancers including melanoma, non-small cell lung cancer, and renal cell carcinoma.
  • VEGF Vascular endothelial growth factor
  • TME tumor microenvironment
  • VEGF inhibition has been shown to interfere with tumor angiogenesis and reduce tumor growth in preclinical and clinical studies (Baraniskin et al., 2019; Garcia et al., 2020; Y. Zhao et al., 2022) .
  • VEGF inhibitors such as Bevacizumab which works by blocking VEGF binding to VEGFR1, VEGFR2 and hence preventing the downstream signaling and formation of new blood vessels, have been approved by the FDA for the treatment of several cancer types, including colorectal cancer, non-small cell lung cancer and hepatocellular carcinoma etc.
  • Cytotoxic lymphocyte-associated molecule-4 (CTLA-4, CD152) is another immune checkpoint protein that regulates T cell function.
  • CTLA4 shares identical ligands with CD28, which are CD80/B7-1 and CD86/B7-2.
  • CTLA-4 is constitutively expressed on Foxp3+T regulatory cells (Tregs) and is upregulated on other T cells upon activation (Jago, Yates, Camara, Lechler, & Lombardi, 2004; Menger, Bergerhoff, Peggs, & Quezada, 2015) .
  • CTLA4 was proposed as dampening T cell activation by outcompeting CD28 in binding with CD80/B7-1 and CD86/B7-2 (Chikuma, 2017) .
  • CTLA-4 inhibitors such as Ipilimumab have been approved by the FDA for the treatment of several cancer types, including melanoma and prostate cancer.
  • Ipilimumab as the best-known anti-CTLA4 antibody, was approved in 2011 for the treatment of advanced melanoma. Ipilimumab has exhibited potent cancer immunotherapeutic effects clinically both as monotherapy (Hodi et al., 2010) and as part of combination therapy with Nivolumab (Larkin et al., 2015) .
  • CTLA-4 therapy demonstrates severe immunotherapy-related adverse effects (irAEs) (Calabrese, Calabrese, & Cappelli, 2018) , especially when combined with Nivolumab treatment due to systemic activation of T cells by blocking the B7-CTLA-4 pathway, resulting in lowering the patient’s tolerance of the antibodies (Bertrand, Kostine, Barnetche, Truchetet, & Schaeverbeke, 2015; Hodi, 2010) . Even so, CTLA-4 remains an important immunotherapy target because of the capacity of inducing long-lasting immunity in cancer patients (Maio et al., 2015; dudorf et al., 2015) . The major challenge of generating CTLA4 antibodies is to improve their safety and efficacy.
  • irAEs severe immunotherapy-related adverse effects
  • PD-1, CTLA-4, and VEGF are essential targets for cancer therapy, and their inhibition has shown promising results in preclinical and clinical studies.
  • PD-1 and CTLA-4 inhibitors enhance T cell activity and promote anti-tumor immune responses
  • VEGF inhibitors interfere with tumor angiogenesis and reduce tumor growth.
  • drugs like Ipilimumab has various side effects. Thus, further studies are needed to optimize the use of these inhibitors and explore their potential in combination with other cancer therapies.
  • the invention provides an isolated antibody or antigen-binding fragment thereof that binds to PD-1 (Programmed cell death protein 1) , comprising: a heavy-chain antibody variable domain (VHH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80%identical to SEQ ID NO: 1, the VHH CDR2 region comprises an amino acid sequence that is at least 80%identical to SEQ ID NO: 2, and the VHH CDR3 region comprises an amino acid sequence that is at least 80%identical to SEQ ID NO: 3.
  • VHH heavy-chain antibody variable domain
  • CDRs complementarity determining regions
  • the VHH comprises CDRs 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.
  • the VHH comprises an amino acid sequence that is at least 80%identical to SEQ ID NO: 4.
  • the VHH comprises the amino acid sequence of SEQ ID NO: 4, or consists of the amino acid sequence of SEQ ID NO: 4.
  • the antibody or antigen-binding fragment specifically binds to PD-1.
  • the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.
  • the invention also provides an isolated antibody or antigen-binding fragment thereof comprising the VHH CDRs 1, 2, and 3, of the antibody or antigen-binding fragment thereof above.
  • the antibody or antigen-binding fragment comprises two or more heavy-chain antibody variable domains.
  • the invention also provides an isolated antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof above.
  • the invention also provides an isolated multi-specific antibody or antigen-binding fragment thereof, comprising a first domain specifically binding to PD-1and a second domain specifically binding to CTLA-4.
  • the antibody or antigen-binding fragment thereof further comprises one or more of additional domains that specifically bind to an antigen other than PD-1 and CTLA-4.
  • the antibody or antigen-binding fragment thereof further comprises a domain specifically binding to VEGF.
  • an anti-PD-1/CTLA-4/VEGF trispecific antibody or antigen-binding fragment thereof comprising a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and a third domain specifically binding to VEGF.
  • the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 1, 2, and 3, respectively.
  • VH1 heavy chain variable region
  • HCDR1 heavy chain complementarity determining regions 1
  • HCDR2 heavy chain complementarity determining regions 2
  • HCDR3 heavy chain complementarity determining regions 3 of SEQ ID NOs: 1, 2, and 3, respectively.
  • the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1) comprising an amino acid sequence that is at least 80%identical to SEQ ID NO: 4. In one embodiment, the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1) of SEQ ID NO: 4, or consists of a heavy chain variable region (VH) of SEQ ID NO: 4.
  • the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 7, 8, and 9, respectively.
  • VH2 heavy chain variable region
  • the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2) comprising an amino acid sequence that is at least 80%identical to SEQ ID NO: 10.
  • the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2) of SEQ ID NO: 10, or consists of a heavy chain variable region (VH) of SEQ ID NO: 10.
  • the third domain specifically binding to VEGF comprises a heavy chain variable region (VH3) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 13, 14, and 15, respectively, and a light chain variable region (VL) comprising light chain complementarity determining regions 1 (LCDR1) , 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs: 19, 20, and 21, respectively.
  • VH3 heavy chain variable region
  • VL light chain variable region
  • the third domain specifically binding to VEGF comprises a heavy chain variable region (VH3) of SEQ ID NO: 16 and a light chain variable region (VL) of SEQ ID NO: 22, or consists of the same.
  • the third domain specifically binding to VEGF comprises a heavy chain of SEQ ID NO: 17 and a light chain of SEQ ID NO: 23, or consists of the same.
  • the third domain specifically binding to VEGF comprises a full length antibody comprised of two heavy chains and two light chains inter-connected by disulfide bonds, or antigen-binding fragment thereof, or consists of the same.
  • the antigen-binding fragment of the full length antibody is a Fab fragment or a scFv fragment.
  • the isolated antibody or antigen-binding fragment thereof further comprises an Fc region of IgG.
  • the Fc region is of IgG1.
  • the Fc region is of IgG1 LALA (IgG1 with Leu234Ala/Leu235Ala mutations) .
  • the antibody or antigen-binding fragment thereof according to the present invention is of an IgG1, IgG2, IgG3, or IgG4 isotype, optionally comprising one, two, three, four, five, six, seven, eight, nine or ten substitutions in an Fc region.
  • the first domain specifically binding to PD-1 is positioned at the N-terminal of the Fc region.
  • the second domain specifically binding to CTLA-4 is positioned at the N-terminal of the Fc region.
  • the third domain specifically binding to VEGF is positioned at the N-terminal of the Fc region.
  • the first domain specifically binding to PD-1 is positioned at the C-terminal of the Fc region.
  • the second domain specifically binding to CTLA-4 is positioned at the C-terminal of the Fc region.
  • the third domain specifically binding to VEGF is positioned at the C-terminal of the Fc region.
  • the first domain specifically binding to PD-1, the second domain specifically binding to CTLA-4, the third domain specifically binding to VEGF, and the Fc region are connected to each other directly or via one or more linkers.
  • the linkers are same or different.
  • the linkers are a flexible connection.
  • the linker is a peptide linker.
  • the linker is GGGGSGGGGS.
  • the third domain specifically binding to VEGF is a Fab fragment or a scFv fragment
  • the anti-PD-1/CTLA-4/VEGF trispecific antibody according to the present invention is shown by the following formula from N to C terminal:
  • the third domain specifically binding to VEGF is a full length antibody.
  • the first domain specifically binding to PD-1 is connected optionally via a linker at the C terminal of either of or both the heavy chains of the full length antibody
  • the second domain specifically binding to CTLA-4 is connected optionally via a linker at the C terminal of the first domain specifically binding to PD-1.
  • the second domain specifically binding to CTLA-4 is connected optionally via a linker at the C terminal of either of or both the heavy chains of the full length antibody
  • the first domain specifically binding to PD-1 is connected optionally via a linker at the C terminal of the second domain specifically binding to CTLA-4.
  • first domain specifically binding to PD-1 and the second domain specifically binding to CTLA-4 are connected in the same or differenent sequence at the C terminal of the two heavy chains of the full length antibody.
  • two second domains specifically binding to CTLA-4 are each connected via a linker at the C terminal of the heavy chains of the full length antibody, and two first domains specifically binding to PD-1 are each connected via a linker at the C terminal of the two second domains specifically binding to CTLA-4.
  • the anti-PD-1/CTLA-4/VEGF trispecific antibody according to the present invention comprises a heavy chain of SEQ ID NO: 27 and a light chain of SEQ ID NO: 23, or conisists of the same.
  • the antibody according to the present invention comprises one or two heavy chains and one or two light chains, or consists of the same. In one embodiment, the antibody according to the present invention comprises two heavy chains and two light chains, or consists of the same. In one embodiment, the anti-PD-1/CTLA-4/VEGF trispecific antibody according to the present invention comprises a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and a third domain specifically binding to VEGF.
  • the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 1, 2, and 3, respectively;
  • the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 7, 8, and 9, respectively;
  • the third domain specifically binding to VEGF comprises a heavy chain variable region (VH3) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 13, 14, and 15, respectively, and a light chain variable region (VL) comprising light chain complementarity determining regions 1 (LCDR1) , 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs: 19, 20, and 21, respectively.
  • the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1) of SEQ ID NO: 4, or consists of a heavy chain variable region (VH1) of SEQ ID NO: 4;
  • the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2) of SEQ ID NO: 10, or consists of a heavy chain variable region (VH2) of SEQ ID NO: 10;
  • the third domain specifically binding to VEGF comprises a heavy chain variable region (VH3) of SEQ ID NO: 16 and a light chain variable region (VL) of SEQ ID NO: 22, or consists of the same.
