EP3411068A2 - Protéines de liaison bispécifiques pour pd-l1 et kdr - Google Patents

Protéines de liaison bispécifiques pour pd-l1 et kdr

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Publication number
EP3411068A2
EP3411068A2 EP17748163.7A EP17748163A EP3411068A2 EP 3411068 A2 EP3411068 A2 EP 3411068A2 EP 17748163 A EP17748163 A EP 17748163A EP 3411068 A2 EP3411068 A2 EP 3411068A2
Authority
EP
European Patent Office
Prior art keywords
seq
nos
antibody
bispecific
kdr
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.)
Withdrawn
Application number
EP17748163.7A
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German (de)
English (en)
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EP3411068A4 (fr
Inventor
Zhenping Zhu
Dan Lu
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Kadmon Corp LLC
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Kadmon Corp LLC
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Publication of EP3411068A2 publication Critical patent/EP3411068A2/fr
Publication of EP3411068A4 publication Critical patent/EP3411068A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • C07K16/2827—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 B7 molecules, e.g. CD80, CD86
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/70—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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C07K2317/90—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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • bispecific binding proteins that comprise a first binding region that binds human PD-Ll and a second binding region that binds human KDR. Also provided are methods of making the bispecific binding proteins and methods of using the bispecific binding proteins to treat diseases or conditions in which it is desirable to reduce or inhibit immunosuppression or to reduce or inhibit angiogenesis.
  • PD-1 Programmed death 1
  • CD28 is a member of the CD28 family of receptors comprising CD28, CTLA-4, PD-1, ICOS, and BTLA (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8).
  • PD-1 is an inducible immunosuppressive receptor mainly upregulated on activated T cells and B cells during the progression of immunopathological conditions.
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • PD-Ll is a cell surface glycoprotein and a major ligand for PD-1. PD-Ll is also inducible on lymphoid tissues and non-lymphoid peripheral tissues following cellular activation.
  • PD-Ll is upregulated in a variety of affected cell types including cancer and stromal cells in addition to immune cells, and plays an active role in immunosuppression during the course of the deterioration of diseases (Iwai et al (2002) PNAS 99:12293-7, OMgashi et al. (2005) Clin Cancer Res 11 :2947-53).
  • PD-Ll upregulation has been linked to poor clinical outcomes in a variety of cancers and viral infection (Hofmeyer et al. (2011) J. BioMed. Biotech. 2011:1-9,
  • VEGFRs are receptor tyrosine kinases and belong to the same family of receptors as those of the PDGFs and fibroblast growth factors (FGFs).
  • KDR also known as VEGFR2
  • VEGFR2 is a receptor that binds VEGF isoforms A, C, D, and E. It plays a role in endothelial cell differentiation and in the mitogenic, angiogenic, and permeability-enhancing effects of VEGFs.
  • KDR is a 200 kDa glycoprotein that consists of 7 Ig-like loops in the extracellular domain, a transmembrane domain, and two intracellular tyrosine kinase domains split by a kinase insert.
  • the second and third Ig-like loops are high-affinity ligand-binding domains for VEGF while the first and fourth Ig-like loops regulate ligand binding and receptor dimerization, respectively.
  • VEGF binds KDR with a Kd of 75-250 pM as compared to a Kd of 25 pM for VEGFR1.
  • KDR is primarily expressed on the cell surface of vascular endothelial cells.
  • KDR is also found on the cell surface of hematopoietic cells, vascular smooth muscle cells (VSMCs), and some malignant cells.
  • Angiogenesis is a highly complex process of developing new blood vessels that involves the proliferation and migration of, and tissue infiltration by, capillary endothelial cells from pre-existing blood vessels, cell assembly into tubular structures, joining of newly forming tubular assemblies to closed-circuit vascular systems, and maturation of newly formed capillary vessels.
  • Angiogenesis is important in normal physiological processes including embryonic development, follicular growth, and wound healing. Undue angiogenesis also leads to neovascularization in neoplastic diseases, and in non-neoplastic diseases such as age-related macular degeneration (AMD), diabetic retinopathy, and neovascular glaucoma.
  • Anti- angiogenic therapy that targets vascular endothelial growth factor (VEGF) with ranibizumab (LUCENTIS®) has been shown to be effective in delaying progression of AMD.