  • the heavy chain comprises an amino acid sequence of SEQ ID NO: 27 and a light chain of SEQ ID NO: 23, or consists of the same.
  • the invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the invention and a pharmaceutically accepted carrier.
  • the invention also provides a polynucleotide encoding the heavy chain variable regions (VHs) or the heavy chain of the antibody or antigen-binding fragment thereof of the invention.
  • VHs heavy chain variable regions
  • the invention also provides a polynucleotide encoding the antibody or antigen-binding fragment thereof of the invention.
  • the invention also provides a vector comprising the polynucleotide of the invention.
  • the invention also provides a host cell comprising the vector of the invention.
  • the invention also provides a method of producing the antibody of the invention, comprising culturing the host cell of the invention in conditions that the antibody or antigen-binding fragment thereof is expressed, and recovering the antibody produced by the host cell.
  • the invention also provides a method of treating a cancer in a subject, comprising administering a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof of the invention to the subject in need thereof for a time sufficient to treat the cancer.
  • the invention also provides use of the isolated antibody or antigen-binding fragment thereof of the invention in the manufacture of a medicament for treating a cancer in a subject in need thereof.
  • the invention also provides the isolated antibody or antigen-binding fragment thereof of the invention, for use in treating a cancer in a subject in need thereof.
  • FIG 1 shows schematic diagram of the structure of the trispecific antibody GBD214-33-03 (anti-PD1/CTLA4/VEGF trispecific antibody) according to the present invention.
  • Figure 2 shows binding specifities of different antibodies to human PD1 protein.
  • the results showed GBD214-33-03 binding to PD1 in HEK293-PD1 cells.
  • GBD214-33-03 showed potent binding to HEK293-PD1 cells with a similar median fluorescence intensity (MFI) to that of its PD1 parental, GBD002-hS019-WS(anti-PD1 antibody) and pembrolizumab.
  • MFI median fluorescence intensity
  • FIG. 3 shows the performance of different antibodies in PD1 reporter assay.
  • the PD1 reporter assay showed GBD214-33-03 has similar maximum luminescence signals compared with pembrolizumab and AK104 (anti-PD1/CTLA4 bispecif antibody by Akesobio, Inc. ) .
  • the result demonstrates that GBD214-33-03 can block the interaction of PD1 to its ligand PD-L1.
  • FIG. 4 shows the performance of different antibodies in mixed lymphocyte reaction (MLR) assay.
  • MLR mixed lymphocyte reaction
  • Figure 5 shows VEGF binding of different antibodies. The result showed GBD214-33-03 has similar VEGF binding compared with bevacizumab.
  • Figure 6 shows VEGF blocking of different antibodies. The result showed GBD214-33-03 can inhibit VEGFR2 binding to VEGF165, which is similar with bevacizumab.
  • Figure 7 shows the performance of different antibodies in VEGF reporter assay. The result showed GBD214-33-03 can inhibit VEGF165 induced downstream NFAT signaling by blocking VEGFR2 binding to VEGF165, which is similar with bevacizumab.
  • Figures 8a and 8b show binding specificities of different antibodies to human CTLA4 protein.
  • the results showed that in CHOK1-CTLA4 cells GBD214-33-03 has lower MFI compared to ipilimumab, which suggests GBD214-33-03 partially binding to CTLA4 in CHOK1-CTLA4 cells.
  • the CTLA4 parental GBD008-hS005-3-2 anti-CTLA4 antibody
  • GBD214-33-03 showed potent binding compared with its PD1 parental, GBD002-hS019-WS.
  • Figures 9a to 9d show blocking of CD80 and CD86 binding to CTLA4 by different antibodies.
  • the results showed that GBD214-33-03 partially inhibited the binding of CD80/CD86 to CTLA4 in CHOK1-CTLA4 and CHOK1-PD1-CTLA4 cells.
  • GBD214-33-03 showed weaker blocking capability compared with ipilimumab.
  • Figure 10 shows PD1 receptor downregulation in CHOK1-PD1-CTLA4 cells by different antibodies. The results showed the PD1 downregulation in the cells due to CTLA4 internalization. The percentage of downregulated PD1 was measured. GBD214-33-03 showed similar PD1 internalization compared with AK104.
  • Figures 11a and 11b show the in vivo anti-tumor efficacy of different antibodies in A375 PBMC mouse model. Results showed that in A375 PBMC model, GBD214-33-03 (1 mg/kg, 5 mg/kg and 20 mg/kg) has better efficacy compared to AK112 (anti-PD1/VEGF bispecifc antibody by Akesobio, Inc.
  • mice (1 mg/kg, 5 mg/kg and 20 mg/kg) , AK104 (1 mg/kg, 5 mg/kg and 20 mg/kg) , the combo of pembrolizumab, bevacizumab and Ipilimumab-LALA (Ipilimumab with Leu234Ala/Leu235Ala mutations) (15 mg/kg+15 mg/kg+15 mg/kg) .
  • the average body weight of mice started to decrease from Day 7 with no more than 15%reduction for all treatment groups.
  • Figures 12a and 12b show in vivo anti-tumor efficacy of different antibodies in HT29 PBMC mouse model. Results showed that in HT29 PBMC model, GBD214-33-03 (1 mg/kg, 5 mg/kg and 20 mg/kg) has better efficacy compared to AK112 (1 mg/kg, 5 mg/kg and 20 mg/kg) , AK104 (1 mg/kg, 5 mg/kg and 20 mg/kg) , the combo of pembrolizumab, bevacizumab and Ipilimumab-LALA (15 mg/kg+15 mg/kg+15 mg/kg) . The average body weight of mice was relatively stable with no more than 15%reduction for all treatment groups.
  • Specific binding or “specifically binds” or “binds” refers to an antibody binding to an antigen or an epitope within the antigen with greater affinity than for other antigens.
  • the antibody binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (K D ) of about 1x10 -8 M or less, for example about 1x10 -9 M or less, about 1x10 -10 M or less, about 1x10 -11 M or less, or about 1x10 -12 M or less, typically with the K D that is at least one hundred fold less than its K D for binding to a non-specific antigen (e.g., BSA, casein) .
  • the dissociation constant may be measured using standard procedures.
  • Antibodies that specifically bind to the antigen or the epitope within the antigen may, however, have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs) , such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) , Pan troglodytes (chimpanzee, chimp) or Callithrixjacchus (common marmoset, marmoset) . While a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two distinct antigens or two distinct epitopes.
  • Antibodies is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen-binding fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity.
  • “Full length antibodies” are comprised of two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds as well as multimers thereof (for example IgM) . Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CH1, hinge CH2 and CH3) .
  • Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL) .
  • the VH and the VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with framework regions (FR) .
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • Complementarity determining regions are “antigen binding sites” in an antibody. CDRs may be defined using various terms: (i) Complementarity Determining Regions (CDRs) , three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat, (1970) J Exp Med 132: 211-50; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) .
  • “Hypervariable regions” , “HVR” , or “HV” three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3) refer to the regions of an antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk, (1987) Mol Biol 196: 901-17) .
  • the International ImMunoGeneTics (IMGT) database http: //www_imgt_org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between CDRs, HVs and IMGT delineations is described in Lefranc et al., (2003) Dev Comparat Immunol 27: 55-77.
  • CDR CDR
  • HCDR1 CDR1
  • HCDR2 CDR3
  • LCDR1 CDR2
  • LCDR3 CDR3
  • Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
  • IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.
  • Antibody light chains of any vertebrate species may assigned to one of two clearly distinct types, namely kappa ( ⁇ ) and lambda ( ⁇ ) , based on the amino acid sequences of their constant domains.
  • Isolated antibody refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding to PD-1 is substantially free of antibodies that specifically bind antigens other than PD-1) .
  • the trispecific antibody specifically binds PD-1, CTLA-4 and VEGF, and is substantially free of antibodies that specifically bind antigens other than PD-1, CTLA-4 and VEGF.
  • isolated antibody encompasses antibodies that are isolated to a higher purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%pure.
  • Recombinant refers to antibodies and other proteins that are prepared, expressed, created or isolated by recombinant means.
  • Epitope refers to a portion of an antigen to which an antibody specifically binds. Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be composed of contiguous and/or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule. Antibody “epitope” depends on the methodology used to identify the epitope.
  • Multispecific refers to an antibody that specifically binds at least two distinct antigens or two distinct epitopes within the antigens, for example three, four or five distinct antigens or epitopes.
  • Trispecific refers to an antibody that specifically binds three distinct antigens or three distinct epitopes within the same antigen.
  • the trispecific antibody may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs) , such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) , Pan troglodytes (chimpanzee, chimp) or Callithrixjacchus (common marmoset, marmoset) , or may bind an epitope that is shared between two or more distinct antigens.
  • homologs such as human or monkey
  • Macaca fascicularis cynomolgus, cyno
  • Pan troglodytes chimpanzee, chimp
  • Callithrixjacchus common marmoset, marmoset
  • Vector refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems.
  • Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system.
  • Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector.
  • the polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid of these.
  • “Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.
  • Polynucleotide refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry.
  • cDNA is a typical example of a polynucleotide.
  • “Overexpress” “overexpressed” and “overexpressing” is used interchangeably and refers to a sample such as a cancer cell, malignant cell or cancer tissue that has measurably higher levels of PD-1, CTLA-4 or VEGF, or a ligand thereof when compared to a reference sample.
  • the overexpression may be caused by gene amplification or by increased transcription or translation.
  • Expression and overexpression of protein in the sample may be measured using well know assays using for example ELISA, immunofluorescence, flow cytometry or radioimmunoassay on live or lysed cells.
  • Expression and overexpression of a polynucleotide in the sample may be measured for example using fluorescent in situ hybridization, Southern blotting, or PCR techniques.
  • a protein or a polynucleotide is overexpressed when the level of the protein or the polynucleotide in the sample at least 1.5-fold higher or statistically significant when compared to the reference sample. Selection of the reference sample is known.
  • sample refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject.
  • exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, ascites fluids such as those associated with non-solid tumors, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage, liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium, lavage fluids and the like, tissue biopsies, fine needle aspirations or surgically resected tumor tissue.
  • biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sput
  • a “cancer cell” or a “tumor cell” refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. Although transformation may arise from infection with a transforming virus and incorporation of new genomic nucleic acid, uptake of exogenous nucleic acid or it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene.
  • Transformation/cancer is exemplified by morphological changes, immortalization of cells, aberrant growth control, foci formation, proliferation, malignancy, modulation of tumor specific marker levels, invasiveness, tumor growth in suitable animal hosts such as nude mice, and the like, in vitro, in vivo, and ex vivo (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994) ) .
  • “About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result or embodiment, “about” means within one standard deviation per the practice in the art, or a range of up to 5%,whichever is larger.
  • Anti-PD-1/CTLA-4/VEGF trispecific antibody refers to a molecule comprising at least one binding domain specifically binding to PD-1, at least one binding domain specifically binding to CTLA-4 and at least one binding domain specifically binding to VEGF.
  • the domains specifically binding to PD-1, CTLA-4 and VEGF are typically VH/VL pairs or only VHs.
  • the trispecific anti-PD-1/CTLA-4/VEGF antibody may be monovalent or divalent in terms of its binding to PD-1, CTLA-4 or VEGF.
  • Value refers to the presence of a specified number of binding sites specific for an antigen in a molecule.
  • the terms “monovalent” , “bivalent” , “tetravalent” , and “hexavalent” refer to the presence of one, two, four and six binding sites, respectively, specific for an antigen in a molecule.
  • single-chain Fv or “scFv” antibody refers to antibody fragments comprising the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the scFv to form the desired structure for antigen-binding.
  • scFv see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also, International Publ. No. WO 88/01649 and U.S. Pat.
  • the scFv comprises from N to C terminal the V H region, the peptide linker and the V L region (VH-VL format) . In another embodiment, the scFv comprises from N to C terminal the V L region, the peptide linker and the V H region (VL-VH format) .
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V H ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H -V L or V L -V H ) .
  • V H heavy chain variable domain
  • V L light chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, e.g., EP 404, 097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.
  • Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136 For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.
  • a “Fab” is comprised of the VH and CH1 regions of a heavy chain and the VL and CL regions of a light chain, which are typically joined together by disulfide bonds and have a single antigen binding site.
  • the VH, CH1, VL and CL regions in a Fab can be arranged in various ways to confer an antigen binding capability according to the present disclosure.
  • the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on a separate polypeptide.
  • VH, CH1, VL and CL regions can all be on the same polypeptide, optionally arranged in different orders.
  • An antigen specific CD4 + or CD8 + T cell refers to a CD4 + or CD8 + T cell activated by a specific antigen, or immunostimulatory epitope thereof.
  • Subject includes any human or nonhuman animal.
  • Nonhuman animal includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms “patient” or “subject” are used interchangeably.
  • Treating” or “treatment” refers to therapeutic treatment wherein the object is to slow down (lessen) an undesired physiological change or disease, such as the development or spread of tumor or tumor cells, or to provide a beneficial or desired clinical outcome during treatment.
  • Beneficial or desired clinical outcomes include alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, lack of metastasis, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable.
  • Treatment may also mean prolonging survival as compared to expected survival if a subject was not receiving treatment.
  • Those in need of treatment include those subjects already with the undesired physiological change or diseases well as those subjects prone to have the physiological change or disease.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • Atherapeutically effective amount of the antibody of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody of the invention to elicit a desired response in the individual.
  • Exemplary indicators of an effective therapeutic or combination of therapeutics include, for example, improved well-being of the patient, reduction in a tumor burden, arrested or slowed growth of a tumor, and/or absence of metastasis of cancer cells to other locations in the body.
  • the present invention provides anti-PD-1 antibodies, anti-PD-1/CTLA-4 antibodies, and especially trispecific antibodies specifically binding to PD-1, CTLA-4 and VEGF, or antigen-binding fragments thereof.
  • the present invention provides polynucleotides encoding the antibodies or antigen-binding fragments thereof of the invention or complementary nucleic acids thereof, vectors, host cells, and methods of making and using them.
  • the antibodies of the invention comprise the antigen-binding fragments thereof where applicable.
  • an isolated anti-PD-1 antibody or antigen-binding fragment thereof may be abbreviated as "an isolated anti-PD-1 antibody” , and so on.
  • the invention provides an isolated antibody or antigen-binding fragment thereof that binds to PD-1 (Programmed cell death protein 1) , comprising: a heavy-chain antibody variable domain (VHH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 1, the VHH CDR2 region comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 2, and the VHH CDR3 region comprises an amino acid sequence that is at least 80%, 81%
  • the VHH comprises CDRs 1, 2, and 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.
  • the VHH comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 4.
  • the VHH comprises the amino acid sequence of SEQ ID NO: 4, or consists of the amino acid sequence of SEQ ID NO: 4.
  • the antibody or antigen-binding fragment thereof that binds to PD-1 comprises a heavy chain comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 5.
  • the antibody or antigen-binding fragment thereof that binds to PD-1 comprises a heavy chain comprising, or consisting of, the amino acid sequence of SEQ ID NO: 5.
  • the antibody or antigen-binding fragment specifically binds to PD-1. In one embodiment, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.
  • the invention also provides an isolated antibody or antigen-binding fragment thereof comprising the VHH CDRs 1, 2, and 3, of the antibody or antigen-binding fragment thereof above.
  • the antibody or antigen-binding fragment thereof comprises the VHH CDRs 1, 2, and 3, each of which comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NOs: 1, 2, and 3, respectively.
  • the antibody or antigen-binding fragment thereof comprises the VHH CDRs 1, 2, and 3 set forth in SEQ ID NOs: 1, 2, and 3, respectively.
  • the antibody or antigen-binding fragment comprises two or more heavy-chain antibody variable domains. In one embodiment, the antibody or antigen-binding fragment comprises two, three, four, five, six, seven, eight, nine or ten VHHs.
  • the invention also provides an isolated antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof above.
  • the antibody or antigen-binding fragment thereof cross-competes with an antibody or antigen-binding fragment thereof comprising the VHH CDRs 1, 2, and 3, each of which comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NOs: 1, 2, and 3, respectively.
  • the antibody or antigen-binding fragment thereof cross-competes with an antibody or antigen-binding fragment thereof comprising the VHH CDRs 1, 2, and 3 set forth in SEQ ID NOs: 1, 2, and 3, respectively.
  • the antibody or antigen-binding fragment thereof cross-competes with an antibody or antigen-binding fragment thereof comprising the VHH comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 4.
  • the antibody or antigen-binding fragment thereof cross-competes with an antibody or antigen-binding fragment thereof comprising the VHH comprising, or consisting of, the amino acid sequence of SEQ ID NO: 4.
  • the invention also provides an isolated multi-specific antibody or antigen-binding fragment thereof, comprising a first domain specifically binding to PD-1 and a second domain specifically binding to CTLA-4.
  • the antibody further comprises one or more of additional domains that specifically bind to an antigen other than PD-1 and CTLA-4.
  • the additional domains that specifically bind to an antigen other than PD-1 and CTLA-4 comprise a third domain specifically binding to VEGF.
  • an isolated anti-PD-1/CTLA-4/VEGF trispecific antibody or antigen-binding fragment thereof comprising a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4 and a third domain specifically binding to VEGF.
  • the antibody or antigen-binding fragment thereof of the invention enhances activation of antigen-specific CD4 + or CD8 + T cells.
  • the antibody or antigen-binding fragment thereof of the invention inhibits PD-1 binding to PD-L1 and PD-L2.
  • the antibody or antigen-binding fragment thereof of the invention inhibits CTLA-4 binding to CD80 and CD86.
  • the antibody or antigen-binding fragment thereof of the invention inhibits VEGF binding to VEGFR1 and/or VEGFR2.
  • the antibody or antigen-binding fragment thereof of the invention induces PD-1 internalization on the surface of cells.
  • the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) , wherein the HCDR1, HCDR2 and HCDR3 each comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NOs: 1, 2, and 3, respectively, or consists of the same.
  • VH1 heavy chain variable region
  • HCDR2 and HCDR3 each comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NOs: 1,
  • the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 1, 2, and 3, respectively, or consists of the same.
  • VH1 heavy chain variable region
  • HCDR1 heavy chain complementarity determining regions 1
  • HCDR2 heavy chain complementarity determining regions 2
  • HCDR3 3 of SEQ ID NOs: 1, 2, and 3, respectively, or consists of the same.
  • the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) , wherein the HCDR1, HCDR2 and HCDR3 each comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NOs: 7, 8, and 9, respectively, or consists of the same.
  • VH2 heavy chain variable region
  • HCDR3 each comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NOs: 7, 8, and 9, respectively,
  • the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 7, 8, and 9, respectively, or consists of the same.
  • VH2 heavy chain variable region
  • the third domain specifically binding to VEGF comprises a heavy chain variable region (VH3) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) , wherein the HCDR1, HCDR2 and HCDR3 each comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NOs: 13, 14, and 15, respectively, and a light chain variable region (VL) comprising light chain complementarity determining regions 1 (LCDR1) , 2 (LCDR2) and 3 (LCDR3) , wherein the LCDR1, LCDR2 and LCDR3 each comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • the third domain specifically binding to VEGF comprises a heavy chain variable region (VH3) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 13, 14, and 15, respectively, and a light chain variable region (VL) comprising light chain complementarity determining regions 1 (LCDR1) , 2 (LCDR2) and 3 (LCDR3) of SEQ ID NOs: 19, 20, and 21, respectively, or consists of the same.
  • VH3 heavy chain variable region
  • VL light chain variable region
  • the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1) of SEQ ID NO: 4, or consists of the same, the VH1 optionally having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions.
  • VH1 heavy chain variable region
  • any substitutions are not within the CDRs.
  • the first domain comprises a heavy chain variable region (VH1) having the amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 4, or consists of the same.
  • VH1 heavy chain variable region
  • any variation from the sequences of the SEQ ID NO: 4 is not within the CDRs.
  • the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2) of SEQ ID NO: 10, or consists of the same, the VH2 optionally having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions.
  • VH2 optionally having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions.
  • any substitutions are not within the CDRs.
  • the second domain comprises a heavy chain variable region (VH2) having the amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 10, or consists of the same.
  • VH2 heavy chain variable region
  • any variation from the sequences of the SEQ ID NO: 10 is not within the CDRs
  • the third domain specifically binding to VEGF comprises a heavy chain variable region (VH3) of SEQ ID NO: 16 and a light chain variable region (VL) of SEQ ID NO: 22, or consists of the same, each of the VH3 and the VL optionally having one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions.
  • VH3 heavy chain variable region
  • VL light chain variable region
  • any substitutions are not within the CDRs.
  • the third domain comprises a heavy chain variable region (VH3) having the amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 16 and a light chain variable region (VL) having the amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 22, or consists of the same.