  • Angiogenesis plays a significant role in the growth and metastasis of primary tumors, since growth and metastasis is dependent on formation of new blood vessels. Without the neovascularization resulting from angiogenesis, tumors become necrotic, apoptotic, or fail to grow to appreciable size.
  • Tumor angiogenesis involves several processes, including endothelial cell activation, proliferation, migration, and tissue infiltration from preexisting blood vessels. These processes are triggered by the production of angiogenic growth factors such as VEGF by tumor cells and their surrounding stroma.
  • KDR has become an important target of anti-cancer therapy. Since many tumors secrete elevated amounts of VEGF while the number of KDR molecules remains relatively constant, targeting KDR increases the probability of suppressing VEGF signaling, thus inhibiting tumor growth.
  • bispecific binding proteins that bind to human PD-Ll and human KDR.
  • the bispecific binding proteins bind to PD-Ll and block the interaction of PD-Ll with PD- 1.
  • bispecific binding proteins are useful to reduce or inhibit immunosuppression.
  • bispecific binding proteins are useful to reduce or inhibit angiogenesis.
  • Bispecific binding proteins are particularly useful in combining, in one agent, the ability to inhibit both immunosuppression and angiogenesis.
  • bispecific antibodies that bind to human PD-Ll and human KDR.
  • the bispecific antibodies comprise a first antigen-binding site that binds to human PD-Ll and a second antigen-binding site that binds to human KDR.
  • nucleic acid molecules encoding the bispecific antibodies as well as expression vectors comprising the nucleic acids and which are capable of expressing the nucleic acids in a prokaryotic or eukaryotic host cell, thus leading to the production of the bispecific antibodies.
  • host cells comprising the expression vectors for the recombinant production of bispecific antibodies.
  • a bispecific antibody comprising an scFv that binds PD-Ll linked to an antibody that binds KDR.
  • the PD-Ll scFv is linked to the carboxy terminal end of the heavy chain constant domain of an IgG that binds KDR.
  • the PD-Ll scFv is linked to the carboxy terminal end of the light chain constant domain of an IgG that binds KDR.
  • the PD-Ll scFv is linked to the amino terminal end of the heavy chain variable domain of an IgG that binds KDR.
  • the PD-Ll scFv is linked to the amino terminal end of the light chain variable domain of an IgG that binds KDR.
  • a bispecific antibody comprising an scFv that binds KDR linked to an antibody that binds PD-Ll.
  • the KDR scFv is linked to the carboxy terminal end of the heavy chain constant domain of an IgG that binds PD-Ll .
  • the KDR scFv is linked to the carboxy terminal end of the light chain constant domain of an IgG that binds PD-L1.
  • the KDR scFv is linked to the amino terminal end of the heavy chain variable domain of an IgG that binds PD-L1.
  • the PD-L1 scFv is linked to the amino terminal end of the light chain variable domain of an IgG that binds PD-L1.
  • amino acid sequence of the scFv that binds PD-L1 is:
  • amino acid sequence of the scFv that binds KDR is:
  • amino acid sequence of the scFv that binds KDR is:
  • amino acid sequence of the heavy chain variable domain of the IgG that binds PD-L1 is:
  • amino acid sequence of the light chain variable domain of the IgG that binds PD-L1 is:
  • amino acid sequence of the heavy chain variable domain of the IgG that binds KDR is:
  • amino acid sequence of the light chain variable domain of the IgG that binds KDR is:
  • amino acid sequence of the heavy chain variable domain of the IgG that binds KDR is:
  • amino acid sequence of the light chain variable domain of the IgG that binds KDR is:
  • the CDRs are underlined.
  • the bispecific antibody comprises a heavy chain variable domain that binds human PD-L1 and has three complementarity determining regions (CDRs) where the amino acid sequence of CDR1 is GFTFSAYRMF (SEQ ID NO: 10), the amino acid sequence of CDR2 is SIYPS GGITFYADS VKG (SEQ ID NO: 11), and the amino acid sequence of CDR3 is IKLGTVTTVDY (SEQ ID NO: 12).
  • CDRs complementarity determining regions
  • the bispecific antibody comprises a light chain variable domain that binds human PD-L1 and has three CDRs where the amino acid sequence of CDR1 is TGTS S D VGA YN Y VS (SEQ ID NO: 13), the amino acid sequence of CDR2 is DVSNRPS (SEQ ID NO: 14), and the amino acid sequence of CDR3 is SSYTSSSTRV (SEQ ID NO: 15).
  • the bispecific antibody comprises a heavy chain variable domain that binds human KDR and has three CDRs where the amino acid sequence of CDR1 is GFTFSWYVMG (SEQ ID NO: 16), the amino acid sequence of CDR2 is SIYPSGGATNYADSVKG (SEQ ID NO: 17), and the amino acid sequence of CDR3 is GNYFDY (SEQ ID NO: 18).
  • the bispecific antibody comprises a light chain variable domain that binds human KDR and has three CDRs where the amino acid sequence of CDR1 is S GEKLGDE Y AS (SEQ ID NO: 19), the amino acid sequence of CDR2 is QDNKRPS (SEQ ID NO: 20), and the amino acid sequence of CDR3 is QAWDSSTLL (SEQ ID NO: 21).
  • the bispecific antibody comprises a heavy chain variable domain that binds human KDR and has three CDRs where the amino acid sequence of CDR1 is GFTFSWYVMG (SEQ ID NO: 22), the amino acid sequence of CDR2 is
  • SIYPQGGATSYADSVK (SEQ ID NO: 23), and the amino acid sequence of CDR3 is GNYFDY (SEQ ID NO: 24).
  • the bispecific antibody comprises a light chain variable domain that binds human KDR and has three CDRs where the amino acid sequence of CDR1 is RASQSVSSNYFG (SEQ ID NO: 25), the amino acid sequence of CDR2 is GASSRAT (SEQ ID NO: 26), and the amino acid sequence of CDR3 is QQFDSLPLT (SEQ ID NO: 27).
  • amino acid sequence of the heavy chain of the IgG that binds PD-L1 is:
  • the CDRs are underlined.
  • the LL bold
  • the LL can be mutated to other residue such as AA.
  • amino acid sequence of the light chain of the IgG that binds PD-L1 is:
  • amino acid sequence of the heavy chain of the IgG that binds KDR is:
  • the CDRs are underlined.
  • the LL bold
  • the LL can be mutated to other residues such as AA.
  • amino acid sequence of the heavy chain of the IgG that binds KDR is:
  • the amino acid sequence of the light chain of the IgG that binds KDR is:
  • amino acid sequence of the scFv that binds PD-L1 is:
  • the bispecific antibody comprises a heavy chain variable domain that binds human PD-L1 and has three complementarity determining regions (CDRs) where the amino acid sequence of CDR1, CDR2 and CDR3 are: GFTFSWYLMK,
  • the bispecific antibody comprises a light chain variable domain that binds human PD-L1 and has three complementarity determining regions (CDRs) where the amino acid sequence of CDR1, CDR2 and CDR3 are: RASQTVSKYFNW, ATSTLQS, and QQSYTTPWT, respectively (SEQ ID NOs: 61-63).
  • CDRs complementarity determining regions
  • amino acid sequence of the heavy chain variable domain of the IgG that binds PD-L1 is:
  • amino acid sequence of the heavy chain of the IgG that binds PD-L1 is:
  • amino acid sequence of the light chain variable domain of the IgG that binds PD-Ll is:
  • amino acid sequence of the light chain of the IgG that binds PD-Ll is:
  • the invention also provides conjugates of the bispecific binding proteins, for example, and without limitation, to imaging agents, therapeutic agents, or cytotoxic agents.
  • the invention further provides compositions comprising the bispecific binding proteins and at least one pharmaceutically acceptable carrier.
  • a fusion protein capable of binding to human PDL1 and also to human KDR.
  • the fusion protein may include a portion that binds to human PD- LI and a portion that binds to human KDR.
  • the portion of the fusion protein that binds to human PD-Ll is an antibody or PD-Ll binding fragment thereof.
  • the portion of the fusion protein that binds to human KDR is an antibody or KDR binding fragment thereof.
  • the portion of the fusion protein that binds to human PD-Ll is an antibody or PD-Ll binding fragment thereof and the portion of the fusion protein that binds to human KDR is an antibody or KDR binding fragment thereof.
  • a method of inhibiting the interaction of human PD1 with human PD-Ll in a subject which comprises administering an effective amount of a bispecific antibody or fragment thereof disclosed herein.
  • a method of inhibiting immunosuppression mediated by human PD-Ll which comprises administering an effective amount of a bispecific antibody or fragment thereof disclosed herein, or a fusion protein disclosed herein.