  • any variation from the sequences of the SEQ ID NO: 16 and/or SEQ ID NO: 22 is not within the CDRs.
  • the first, second and third domains are a chimeric antibody, a humanized antibody, a human antibody, a single chain antibody, Fv, Fab, F (ab') 2, Fd, single chain Fv molecule (scFv) , diabody, or single domain antibody (dAb) .
  • the first, second and third domains, independently of each other, are humanized. In one embodiment, the first and second domains, independently of each other, are a humanized VHH.
  • the antibody according to the present invention comprises only one heavy chain. In one embodiment, the antibody according to the present invention comprises one heavy chain and one light chain. In one embodiment, the antibody according to the present invention comprises two heavy chains. In one embodiment, the antibody according to the present invention comprises two heavy chains and two light chains.
  • the antibody according to the present invention consists of one heavy chain. In one embodiment, the antibody according to the present invention consists of one heavy chain and one light chain. In one embodiment, the antibody according to the present invention consists of two heavy chains. In one embodiment, the antibody according to the present invention consists of two heavy chains and two light chains.
  • the anti-PD-1/CTLA-4/VEGF trispecific antibody comprises a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and/or a third domain specifically binding to VEGF.
  • the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 1, 2, and 3, respectively;
  • the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 7, 8, and 9, respectively;
  • the third domain specifically binding to VEGF comprises a heavy chain variable region (VH3) comprising heavy chain complementarity determining regions 1 (HCDR1) , 2 (HCDR2) and 3 (HCDR3) of SEQ ID NOs: 13, 14, and 15, respectively, and a light chain variable region (VH1) comprising
  • the first domain specifically binding to PD-1 comprises a heavy chain variable region (VH1) of SEQ ID NO: 4, or consists of a heavy chain variable region (VH1) of SEQ ID NO: 4;
  • the second domain specifically binding to CTLA-4 comprises a heavy chain variable region (VH2) of SEQ ID NO: 10, or consists of a heavy chain variable region (VH2) of SEQ ID NO: 10;
  • the third domain specifically binding to VEGF comprises a heavy chain variable region (VH3) of SEQ ID NO: 16 and a light chain variable region (VL) of SEQ ID NO: 22, or consists of the same.
  • the third domain specifically binding to VEGF comprises a heavy chain of SEQ ID NO: 17 and a light chain of SEQ ID NO: 23, or consists of the same.
  • the third domain specifically binding to VEGF comprises a full length antibody comprised of two heavy chains and two light chains inter-connected by disulfide bonds, or antigen-binding fragment thereof, or consists of the same.
  • the antigen-binding fragment of the full length antibody is a Fab fragment or a scFv fragment.
  • the antibody or antigen-binding fragment thereof further comprises an Fc region of IgG.
  • the antibody or antigen-binding fragment thereof is an IgG1, IgG2, IgG3 or IgG4 isotype.
  • the antibody or antigen-binding fragment thereof is an IgG1 LALA isotype.
  • LALA refers to amino acid substitutions L234A/L235A (Kabat nomenclature) to be introduced, as well known in the art.
  • the first domain specifically binding to PD-1 is positioned at the N-terminal of the Fc region.
  • the second domain specifically binding to CTLA-4 is positioned at the N-terminal of the Fc region.
  • the third domain specifically binding to VEGF is positioned at the N-terminal of the Fc region.
  • the first domain specifically binding to PD-1 is positioned at the C-terminal of the Fc region.
  • the second domain specifically binding to CTLA-4 is positioned at the C-terminal of the Fc region.
  • the third domain specifically binding to VEGF is positioned at the C-terminal of the Fc region.
  • the first domain specifically binding to PD-1, the second domain specifically binding to CTLA-4, the third domain specifically binding to VEGF, and the Fc region are connected to each other directly or via one or more linkers, such as flexible linkers.
  • the linkers are same or different.
  • the linker is a peptide linker, and most preferably by a peptide linker that lacks a proteolytic cleavage site.
  • the amino acid residues of the linker are selected from G, A, S, P, E, T, D, and K.
  • the linker is GGGGSGGGGS.
  • the third domain specifically binding to VEGF is a Fab fragment or a scFv fragment
  • the anti-PD-1/CTLA-4/VEGF trispecific antibody according to the present invention is shown by the following formula from N to C terminal:
  • the third domain specifically binding to VEGF is a Fab fragment or a scFv fragment
  • the anti-PD-1/CTLA-4/VEGF trispecific antibody according to the present invention is shown by the following formula from N to C terminal:
  • the third domain specifically binding to VEGF is a Fab fragment or a scFv fragment
  • the anti-PD-1/CTLA-4/VEGF trispecific antibody according to the present invention is shown by the following formula from N to C terminal:
  • the third domain specifically binding to VEGF is a Fab fragment or a scFv fragment
  • the anti-PD-1/CTLA-4/VEGF trispecific antibody according to the present invention is shown by the following formula from N to C terminal:
  • the third domain specifically binding to VEGF is a Fab fragment or a scFv fragment
  • the anti-PD-1/CTLA-4/VEGF trispecific antibody according to the present invention is shown by the following formula from N to C terminal:
  • the third domain specifically binding to VEGF is a Fab fragment or a scFv fragment
  • the anti-PD-1/CTLA-4/VEGF trispecific antibody according to the present invention is shown by the following formula from N to C terminal:
  • the third domain specifically binding to VEGF is a full length antibody.
  • the first domain specifically binding to PD-1 is connected optionally via a linker at the C terminal of either of or both the heavy chains of the full length antibody
  • the second domain specifically binding to CTLA-4 is connected optionally via a linker at the C terminal of the first domain specifically binding to PD-1.
  • the second domain specifically binding to CTLA-4 is connected optionally via a linker at the C terminal of either of or both the heavy chains of the full length antibody
  • the first domain specifically binding to PD-1 is connected optionally via a linker at the C terminal of the second domain specifically binding to CTLA-4.
  • the first domain specifically binding to PD-1 and the second domain specifically binding to CTLA-4 are connected in the same or differenent sequence at the C terminal of the two heavy chains of the full length antibody.
  • two second domains specifically binding to CTLA-4 are each connected via a linker at the C terminal of the heavy chains of the full length antibody, and two first domains specifically binding to PD-1 are each connected via a linker at the C terminal of the two second domains specifically binding to CTLA-4 (see Fig. 1) .
  • the anti-PD-1/CTLA-4/VEGF trispecific antibody according to the present invention comprises one heavy chain and one light chain, or consists of the same, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO: 27 and the light chain comprises an amino acid sequence of SEQ ID NO: 23.
  • the anti-PD-1/CTLA-4/VEGF trispecific antibody according to the present invention comprises two heavy chains and two light chains, or consists of the same, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO: 27 and the light chain comprises an amino acid sequence of SEQ ID NO: 23.
  • the heavy chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 27.
  • the light chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 23.
  • the heavy chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 27, and the light chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 23.
  • sequence alignment preferably relates to the percentage of the amino acid or nucleotide residues of the shorter sequence which are identical with the amino acid or nucleotide residues of the longer sequence. Sequence alignment can be determined conventionally with the use of computer programs. The deviations appearing in the comparison between a given sequence and the above-described sequences of the disclosure may be caused for instance by addition, deletion, substitution, insertion or recombination.
  • the CDR sequences of the antibody of the invention may comprise any conservative modifications.
  • Constant modification refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequences.
  • Conservative modifications include amino acid substitutions, additions and deletions.
  • Conservative substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain.
  • amino acids with acidic side chains for example, aspartic acid, glutamic acid
  • basic side chains for example, lysine, arginine, histidine
  • nonpolar side chains for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • uncharged polar side chains for example, glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan
  • aromatic side chains for example, phenylalanine, tryptophan, histidine, tyrosine
  • aliphatic side chains for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine
  • amide for example, asparagine, glutamine
  • beta-branched side chains for example, asparagine, glutamine
  • any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al., Acta Physiol. Scand. Suppl. 643: 55-67, 1998; Sasaki et al., Adv. Biophys. 35: 1-24, 1998) .
  • Amino acid substitutions to the antibodies of the invention may be made by well-known methods for example by PCR mutagenesis (US Pat. No. 4,683,195) .
  • libraries of variants may be generated using known methods, for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp) .
  • NNK random
  • DVK codons which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp) .
  • the resulting antibody variants may be tested for their characteristics using assays described herein.
  • the antibodies of the invention are human.
  • the antibodies of the invention are humanized.
  • Monospecific antibodies of the invention described herein may be generated using various technologies. For example, the hybridoma method of Kohler and Milstein, Nature 256: 495, 1975 may be used to generate monoclonal antibodies.
  • a mouse or other host animal such as a hamster, rat, alpaca or monkey
  • spleen cells from immunized animals with myeloma cells using standard methods to form hybridoma cells
  • Colonies arising from single immortalized hybridoma cells are screened for production of antibodies with desired properties, such as specificity of binding, cross-reactivity or lack thereof, and affinity for the antigen.
  • Balb/c mice may be used to generate mouse anti-human PD-1, CTLA-4 or VEGF antibodies.
  • Alpaca may be used to generate anti-human PD-1 or CTLA-4 VHH.
  • the antibodies made in Balb/c mice and other non-human animals may be humanized using various technologies to generate more human-like sequences.
  • Exemplary humanization techniques including selection of human acceptor frameworks are known and include CDR grafting (U.S. Patent No. 5,225,539) , SDR grafting (U.S. Patent No. 6,818,749) , Resurfacing (Padlan, (1991) Mol Immunol 28: 489-499) , Specificity Determining Residues Resurfacing (U.S. Patent Publ. No. 2010/0261620) , human framework adaptation (U.S. Patent No. 8,748,356) or superhumanization (U.S. Patent No. 7,709,226) .
  • CDRs of parental antibodies are transferred onto human frameworks that may be selected based on their overall homology to the parental frameworks, based on similarity in CDR length, or canonical structure identity, or a combination thereof.
  • Humanized antibodies may be further optimized to improve their selectivity or affinity to a desired antigen by incorporating altered framework support residues to preserve binding affinity (backmutations) by techniques such as those described in Int. Patent Publ. Nos. WO1090/007861 and WO1992/22653, or by introducing variation at any of the CDRs for example to improve affinity of the antibody.
  • Transgenic animals such as mice or rats carrying human immunoglobulin (Ig) loci in their genome may be used to generate human antibodies against a target protein, and are described in for example U.S. Patent No. 6,150,584, Int. Patent Publ. No. WO99/45962, Int. Patent Publ. Nos. WO2002/066630, WO2002/43478, WO2002/043478 and WO1990/04036, Lonberg et al (1994) Nature 368: 856-9; Green et al (1994) Nature Genet. 7: 13-21; Green & Jakobovits (1998) Exp. Med.
  • the endogenous immunoglobulin loci in such animal may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the genome of the antimal using homologous or non-homologous recombination, using transchromosomes, or using minigenes.
  • Companies such as Regeneron (http: //www. regeneron. com) , Harbour Antibodies (http: //www. harbourantibodies. com) , Open Monoclonal Technology, Inc. (OMT) (http: //www. omtinc. net) , KyMab (http: //www. kymab. com) , Trianni (http: //www. trianni. com) and Ablexis (http: //www. ablexis. com) may be engaged to provide human antibodies directed against a selected antigen using technologies as described above.
  • Human antibodies may be selected from a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv) , or unpaired or paired antibody variable regions (Knappik et al., (2000) J Mol Biol 296: 57-86; Krebs et al., (2001) J Immunol Meth 254: 67-84; Vaughan et al., (1996) Nature Biotechnology 14: 309-314; Sheets et al., (1998) PITAS (USA) 95: 6157-6162; Hoogenboom and Winter (1991) J Mol Biol 227: 381; Marks et al., (1991) J Mol Biol 222: 581) .
  • human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv) , or unpaired or paired antibody variable regions
  • the antibodies of the invention may be isolated for example from phage display library expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et al., (2010) J Mol Biol 397: 385-96, and Int. Patent Publ. No. WO09/085462) .
  • the libraries may be screened for phage binding to human and/or cyno PD-1, CTLA-4 or VEGF and the obtained positive clones may be further characterized, the Fabs isolated from the clone lysates, and expressed as full length IgGs.
  • Such phage display methods for isolating human antibodies are described in for example: U.S. Patent Nos.
  • immunogenic antigens and monoclonal antibody production may be performed using any suitable technique, such as recombinant protein production.
  • the immunogenic antigens may be administered to an animal in the form of purified protein, or protein mixtures including whole cells or cell or tissue extracts, or the antigen may be formed de novo in the animal’s body from nucleic acids encoding said antigen or a portion thereof.
  • the anti-PD-1 antibody GBD002-hS019-WS obtained from alpaca in the present disclosure has the amino acid sequences and nucleotide sequences as shown in table 2.
  • the anti-CTLA-4 antibody GBD008-hS005-3-2 obtained from alpaca in the present disclosure has the amino acid sequences and nucleotide sequences as shown in table 3.
  • the anti-VEGF antibody in the present disclosure has the amino acid sequences and nucleotide sequences as shown in table 4.
  • the bispecific PD-1/CTLA-4 antibodies of the invention may be generated by combining PD-1 binding VH (or VH/VL) domains with CTLA-4 binding VH (or VH/VL) domains isolated and characterized herein.
  • the bispecific PD-1/CTLA-4 antibodies may be engineered using VH (or VH/VL) domains from publicly available monospecific anti-PD-1 or anti-CTLA antibodies, and/or by mix-matching the PD-1 or CTLA-4 VEGF binding VH (or VH/VL) domains identified herein with publicly available PD-1 or CTLA-4 binding VH (or VH/VL) domains.
  • the trispecific, tetraspecific antibodies, and so forth can be generated in a similar way.
  • the trispecific PD-1/CTLA-4/VEGF antibodies of the invention may be generated by combining PD-1 binding VH (or VH/VL) domains, CTLA-4 binding VH (or VH/VL) domains with VEGF binding VH (or VH/VL) domains, isolated and characterized herein.
  • the trispecific PD-1/CTLA-4/VEGF antibodies may be engineered using VH (or VH/VL) domains from publicly available monospecific anti-PD-1, anti-CTLA or anti-VEGF antibodies, and/or by mix-matching the PD-1, CTLA-4 or VEGF binding VH (or VH/VL) domains identified herein with publicly available PD-1, CTLA-4 or VEGF binding VH (or VH/VL) domains.
  • Exemplary anti-PD-1 antibodies that may be used to engineer trispecific PD-1/CTLA-4/VEGF antibodies include for example pembrolizumab, and Nivolumab.
  • Exemplary anti-CTLA-4 antibodies that may be used to engineer trispecific PD-1/CTLA-4/VEGF antibodies include for example Ipilimumab.
  • Exemplary anti-VEGF antibodies that may be used to engineer trispecific PD-1/CTLA-4/VEGF antibodies include for example Bevacizumab.
  • Exemplary monospecific domains can also be derived from bispecific antibodies, for example, anti-PD-1xCTLA-4 bispecific antibodies Cadonilimab and MEDI5752, anti-PDF-1/CTLA-4 bispecific antibody AK104, and anti-PDF-1/VEGF bispecific antibody AK112.
  • the generated trispecific PD-1/CTLA-4/VEGF antibodies may be tested for their binding to PD-1, CTLA-4 and VEGF, and for their desired functional characteristics, such as enhancement of activation of antigen specific CD4 + and CD8 + T cells using methods described herein.
  • Mutations are typically made at the DNA level to a molecule such as the constant domain of the antibody using standard methods.
  • the trispecific PD-1/CTLA-4/VEGF antibody GBD214-33-03 constructed in the present disclosure has the amino acid sequences and nucleotide sequences as shown in table 5.
  • the invention also provides an antibody or antigen-binding fragment thereof, wherein the antibody VH and/or VL are encoded by a polynucleotide.
  • the polynucleotide may be a complementary deoxynucleic acid (cDNA) , and may be codon optimized for expression in a suitable host. Codon optimization is a well-known technology.
  • the invention also provides an isolated polynucleotide encoding the VH of the antibody of the invention, the VL of the antibody of the invention, the heavy chain of the antibody of the invention and/or the light chain of the antibody of the invention.
  • the invention also provides an isolated polynucleotide encoding the VH, the VL, or the VH and the VL of the antibody of the invention.
  • the invention also provides an isolated polynucleotide encoding the VHs of SEQ ID NOs: 4, 10, and/or 16.
  • the invention also provides an isolated polynucleotide encoding the heavy chain and/or light chain of the antibody of the invention.
  • the invention provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO: 5 or 11. In one embodiment, the invention provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO: 17 or 27 and/or the light chain of SEQ ID NO: 23.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 6 or 12.
  • the invention provides an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6 or 12.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: SEQ ID NO: 18 or 28, and/or an isolated polynucleotide comprising a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: SEQ ID NO: 24.
  • the invention provides an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 18 or 28 and/or the nucleotide sequence of SEQ ID NO: 24.
  • polynucleotide sequences encoding the VH or the VL or an antigen-binding fragment thereof of the antibodies of the invention, or the heavy chain or the light chain of the antibodies of the invention may be operably linked to one or more regulatory elements, such as a promoter or enhancer, that allow expression of the nucleotide sequence in the intended host cell.
  • the polynucleotide may be a cDNA.
  • the invention also provides a vector comprising the polynucleotide of the invention.
  • vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the synthetic polynucleotide of the invention into a given organism or genetic background by any means.
  • polynucleotides encoding light and/or heavy chain variable regions of the antibodies of the invention, optionally linked to constant regions are inserted into expression vectors.
  • the light and/or heavy chains may be cloned in the same or different expression vectors.
  • the DNA segments encoding immunoglobulin chains may be operably linked to control sequences in the expression vector (s) that ensure the expression of immunoglobulin polypeptides.
  • control sequences include signal sequences, promoters (e.g. naturally associated or heterologous promoters) , enhancer elements, and transcription termination sequences, and are chosen to be compatible with the host cell chosen to express the antibody.
  • Suitable expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers such as ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance to permit detection of those cells transformed with the desired DNA sequences.
  • Suitable promoter and enhancer elements are known in the art.
  • exemplary promoters include light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various known tissue specific promoters. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • Exemplary vectors that may be used are Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA) ; pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden) .
  • Eukaryotic pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia) , pEE6.4 (Lonza) and pEE12.4 (Lonza) .
  • the invention also provides a host cell comprising one or more vectors of the invention.
  • “Host cell” refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but to the progeny of such a cell, and also to a stable cell line generated from the particular subject cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells or archeal cells.
  • Escherichia coli, bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species are examples of prokaryotic host cells.
  • Other microbes, such as yeast, are also useful for expression. Saccharomyces (for example, S. cerevisiae) and Pichia are examples of suitable yeast host cells.
  • Exemplary eukaryotic cells may be of mammalian, insect, avian or other animal origins.
  • Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines such as SP2/0 (American Type Culture Collection (ATCC) , Manassas, VA, CRL-1581) , NS0 (European Collection of Cell Cultures (ECACC) , Salisbury, Wiltshire, UK, ECACC No. 85110503) , FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines.
  • An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196) .
  • CHOK1SV Longza Biologics, Walkersville, MD
  • CHOK2SV Longza
  • CHO-K1 ATCC CRL-611
  • DG44 DG44
  • the invention also provides a method of producing an antibody of the invention comprising culturing the host cell of the invention in conditions that the antibody is expressed, and recovering the antibody produced by the host cell.
  • Methods of making antibodies and purifying them are well known in the art. Once synthesized (either chemically or recombinantly) , the whole antibodies, their dimers, individual light and/or heavy chains, or other antibody fragments such as VH and/or VL, may be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., (1982) ) .
  • a subject antibody may be substantially pure, for example, at least about 80%to 85%pure, at least about 85%to 90%pure, at least about 90%to 95%pure, or at least about 98%to 99%, or more, pure, for example, free from contaminants such as cell debris, macromolecules, etc. other than the subject antibody.
  • polynucleotide sequences of the invention may be incorporated into vectors using standard molecular biology methods. Host cell transformation, culture, antibody expression and purification are done using well known methods.
  • Another embodiment of the invention is a method of producing the antibody, or an antigen-binding fragment thereof of the invention, comprising:
  • the invention provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof of the invention and a pharmaceutically acceptable carrier.
  • the antibodies or antigen-binding fragments thereof of the invention may be prepared as pharmaceutical compositions containing an effective amount of the antibody or antigen-binding fragment thereof as an active ingredient in a pharmaceutically acceptable carrier.
  • Carrier refers to a diluent, adjuvant, excipient, or vehicle with which the antibody of the invention is administered.
  • Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4%saline and 0.3%glycine may be used.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc.
  • concentration of the antibodies or antigen-binding fragments thereof of the invention in such pharmaceutical formulation may vary, from less than about 0.5%, usually to at least about 1%to as much as 15 or 20%by weight and may be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21 st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.