  • a method of stimulating an immune response against a cell or tissue that expresses human PD-Ll which comprises administering to a subject an effective amount of a bispecific antibody or fragment thereof disclosed herein, or a fusion protein disclosed herein.
  • the cell or tissue the expresses human PD-Ll is a neoplastic cell or an infected cell.
  • a method of neutralizing activation of human KDR or murine KDR comprising contacting a cell with an effective amount of a bispecific antibody or fragment thereof of the present invention.
  • Also provided is a method of inhibiting angiogenesis comprising administering to a subject an effective amount of a bispecific antibody or fragment thereof of the present invention.
  • a method of reducing tumor growth comprising administering to a subject an effective amount of a bispecific antibody or fragment thereof of the present invention.
  • a method of treating a neoplastic disease in a subject comprising administering to a subject an effective amount of a bispecific antibody or fragment thereof as disclosed herein, wherein the neoplastic diseases is selected from the group consisting of lung cancer, colorectal cancer renal cell carcinoma, glioblastoma, ovarian cancer, bladder cancer, gastric cancer, multiple myeloma, non-small cell lung cancer and pancreatic cancer.
  • a bispecific binding protein comprising a first region that binds to human PD-Ll and a second region that binds to human KDR.
  • bispecific binding protein of embodiment 1 which is a bispecific antibody.
  • a bispecific antibody comprising an IgG, IgA, IgE, or IgD and an scFv.
  • the bispecific antibody of embodiment 4 comprising an IgG that binds PD-Ll and an scFv that binds KDR.
  • the bispecific antibody of embodiment 4 comprising an IgG that binds KDR and an scFv that binds PD-Ll.
  • the bispecific antibody of embodiment 5 or 6 comprising a heavy chain variable domain that binds human PD-L1 and has three complementarity determining regions (CDRs) where the amino acid sequences of CDR1, CDR2, and CDR3 are SEQ ID NOs: 10-12, respectively or SEQ ID NOs: 58-60, respectively.
  • CDRs complementarity determining regions
  • the bispecific antibody of embodiment 5 or 6 comprising a light chain variable domain that binds human PD-L1 and has three CDRs where the amino acid sequences of CDR1, CDR2, and CDR3 are SEQ ID NOs: 13-15, respectively, or SEQ ID NOs: 61-63, respectively.
  • bispecific antibody of embodiment 5 or 6 comprising a heavy chain variable domain that binds human KDR and has three CDRs where the amino acid sequences of
  • CDR1, CDR2, and CDR3 are SEQ ID NOs: 16-18, respectively.
  • the bispecific antibody of embodiment 5 or 6 comprising a light chain variable domain that binds human KDR and has three CDRs where the amino acid sequences CDR1, CDR2, and CDR3 are SEQ ID NOs: 19-21, respectively.
  • the bispecific antibody of embodiment 5 or 6 comprising a heavy chain variable domain that binds human KDR and has three CDRs where the amino acid sequences CDR1, CDR2, and CDR3 are SEQ ID NOs: 22-24, respectively.
  • the bispecific antibody of embodiment 5 or 6 comprising a light chain variable domain that binds human KDR and has three CDRs where the amino acid sequences CDR1, CDR2, and CDR3 are SEQ ID NOs: 25-27, respectively.
  • the bispecific antibody of embodiment 5 or 6 comprising a heavy chain and a light chain, wherein the heavy chain and the light chain comprise the respective sequences of a heavy chain/light chain pair selected from the group consisting of SEQ ID NOs: 34 and 31, SEQ ID NOs: 30 and 35, SEQ ID NOs: 36 and 31, SEQ ID NOs: 30 and 37, SEQ ID NOs: 38 and 33, SEQ ID NOs: 32 and 39, SEQ ID NOs: 40 and 33, SEQ ID NOs: 32 and 41, SEQ ID NOs: 42 and 29, SEQ ID NOs: 28 and 43, SEQ ID NOs: 44 and 29, SEQ ID NOs: 28 and 45, SEQ ID NOs: 46 and 29, SEQ ID NOs: 28 and 47, SEQ ID NOs: 48 and 29, SEQ ID NOs: 28 and 49, SEQ ID NOs: 51 and 50, SEQ ID NOs: 52 and 50, SEQ ID NOs: 53 and 50, SEQ ID NOs: 54 and 29, SEQ ID NOs: 55 and 29, and
  • a method of treating a patient in need of reducing immunosuppression or reducing angiogenesis comprising administering to a patient in need of such reduction of immunosuppression or angiogenesis a bispecific binding protein of any one of embodiments 1-3 or a bispecific antibody of any one of embodiments 4-13.
  • a method of treating cancer comprising administering to a patient in need thereof a bispecific binding protein of any one of embodiments 1-3 or a bispecific antibody of any one of embodiments 4-13.
  • cancer selected from the group consisting of lung cancer, colorectal cancer renal cell carcinoma, glioblastoma, ovarian cancer, bladder cancer, gastric cancer, multiple myeloma, non-small cell lung cancer, and pancreatic cancer.
  • An isolated nucleic acid molecule encoding a bispecific binding protein of any one of embodiments 1-3, a bispecific antibody of any one of embodiments 4-13, or a polypeptide chain thereof.
  • a vector comprising the nucleic acid molecule of embodiment 17.
  • a cultured host cell comprising the vector of embodiment 18.
  • a method for producing a polypeptide comprising culturing the host cell of embodiment 19 under conditions permitting expression of the nucleic acid molecule.
  • a pharmaceutical composition comprising
  • a bispecific binding protein of any one of embodiments 1-3, a bispecific antibody of any one of embodiments 4-13, or a conjugate of embodiment 21, and a pharmaceutically acceptable carrier e.g., a bispecific binding protein of any one of embodiments 1-3, a bispecific antibody of any one of embodiments 4-13, or a conjugate of embodiment 21, and a pharmaceutically acceptable carrier.
  • Figures 1A, IB, 1C, and ID show four possible ways in which a bispecific antibody comprising a PD-Ll -binding region and a KDR-binding region may be constructed.
  • the bispecific antibody comprises an IgG comprising one of the binding regions that is covalently linked to an scFv comprising the other binding region.
  • the IgG is shown using the conventional two-armed antibody depiction; the scFv is the elongated oval.
  • Figure 1 A depicts the scFv linked to the carboxy terminal end of the IgG heavy chain constant domain.
  • Figure IB depicts the scFv linked to the carboxy terminal end of the IgG light chain constant domain.
  • Figure 1C depicts the scFv linked to the amino terminal end of the IgG light chain variable domain.
  • Figure ID depicts the scFv linked to the amino terminal end of the IgG heavy chain variable domain.
  • Figure 2 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 34 and 31) of a bispecific antibody in which a PD-L1 -specific scFv referred to as D7A8 is linked to the carboxy terminal end of the heavy chain constant domain of a KDR-specific IgG antibody referred to as B1A1.
  • Figure 3 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 30 and 35) of a bispecific antibody in which a PD-L1 -specific scFv referred to as D7A8 is linked to the carboxy terminal end of the light chain constant domain of a KDR-specific IgG antibody referred to as B1A1.
  • Figure 4 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 36 and 31) of a bispecific antibody in which a PD-L1 -specific scFv referred to as D7A8 is linked to the amino terminal end of the heavy chain variable domain of a KDR-specific IgG antibody referred to as B 1 A 1.
  • Figure 5 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 30 and 37) of a bispecific antibody in which a PD-L1 -specific scFv referred to as D7A8 is linked to the amino terminal end of the light chain variable domain of a KDR-specific IgG antibody referred to as B1A1.
  • FIG. 6 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 38 and
  • Figure 7 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 32 and 39) of a bispecific antibody in which a PD-L1 -specific scFv referred to as D7A8 is linked to the carboxy terminal end of the light chain constant domain of a KDR-specific IgG antibody referred to as B1C4A7.
  • Figure 8 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 40 and 33) of a bispecific antibody in which a PD-L1 -specific scFv referred to as D7A8 is linked to the amino terminal end of the heavy chain variable domain of a KDR-specific IgG antibody referred to as B1C4A7.
  • Figure 9 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 32 and 41) of a bispecific antibody in which a PD-L1 -specific scFv referred to as D7A8 is linked to the amino terminal end of the light chain variable domain of a KDR-specific IgG antibody referred to as B1C4A7.
  • Figure 10 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 42 and 29) of a bispecific antibody in which a KDR-specific scFv referred to as BlAl is linked to the carboxy terminal end of the heavy chain constant domain of a PD-L1 -specific IgG antibody referred to as D7A8.