  • the mode of administration for therapeutic use of the antibodies or antigen-binding fragments thereof of the invention may be any suitable route that delivers the antibody or antigen-binding fragments thereof to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal) , using a formulation in a tablet, capsule, solution, powder, gel, particle; and contained in a syringe, an implanted device, osmotic pump, cartridge, micropump; or other means appreciated by the skilled artisan, as well known in the art.
  • parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous
  • pulmonary, transmucosal oral, intranasal, intravaginal, rectal
  • a formulation in a tablet, capsule, solution, powder, gel, particle and contained in a s
  • Site specific administration may be achieved by for example intratumoral, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.
  • the antibodies or antigen-binding fragments thereof of the invention may be administered to a subject by any suitable route, for example parentally by intravenous (i.v. ) infusion or bolus injection, intramuscularly or subcutaneously or intraperitoneally.
  • i.v. infusion may be given over for example 15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.
  • the dose given to a subject is sufficient to alleviate or at least partially arrest the disease being treated ( “therapeutically effective amount” ) and may be sometimes 0.005 mg to about 100 mg/kg, e.g. about 0.05 mg to about 30 mg/kg or about 5 mg to about 25 mg/kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.
  • a fixed unit dose may also be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's surface area, e.g., 500, 400, 300, 250, 200, or 100 mg/m 2 .
  • 1 and 8 doses e.g., 1, 2, 3, 4, 5, 6, 7 or 8
  • 1 and 8 doses may be administered to treat the patient, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may be given.
  • the administration of the antibodies or antigen-binding fragments thereof of the invention may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration.
  • the repeated administration may be at the same dose or at a different dose.
  • the antibodies or antigen-binding fragments thereof of the invention may be administered at 8 mg/kg or at 16 mg/kg at weekly interval for 8 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every two weeks for an additional 16 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every four weeks by intravenous infusion.
  • the antibodies or antigen-binding fragments thereof of the invention may be provided as a daily dosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
  • 0.1-100 mg/kg such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,
  • the antibodies or antigen-binding fragments thereof of the invention may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission.
  • the antibodies or antigen-binding fragments thereof of the invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional protein preparations and well known lyophilization and reconstitution techniques can be employed.
  • the antibodies or antigen-binding fragments thereof of the invention have in vitro and in vivo diagnostic, as well as therapeutic and prophylactic utilities.
  • the antibodies or antigen-binding fragments thereof of the invention may be administered to cells in culture, in vitro or ex vivo, or to a subject to treat, prevent, and/or diagnose a variety of disorders, such as cancers and infectious disorders.
  • the invention provides a method of modifying an immune response in a subject comprising administering to the subject the antibody or antigen-binding fragment thereof of the invention for a time sufficient to modify the immune response.
  • the immune response is enhanced, stimulated or up-regulated.
  • the subject is a human patient.
  • the subject is a human patient in need of enhancement of the immune response.
  • the subject is immunocompromised.
  • the subject is at risk of being immunocompromised.
  • Immunocompromised subject may be undergoing, or has undergone a chemotherapeutic or radiation therapy.
  • the subject is or is at risk of being immunocompromised as a result of an infection.
  • the antibodies or antigen-binding fragments thereof of the invention are suitable for treating a subject having a disorder that may be treated by augmenting T-cell mediated immune responses.
  • the antibodies or antigen-binding fragments thereof used in the methods of the invention are those as defined in the present disclosure.
  • the trispecific PD-1/CTLA-4/VEGF antibody used in the methods of the invention is GBD214-33-03.
  • the amino acid sequences and nucleotide sequences of the antibody is shown in Table 5.
  • the invention also provides a method of inhibiting growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of the invention for a time sufficient to inhibit growth of tumor cells.
  • the invention also provides a method of treating a cancer by administering to the subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof of the invention for a time sufficient to treat the cancer.
  • Cancer may be a hyperproliferative condition or disorder, a solid tumor, a hematological malignancy, a soft tissue tumor, or a metastatic lesion.
  • Cancer is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathology type or stage of invasiveness.
  • cancers include solid tumors, hematological malignancies, soft tissue tumors, and metastatic lesions.
  • Exemplary solid tumors include malignancies, e.g., sarcomas, and carcinomas (including adenocarcinomas and squamous cell carcinomas) of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon) , genitourinary tract (e.g., renal, urothelial cells) , prostate and pharynx.
  • Adenocarcinomas include malignancies such as most colon cancers, a rectal cancer, a renal-cell carcinoma, a liver cancer, a non-small cell carcinoma of the lung, a cancer of the small intestine and a cancer of the esophagus.
  • Squamous cell carcinomas include malignancies, e.g., in the lung, esophagus, skin, head and neck region, oral cavity, anus, and cervix.
  • the cancer is a melanoma.
  • Metastatic lesions of the aforementioned cancers may also be treated or prevented using the methods and antibodies of the invention described herein.
  • Exemplary cancers whose growth may be inhibited or reduced using the antibodies or antigen-binding fragments thereof of the invention include cancers that may be responsive to immunotherapy.
  • Examples of such cancers include a melanoma, a renal cancer, a prostate cancer, a breast cancer, a colon cancer, a gastrointestinal cancer, a stomach cancer, an esophageal cancer, a lung cancer, a metastatic malignant melanoma, a clear cell carcinoma, a hormone refractory prostate adenocarcinoma, a non-small cell lung cancer or cancer of the head and neck.
  • Refractory or recurrent malignancies may be treated using the antibodies or antigen-binding fragments thereof of the invention described herein.
  • exemplary cancers that may be treated with the antibodies or antigen-binding fragments thereof of the invention include an anal cancer, a basal cell carcinoma, a biliary tract cancer, a bladder cancer, a bone cancer, brain and CNS cancers, a carcinoma of the fallopian tubes, carcinoma of the vagina, a carcinoma of the vulva, a cutaneous or intraocular malignant melanoma, a astro-esophageal cancer, a testicular cancer, an ovarian cancer, a pancreatic cancer, a rectal cancer, an uterine cancer, a primary CNS lymphoma; a neoplasm of the central nervous system (CNS) , a cervical cancer, a choriocarcinoma, a rectum cancer, a connective tissue cancer, a cancer of the digestive system, an endometrial cancer, an eye cancer; an intra-epithelial neoplasm, a kidney cancer, a larynx cancer, a liver
  • Exemplary hematological malignancies that may be treated with the antibodies or antigen-binding fragments thereof of the invention include leukemias, lymphomas and myeloma, such as a precursor B-cell lymphoblastic leukemia/lymphoma and a B-cell non-Hodgkin's lymphoma, an acute promyelocytic leukemia, an acute lymphoblastic leukemia (ALL) , a B-cell chronic lymphocytic leukemia (CLL) /small lymphocytic lymphoma (SLL) , a B-cell acute lymphocytic leukemia, a B-cell prolymphocytic leukemia, a lymphoplasmacytic lymphoma, a mantle cell lymphoma (MCL) , a follicular lymphoma (FL) , including low-grade, intermediate-grade and high-grade FL, a cutaneous follicle center lymphoma, a marginal zone B-cell
  • a pro-lymphocytic/myelocytic leukemia an acute myeloid leukemia (AML) , a chronic myeloid leukemia (CML) , a large granular lymphocytic (LGL) leukemia, a NK-cell leukemia and Hodgkin’s lymphoma.
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • LGL large granular lymphocytic
  • NK-cell leukemia a NK-cell leukemia and Hodgkin’s lymphoma.
  • “Plasma cell disorder” refers to disorders characterized by clonal plasma cells, and includes a multiple myeloma, a light chain amyloidosis and Waldenstrom’s macroglobulinemia.
  • Light chain amyloidosis and Waldenstrom’s macroglobulinemia can arise independently from multiple myeloma. They may also present simultaneously with multiple myeloma, and develop either before or after the development of multiple myeloma.
  • Exemplary B-cell non-Hodgkin's lymphomas are a lymphomatoid granulomatosis, a primary effusion lymphoma, an intravascular large B-cell lymphoma, a mediastinal large B-cell lymphoma, heavy chain diseases (including ⁇ , ⁇ , and a disease) , lymphomas induced by therapy with immunosuppressive agents, such as cyclosporine-induced lymphoma, and methotrexate-induced lymphoma.
  • the subject has a tumor that expresses PD-L1.
  • the subject has a tumor that expresses CTLA-4.
  • the subject has a tumor that expresses VEGF.
  • the subject has been treated with an anti-PD-1 antibody.
  • the subject is refractory to treatment with the anti-PD-1 antibody.
  • the subject has a relapsed tumor after treatment with the anti-PD-1 antibody.
  • the subject has been treated with the anti-PD-1 antibody (e.g. (pembrolizumab) ) .
  • the anti-PD-1 antibody e.g. (pembrolizumab)
  • the subject has been treated with the anti-PD-1 antibody (e.g. (nivolumab) ) .
  • the anti-PD-1 antibody e.g. (nivolumab)
  • the subject is refractory to treatment with the anti-PD-1 antibody (e.g. (pembrolizumab) ) .
  • the anti-PD-1 antibody e.g. (pembrolizumab)
  • the subject is refractory to treatment with the anti-PD-1 antibody (e.g. (nivolumab) ) .
  • the anti-PD-1 antibody e.g. (nivolumab)
  • the subject has a relapsed tumor after treatment with the anti-PD-1 antibody (e.g. (pembrolizumab) .
  • the anti-PD-1 antibody e.g. (pembrolizumab)
  • the subject has a relapsed tumor after treatment with the anti-PD-1 antibody (e.g. (nivolumab) ) .
  • the anti-PD-1 antibody e.g. (nivolumab)
  • the subject has been treated or is being treated with an anti-PD-L1 antibody (e.g. MEDI-4736, MDX-1105, Avelumab or Atezolizumab) .
  • an anti-PD-L1 antibody e.g. MEDI-4736, MDX-1105, Avelumab or Atezolizumab
  • the subject is refractory to treatment with the anti-PD-L1 antibody (e.g. MEDI-4736, MDX-1105, Avelumab or Atezolizumab) .
  • the anti-PD-L1 antibody e.g. MEDI-4736, MDX-1105, Avelumab or Atezolizumab
  • the subject has a relapsed tumor after treatment with the anti-PD-L1 antibody (e.g. MEDI-4736, MDX-1105, Avelumab or Atezolizumab) .