  • Figure 11 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 28 and 43) of a bispecific antibody in which a KDR-specific scFv referred to as BlAl is linked to the carboxy terminal end of the light chain constant domain of a KDR-specific IgG antibody referred to as D7A8.
  • Figure 12 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 44 and 29) of a bispecific antibody in which a KDR-specific scFv referred to as BlAl is linked to the amino terminal end of the heavy chain variable domain of a KDR-specific IgG antibody referred to as D7A8.
  • Figure 13 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 28 and 45) of a bispecific antibody in which a KDR-specific scFv referred to as BlAl is linked to the amino terminal end of the light chain variable domain of a KDR-specific IgG antibody referred to as D7A8.
  • Figure 14 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 46 and 29) of a bispecific antibody in which a KDR-specific scFv referred to as B1C4A7 is linked to the carboxy terminal end of the heavy chain constant domain of a PD-L1 -specific IgG antibody referred to as D7A8.
  • Figure 15 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 28 and 47) of a bispecific antibody in which a KDR-specific scFv referred to as B1C4A7 is linked to the carboxy terminal end of the light chain constant domain of a KDR-specific IgG antibody referred to as D7A8.
  • Figure 16 shows the heavy and light chain amino acid sequences (SEQ ID NOs: 48 and 29) of a bispecific antibody in which a KDR-specific scFv referred to as B1C4A7 is linked to the amino terminal end of the heavy chain variable domain of a KDR-specific IgG antibody referred to as D7A8.
  • Figure 17 shows the heavy and light chain amino acid sequences (SEQ ID Nos: 28 and 49) of a bispecific antibody in which a KDR-specific scFv referred to as B1C4A7 is linked to the amino terminal end of the light chain variable domain of a KDR-specific IgG antibody referred to as D7A8.
  • FIG. 18 shows structures of bi-specific antibodies (BsAb) against VEGFR2 and PDL1.
  • Anti-VEGFR2 antibody BlAl binding to human, rat and monkey VEGFR2
  • B1C4A7 binding to human, mouse, rat and monkey VEGFR2
  • Anti-PDLl antibody D7A8 (binding to mouse, rat and monkey PDL1) was reformatted to single chain variable fragment and appended to c-terminal of either BlAl or B1C4A7.
  • a "LALA version” refers to an antibody whose CH2 region was mutated by replacing the two leucine residues (LL bold in, e.g., SEQ ID NOs: 28, 30, and 32 above) with two alanines (AA).
  • FIG. 19 shows SDS-PAGE analysis of bi-specific antibodies after Protein A purification.
  • Figure 20 shows size exclusion chromatograms (UV trace at 280 nm) of protein A- purified bi-specific antibodies in a UPLC system.
  • Figure 21 shows DSC scanning results of bi-specific antibodies in a PBS buffer.
  • Figure 22 shows western blotting results of bi-specific antibodies treated at 37°C for 5 days in the mouse serum.
  • Figure 23 shows dose response ELISA results of bi-specific antibody A7-A8 to determinate the EC50 to human and mouse VEGFR2, and human and mouse PDL1.
  • Figure 24 shows dose response blocking ELISA results to determinate IC50 of bi- specific antibody A7-A8 for the VEGF-VEGFR2 and PDL1-PD1.
  • Figure 25 shows binding to cell expressed human or mouse VEGFR2 and human or mouse PDL1.
  • Figure 26 shows interaction of bi-specific antibody A7-A8 with receptor hVEGFR2 and hPDLl examined by surface plasmon resonance (Biacore).
  • Figure 27 shows a scheme where complexes on an immobilized hVEGFR2 surface and in a solution in a cross binding ELISA.
  • Figure 28 shows bi-specific antibody A7-A8 can bind to VEGFR2 and PDL1 simultaneously examining by a cross binding ELISA.
  • Figure 29 shows inhibition of VEGF- stimulated phosphorylation of VEGFR2 and downstream molecules in KDR-PAE (hVEGFR2) and EOMA (mVEGFR2) by bi-specific antibody A7-A8.
  • Figure 30 shows secretion of cytokine IL2 and INFy in the present of bi-specific antibody A7-A8.
  • Figure 31 shows dose response ELISA results of bi-specific antibody A1-A8 to determinate the EC50 to human and mouse VEGFR2, and human and mouse PDL1.
  • Figure 32 shows dose response blocking ELISA results to determinate IC50 of bispecific antibody A1-A8 for the VEGF-VEGFR2 and PDL1-PD1.
  • Figure 33 shows binding to cell expressed human or mouse VEGFR2 and human or mouse PDL1.
  • Figure 34 shows interaction of bi-specific antibody A1-A8 with receptor hVEGFR2 and hPDLl examined by surface plasmon resonance (Biacore).
  • Figure 35 shows bi-specific antibody A1-A8 can bind to VEGFR2 and PDL1 simultaneously examined by a cross binding ELISA.
  • Figure 37 shows secretion of cytokine IL2 and INFy in the present of bi-specific antibody A1-A8 and A7-A8.
  • Figure 38 shows CT26 study results for BsAb (A7-A8).
  • Figures 39A and 39 B show MC38 study results for BsAb (A7-A8).
  • Figure 40 shows SDS-PAGE results for BsAb (B1A1-A11) and BsAb (B1C4A7-A11) variants where degraded bands were observed.
  • Figure 41 shows SDS-PAGE results for BsAb (A11-B1A1) and BsAb (D7A8-B1A1) variants where no degraded bands were observed after orientation was changed.
  • Figure 42 shows binding to PDL1 and VEGFR2.
  • Figure 43 shows blocking interaction of ligands and receptors.
  • Figure 44 shows the common light chain sequence (SEQ ID NO: 50) and three heavy chain sequences for BsAb (A11-B1A1), BsAb (A11-B1A1)_30 and BsAb (Al l-BlAl)_30cc (SEQ ID NOs: 51-53).
  • Figure 45 shows the common light chain sequence (SEQ ID NO: 29) and three heavy chain sequences for BsAb (D7A8-B1A1), BsAb (D7A8-B1A1)_30 and BsAb (D7A8- BlAl)_30cc (SEQ ID NOs: 54-56).
  • PD-1 The interaction of PD-1 on immune cells with PD-Ll inhibits proliferation and cytokine production by immune cells.
  • PD-Ll is also inducible and upregulated in various tissues, including cancer. Together, PD-1 and PD-Ll play a role in immunosuppression.
  • novel bispecific binding proteins such as bispecific antibodies or antigen binding fragments of such bispecific antibodies that bind to human PD-L1 and block its interaction with human PD-1.
  • the bispecific antibodies also bind to human KDR and block its interaction with human VEGFs.
  • the bispecific antibodies block ligand binding (e.g., binding of one or more of VEGF- A, VEGF-C, VEGF-D, or VEGF-E) to KDR.
  • the bispecific antibodies neutralize activation of KDR.
  • the bispecific antibodies may be used for treating neoplastic diseases, including, for example, solid and non-solid tumors, and hyperproliferative disorders. Accordingly, provided are methods of neutralizing the activation of KDR, methods of inhibiting tumor growth, including inhibition of tumor associated angiogenesis, and methods of treating angiogenesis related disorders.
  • kits containing bispecific antibodies or antibody fragments that bind to PDL1 and KDR are also provided.
  • the bispecific antibodies are not limited by any particular mechanism of KDR inhibition.
  • the mechanism followed by one bispecific antibody is not necessarily the same as that followed by another.
  • Some possible mechanisms include preventing binding of the VEGF ligand to the extracellular binding domain of KDR and preventing dimerization or oligomerization of receptors. Other mechanisms cannot, however, be ruled out.
  • the bispecific antibodies inhibit activation of KDR.
  • KDR inhibition is reduced tyrosine kinase activity of the receptor.
  • Tyrosine kinase inhibition can be determined using well-known methods, such as measuring the autophosphorylation level of the receptor. Inhibition of KDR can also be observed through inhibition or regulation of phosphorylation events of natural or synthetic KDR substrates and other components of the KDR signal transduction pathway. Phosphorylation can be detected, for example, using an antibody specific for phosphotyrosine in an ELISA assay or on a western blot.
  • In vivo assays can also be utilized.
  • receptor tyrosine kinase inhibition can be observed by mitogenic assays using cell lines stimulated with receptor ligand in the presence and absence of inhibitor.