  • the anti-PD-L1 antibody e.g. MEDI-4736, MDX-1105, Avelumab or Atezolizumab
  • the subject has been treated or is being treated with an anti-PD-L2 antibody.
  • the subject is refractory to treatment with an anti-PD-L2 antibody.
  • the subject has a relapsed tumor after treatment with an anti-PD-L2 antibody.
  • Symptoms that may be associated with relapse or resistance are, for example, a decline or plateau of the well-being of the patient or re-establishment or worsening of various symptoms associated with solid tumors, and/or the spread of cancerous cells in the body from one location to other organs, tissues or cells.
  • the invention also provides a method of treating a cancer in a subject, comprising administering to the subject a therapeutically effective amount of the antibody of the invention, wherein the subject is being treated or has been treated with an anti-PD-1 antibody.
  • the invention also provides a method of treating a cancer in a subject, comprising administering to the subject a therapeutically effective amount of the antibody of the invention, wherein the subject is being treated or has been treated with an anti-PD-L1 antibody.
  • the invention also provides a method of treating a cancer in a subject, comprising administering to the subject a therapeutically effective amount of the antibody of the invention, wherein the subject is being treated or has been treated with an anti-PD-L2 antibody.
  • the antibodies of the invention may be administered in combination with a second therapeutic agent.
  • the antibodies or antigen-binding fragments thereof of the invention may be administered in combination with one, two, three, four, five or six additional therapeutic agents.
  • “In combination with” refers to administering of the antibodies of the invention and at least one second therapeutic agent concurrently as single agents or sequentially as single agents in any order. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • the second therapeutic agent modulates activity of a molecule involved in the cancer-immunity cycle, e.g. a molecule involved in stimulatory or inhibitory pathways functioning in release of cancer cell antigens, cancer antigen presentation, T cell priming and activation, trafficking of T cells to tumors, infiltration of T cells into tumors, recognition of cancer cells by T cells, and killing of cancer cells.
  • a molecule involved in the cancer-immunity cycle e.g. a molecule involved in stimulatory or inhibitory pathways functioning in release of cancer cell antigens, cancer antigen presentation, T cell priming and activation, trafficking of T cells to tumors, infiltration of T cells into tumors, recognition of cancer cells by T cells, and killing of cancer cells.
  • the cancer-immunity cycle is described in Chen and Mellman (2013) Immunity 39: 1-10.
  • the second therapeutic agend modulates activity of a molecule involved in regulation of activity of T regulatory cells (Treg) , co-stimulatory or co-inhibitory ligands expressed on tumors, activating or inhibitory receptors on natural killer (NK) cells, or immunosuppressive factors in the tumor microenvironment.
  • T regulatory cells T regulatory cells
  • NK natural killer cells
  • the second therapeutic agent typically enhances the activity of stimulatory molecules and suppresses the activity of inhibitory molecules, as is well known.
  • modulate refers to the enhancement of immune response by the second therapeutic agent, wheatear the agent itself is agonist or antagonist of a specific molecule.
  • Example 1 Generation of anti-PD-1 antibody, anti-CTLA-4 antibody, and anti-VEGF antibody
  • Anti-human PD-1 antibody and anti-human CTLA-4 antibody were generated by immunizing alpaca with recombinant, Human PD-1and human CTLA-4 extracellular domain (ECD) proteins, respectively.
  • the total RNAs of PBMC were extracted and the cDNAs were synthesized and amplified.
  • the framework regions of alpaca VH genes were replaced with human frameworks by CDR-grafting technique and were cloned into expression vectors to create corresponding clones of humanized antibodies.
  • the resulting anti-PD-1 antibody GBD002-hS019-WS had a heavy chain variable region of SEQ ID NO: 4.
  • the resulting anti-CTLA-4 antibody had a heavy chain variable region of SEQ ID NO: 10 (GBD008-hS005-3-2) .
  • the amino acids of the heavy and light chain variable regions of the anti-VEGF antibody can be referenced to the commercial antibody Bevacizumab (Avastin) , and shown in SEQ ID NO: 16 and SEQ ID NO: 22, respectively. Biointron was entrusted to synthesize the anti-VEGF antibody, as a variant of Bevacizumab, with the sequences shown in Table 4.
  • the anti-VEGF antibody comprises two heavy chains having the amino acid sequence of SEQ ID NO: 17 and two light chains having the amino acid sequence of SEQ ID NO: 23. In this anti-VEGF antibody, the Fc fragment is different from that of Bevacizumab.
  • trispecific antibody GBD214-33-03 the DNA sequence encoding the heavy chain comprising, from N terminal to C terminal, the heavy chain (SEQ ID NO: 17) of the anti-VEGF antibody, the heavy chain variable region (SEQ ID NO: 10) of the anti-CTLA-4 antibody and the heavy chain variable region (SEQ ID NO: 4) of the anti-PD-1 antibody, and the DNA sequence encoding the light chain (SEQ ID NO: 23) of the anti-VEGF antibody were cloned into pcDNA3.3 expression vector, respectively, to obtain the trispecific antibody.
  • the linker 1 and/or linker 2 were added as a flexible connection in the heavy chain of GBD214-33-03.
  • the obtained trispecific antibody was named “GBD214-33-03” .
  • the schematic diagram of GBD214-33-03 structure was shown in Figure 1.
  • Example 3 FACS analysis of binding specificities of anti-PD1 antibodies to human PD1 protein on the cell surface
  • HEK293-PD1 cells (1x10 5 /well) were washed twice in FACS buffer (PBS+2%BSA) , and resuspended in 100 ⁇ l of FACS buffer containing serially diluted (1: 5) anti-PD1 mAbs, and incubated at 4°C for 1 hour. Cells were then washed twice in FACS buffer and the bound antibodies were detected by incubation with APC anti-human IgG Fc (Biolegend, Catalog #410712) for 1 hour at 4°C. Afterward, cells were washed twice in FACS buffer and then collected and analyzed using the Fortessa flow cytometer (BD Bioscience) . The result is shown in Fig. 2.
  • GBD214-33-03 binding to PD1 in HEK293-PD1 cells.
  • GBD214-33-03 showed potent binding to HEK293-PD1 cells with a similar median fluorescence intensity (MFI) to that of its PD1 parental, GBD002-hS019-WS and pembrolizumab.
  • MFI median fluorescence intensity
  • CHOK1-PD-L1 cells (4x10 4 /well) were harvested and suspended in PRMI 1640 media (Gibco, Catalog #31800022) with 10%FBS (Gibco, Catalog #10099-141) to grow overnight at 37°C in 96 well plate.
  • PRMI 1640 media Gibco, Catalog #31800022
  • 10%FBS Gibco, Catalog #10099-141
  • supernatants were discarded
  • Bio-Glo Luciferenase Assay buffer (Promega, Catalog #G7940) was added to cells, cells were incubated at room temperature for up to 10 minutes before reading using EnVision 2105 multimode plate reader (PerkinElmer) .
  • the result is shown in Fig. 3.
  • the PD1 reporter assay showed GBD214-33-03 has similar maximum luminescence signals compared with pembrolizumab and AK104.
  • the result demonstrates that GBD214-33-03 can block the interaction of PD1 to its ligand PD-L1.
  • CD14+cells were isolated from PBMC by isolation kit (Stem Cell, Catalog #17858) , and manufacturer's instructions were followed. Afterwards, CD14+cells were seeded at 1x10 6 cells/ml, 3 ⁇ 5ml in 6-well plate (3 ⁇ 5x10 6 /well) in culture media added with 50 ng/ml IL4 and 50 ng/ml GMCSF. Cytokines were refreshed every 2-3 days. On day 6, to induce mature DC, 1ug/ml LPS (Sigma-Aldrich, Catalog #L6529) was added to the culture media, cells were incubated at 37°C for 24 hours and then mature DC cells were collected on day 7 for MLR assay.
  • MLR assay fresh T cells were isolated following manufacturer's instructions (Stem Cell, Catalog #17951) , and the cells were seeded at 2x10 5 cells/well to the assay plate. Serially diluted antibodies were added to assay plates, before which mature DC cells were seeded to assay plate at 1x10 4 cells/well. Assay plates were incubated at 37°C for 5 days, supernatants were collected for IFN ⁇ detection using HTRF kit (Cisbio, Catalog #62HIFNGPEH) , and manufacturer's instructions were followed. The result is shown in Fig. 4.
  • VEGF165 protein (1 ⁇ l/ml) (Kactus, Catalog #VEG-HM065) was coated onto 96 well flat bottomed microplates for 2 hours at 37°C. Plates were washed six times using PBST and blocked by adding 300 ⁇ l blocking buffer for 2 hours at 37°C. Supernatants were aspirated and plates were washed with PBST for three times. Serially diluted antibodies were added to each well. Plates were incubated at 37°Cfor 1 hour and then washed with PBST for six times.
  • VEGF165 protein (1 ⁇ l/ml) (Kactus, Catalog #VEG-HM065) was coated onto 96 well flat bottomed microplates for 2 hours at 37°C. Plates were washed six times using PBST and blocked by adding 300 ⁇ l blocking buffer for 2 hours at 37°C. Supernatants were aspirated and plates were washed with PBST for three times. Serially diluted antibodies and VEGFR2-mFc-biotin (Kactus, Catalog #VGF-HM3R2B) were added to each well. Plates were incubated at 37°C for 1 hour and then washed with PBST for six times.
  • HEK293-NFAT-KDR-Luc cells (4x10 4 /well) were harvested and resuspended in PRMI 1640 media (Gibco, Catalog #31800022) with 10%FBS (Gibco, Catalog #10099-141) , and the cells were inoculated at 4x10 4 cell/well to grow in 96-well white plate at 37°C overnight. On the second day, supernatants were discarded, VEGF165 (15ng/ml) (Kactus, Catalog #VEG-HM065) and serially diluted antibodies were added to each well. Cells were incubated at 37°C for 5 hours.
  • Example 9 FACS analysis of binding specificities of anti-CTLA4 antibodies to human CTLA4 protein on the cell surface
  • CHOK1-CTLA4 cells (1x10 5 /well) and CHOK1-PD1-CTLA4 cells (1x10 5 /well) were harvested and collected, and resuspended in 100 ⁇ l of FACS buffer (PBS+2%BSA) .
  • FACS buffer PBS+2%BSA
  • Serially diluted anti-CTLA4 antibodies were added to plates and cells were incubated at 4°C for 1 hour. Cells were then washed twice with FACS buffer and the bound antibodies were detected by incubation with APC anti-human IgG Fc (Biolegend, Catalog #410712) for 1 hour at 4°C. Cells were washed twice with FACS buffer and then collected. Cells were analyzed using the Fortessa flow cytometer (BD Bioscience) . Results are shown in Figs.
  • CHOK1-CTLA4 or CHOK1-PD1-CTLA4 cells (1x10 5 /well) were harvested and collected, and the cells were resuspended in 100 ⁇ l of FACS buffer containing serially diluted anti-CTLA4 antibodies with either CD80-Biotin (SinoBiological, Catlog #10698-H49H-B) or CD86-Biotin (SinoBiological, Catlog #10699-H03H-B) . Cells were incubated at 4°C for 30min and then washed twice using FACS buffer. The bound antibodies were detected by incubation with APC anti-human IgG Fc (Biolegend, Catalog #410712) for 30min at 4°C.
  • APC anti-human IgG Fc Biolegend, Catalog #410712
  • Binding dynamics of antibodies and recombinant human PD1, CTLA-4 or VEGF were analyzed qualitatively and quantitatively by Octet method for protein interaction (Octect RH-16, Sartorius) .
  • Binding rate (Kon) and decoupling rate (Koff) were calculated using a simple one-to-one binding model (Octet evaluation software version 12.2) .
  • the equilibrium dissociation constant (kD) is calculated as the ratio Koff/Kon.
  • Example 12 PD-1 Receptor Downregulation in CHOK1-PD1-CTLA4 Cells Determined by Flow Cytometry.
  • CHOK1-PD1-CTLA4 cells were harvested and cell density was adjusted to 5x10 6 /ml.
  • Cells were added to 96-well U plate at 50 ⁇ l/well in F12K+10%FBS media. Serially diluted antibodies were added to the cells. Cells were incubated at 4°C for 30min and then washed twice using 4°C FACS buffer. From here on, all procedures were performed at 4°C including pipetting and centrifuging. Afterwards, non-competitive PD-1-AF647 antibody was added to plates at working concentration of 10 ⁇ g/ml and cells were incubated at 4°C for another 30min.
  • Example 13 In vivo anti-tumor efficacy of GBD214-33-03 antibody in A375 PBMC mouse model.
  • tumors were established (about 220 mm 3 )
  • pembrolizumab (15 mg/kg)
  • bevacizumab (15 mg/kg)
  • ipilimumab-LALA 15 mg/kg
  • three doses of AK112 (1 mg/kg, 5 mg/kg and 20 mg/kg)
  • three doses of AK104 (1 mg/kg, 5 mg/kg and 20 mg/kg)
  • three doses of GBD214-33-03 (1 mg/kg, 5 mg/kg and 20 mg/kg)
  • the combo of pembrolizumab, bevacizumab and Ipilimumab-LALA (15 mg/kg+15 mg/kg+15 mg/kg) were given by intraperitoneal injection respectively.
  • mice The treatment was given twice a week for five doses. Tumor growth and mouse body weight were monitored every three days and reported as the mean tumor volume and average body weight. Result are shown in Figs. 11a and 11b. Results showed that in A375 PBMC model, GBD214-33-03 (1 mg/kg, 5 mg/kg and 20 mg/kg) has better efficacy compared to AK112 (1 mg/kg, 5 mg/kg and 20 mg/kg) , AK104 (1 mg/kg, 5 mg/kg and 20 mg/kg) , the combo of pembrolizumab, bevacizumab and Ipilimumab-LALA (15 mg/kg+15 mg/kg+15 mg/kg) . The average body weight of mice started to decrease from Day 7 with no more than 15%reduction for all treatment groups.
  • Example 14 In vivo anti-tumor efficacy of GBD214-33-03 antibody in HT29 PBMC mouse model.
  • tumors were established (about 250 mm 3 )
  • pembrolizumab (15 mg/kg)
  • bevacizumab (15 mg/kg)
  • ipilimumab-LALA 15 mg/kg
  • three doses of AK112 (1 mg/kg, 5 mg/kg and 20 mg/kg)
  • three doses of AK104 (1 mg/kg, 5 mg/kg and 20 mg/kg)
  • three doses of GBD214-33-03 (1 mg/kg, 5 mg/kg and 20 mg/kg)
  • the combo of pembrolizumab, bevacizumab and Ipilimumab-LALA (15 mg/kg+15 mg/kg+15 mg/kg) were given by intraperitoneal injection respectively.
  • mice The treatment was given twice a week for five doses. Tumor growth and mouse body weight were monitored every three days and reported as the mean tumor volume and average body weight. The result is shown in Figs. 12a and 12b. Results showed that in HT29 PBMC model, GBD214-33-03 (1 mg/kg, 5 mg/kg and 20 mg/kg) has better efficacy compared to AK112 (1 mg/kg, 5 mg/kg and 20 mg/kg) , AK104 (1 mg/kg, 5 mg/kg and 20 mg/kg) , the combo of pembrolizumab, bevacizumab and Ipilimumab-LALA (15 mg/kg+15 mg/kg+15 mg/kg) . The average body weight of mice was relatively stable with no more than 15%reduction for all treatment groups.
  • this invention developed trispecific antibodies specifically binding to PD-1, CTLA-4 and VEGF to block checkpoints expressed on T cells.
  • the TsAbs have potent receptor binding and ligand blocking activity to PD-1 and VEGF, while only partially blocking interaction between CTLA-4 and its ligands.
  • the designed partial blocking activity to CTLA-4 aims to reduce the peripheral irAE toxicity.
  • the trispecific antibody of the invention showed potent anti-tumor activity.
  • VEGF Vascular endothelial growth factor
  • VEGF/VEGFR-Targeted Therapy and Immunotherapy in Non-small Cell Lung Cancer Targeting the Tumor Microenvironment. Int J Biol Sci, 18(9) , 3845-3858. doi: 10.7150/ijbs.70958

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des anticorps anti-PD-1 et des anticorps trispécifiques se liant spécifiquement à PD-1, CTLA-4 et VEGF, ou des fragments de liaison à l'antigène de ceux-ci, des polynucléotides codant pour les anticorps ou des fragments de liaison à l'antigène de ceux-ci, et des procédés de fabrication et d'utilisation de ceux-ci.
PCT/CN2024/089982 2023-07-14 2024-04-26 Anticorps anti-pd-1/ctla-4/vegf et leur utilisation Pending WO2025016011A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2023107464 2023-07-14
CNPCT/CN2023/107464 2023-07-14

Publications (1)

Publication Number Publication Date
WO2025016011A1 true WO2025016011A1 (fr) 2025-01-23

Family

ID=94281153

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/089982 Pending WO2025016011A1 (fr) 2023-07-14 2024-04-26 Anticorps anti-pd-1/ctla-4/vegf et leur utilisation

Country Status (1)

Country Link
WO (1) WO2025016011A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104974253A (zh) * 2014-04-01 2015-10-14 上海中信国健药业股份有限公司 抗ctla-4/pd-1双特异性抗体、其制备方法及应用
US20170275353A1 (en) * 2016-03-24 2017-09-28 Gensun Biopharma Inc Trispecific inhibitors for cancer treatment
CN108285485A (zh) * 2018-01-08 2018-07-17 乌鲁木齐恒康致远生物技术有限公司 一种抗pd-1的单域抗体及其应用
CN109476762A (zh) * 2016-07-20 2019-03-15 南京传奇生物科技有限公司 多特异性抗原结合蛋白及其使用方法
CN111699200A (zh) * 2018-01-15 2020-09-22 南京传奇生物科技有限公司 针对pd-1的单域抗体和其变体
CN114685655A (zh) * 2020-12-28 2022-07-01 浙江纳米抗体技术中心有限公司 Pd-1结合分子及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104974253A (zh) * 2014-04-01 2015-10-14 上海中信国健药业股份有限公司 抗ctla-4/pd-1双特异性抗体、其制备方法及应用
US20170275353A1 (en) * 2016-03-24 2017-09-28 Gensun Biopharma Inc Trispecific inhibitors for cancer treatment
CN109476762A (zh) * 2016-07-20 2019-03-15 南京传奇生物科技有限公司 多特异性抗原结合蛋白及其使用方法
CN108285485A (zh) * 2018-01-08 2018-07-17 乌鲁木齐恒康致远生物技术有限公司 一种抗pd-1的单域抗体及其应用
CN111699200A (zh) * 2018-01-15 2020-09-22 南京传奇生物科技有限公司 针对pd-1的单域抗体和其变体
CN114685655A (zh) * 2020-12-28 2022-07-01 浙江纳米抗体技术中心有限公司 Pd-1结合分子及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Master's Theses", 1 January 2018, PLA MEDICAL COLLEGE, China, article CHEN SHIXUE: "Computational design of IgG-like multispecific antibodies targeting EGFR, VEGF, PD-1 and CTLA-4", pages: 1 - 72, XP093262746 *

Similar Documents

Publication Publication Date Title
US12269896B2 (en) Antibodies specific for GUCY2c and uses thereof
JP6432121B2 (ja) Ｐｄｌ−１抗体、その医薬組成物及びその使用
CN112088167B (zh) 特异性针对人类连接蛋白4的抗体
JP7384835B2 (ja) Ｃｄ３に特異的な抗体及びその使用
KR20200110358A (ko) 항-pd-1 항체 및 치료 방법
TW201900674A (zh) 含有ＴＧＦ－β受體的融合蛋白及其醫藥用途
JP2022509156A (ja) 抗ｃｄ４０抗体、その抗原結合フラグメント、およびその医学的使用
CN114907479B (zh) 抗cd112r抗体及其用途
JP2024528682A (ja) 薬物組成物及び用途
TW202313682A (zh) 抗icos抗體之用途
CN119371536A (zh) 抗体和使用方法
KR20230022246A (ko) 치료 항체 및 그의 용도
WO2021000813A1 (fr) Anticorps monoclonal ciblant pd-1 et son utilisation
TW202334215A (zh) 靶向cldn18.2之抗體、雙特異性抗體及其應用
TW202128763A (zh) 含有ＴＧＦ－β受體的融合蛋白及其醫藥用途
WO2025016011A1 (fr) Anticorps anti-pd-1/ctla-4/vegf et leur utilisation
WO2025016012A1 (fr) Anticorps trispécifiques anti-pd-1/ctla-4/tigit et leur utilisation
CN120230216B (zh) 抗lair1抗体及其用途
RU2780537C2 (ru) Cd3-специфические антитела и их применение
HK40046552A (en) Antibodies specific for cd3 and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24842011

Country of ref document: EP

Kind code of ref document: A1