  • HUVEC cells ATCC
  • VEGF vascular endothelial growth factor
  • Another method involves testing for inhibition of growth of VEGF-expressing tumor cells, using for example, human tumor cells injected into a mouse. See, e.g., U.S. Patent No. 6,365,157 (Rockwell et al.).
  • the bispecific binding proteins (e.g., bispecific antibodies) disclosed herein may be made by methods known in the art.
  • Such methods may involve the use of nucleic acids encoding the binding proteins (e.g., bispecific antibodies) or portions thereof.
  • Vectors that may be used to make the binding proteins (e.g., bispecific antibodies) disclosed herein include transient expression vectors, suitable for expressing proteins in HEK293 cells, such as pBhl (Dyax) or pcDNATM 3.4 TOPO® vector (Thermo Fisher Scientific).
  • Stable expression vectors suitable for expressing proteins in mammalian cells, such as pCHO.l in CHO-S (Thermo Fisher Scientific) or GS vector in CHO-K (Lonza).
  • Those skilled in the art may consult the following publication for guidance in making the binding proteins (e.g., bispecific antibodies) or portions thereof disclosed herein: Lu D and Zhu Z. (2014);
  • IgG-like tetravalent bispecific antibody IgG-single-chain Fv fusion.
  • Amino acid sequences of heavy and light chain CDRs set forth herein are identified according to the identification systems of Kabat and Chothia.
  • the first two heavy chain CDRs are identified according to the common systems of Kabat and Chothia, which provide distinct, but overlapping locations for the CDRs.
  • a comparison of the numerous heavy and light chains shows a significant similarity among many of the CDR sequences. Accordingly, it would be expected that many of the CDRs can be mixed and matched among the sequences.
  • the bispecific binding proteins or bispecific antibodies described herein can have one or more amino acid substitutions, deletions, insertions, and/or additions.
  • the bispecific antibodies or proteins comprise one of the above-disclosed heavy chain variable domains and one of the above-mentioned light chain variable domains.
  • the bispecific antibodies or binding fragments thereof comprise one or more of the above-disclosed variable domains with an amino acid sequence at least 85% at least 90%, at least 95 %, at least 96%, at least 97%, at least 98%, or at least 99%, identical to one of the above-disclosed variable domain sequences.
  • leucine at position corresponding to 234 or 235, or both, of IgGl can be mutated to alanine (e.g., the above-described LALA version or LALA mutation).
  • mutations can be made to introduce one or more disulfide bonds between two domains, such as two variable domains, so as to improve thermo stability.
  • residues corresponding to those at positions 44 and 248 of SEQ ID NO: 57 can be mutated to cysteines so as to introduce a disulfide bond.
  • the term “antibody” as used to herein may include whole antibodies and any antigen-binding fragments (i.e. , “antigen- binding portions”) or single chains thereof.
  • An “antibody” refers, in one embodiment, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds, or an antigen binding fragment thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • VH heavy chain variable region
  • the heavy chain constant region is comprised of three domains, CHI , CH2 and CH3.
  • each light chain is comprised of a light chain variable region
  • VL light chain constant region
  • the light chain constant region is comprised of one domain, CL.
  • CL complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four framework regions (FRs), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. , effector cells) and the first component (Clq) of the classical complement system.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g. , bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. Examples include human antibodies, humanized antibodies, and chimeric antibodies.
  • antibody fragments may comprise a portion of an intact antibody, generally including the antigen binding and/or variable region of the intact antibody and/or the Fc region of an antibody which retains FcR binding capability. Examples of antibody fragments include linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Preferably, the antibody fragments retain the entire constant region of an IgG heavy chain, and include an IgG light chain.
  • antigen-binding portion or "antigen binding fragment” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g. , human PD-L1 or KDR).
  • an antigen e.g. , human PD-L1 or KDR
  • binding fragments encompassed within the term "antigen-binding portion/fragment" of an antibody include (i) a Fab fragment - a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment - a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, and (v) a dAb fragment (Ward et al. (1989) Nature 341 :544-546) consisting of a VH domain.
  • An isolated complementarity determining region (CDR), or a combination of two or more isolated CDRs joined by a synthetic linker, may comprise and antigen binding domain of an antibody if able to bind antigen.
  • Human antibody refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g. , mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term "human antibody,” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the terms "human” antibodies and “fully human” antibodies are used synonymously.
  • a “humanized” antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody, e.g. a mouse antibody, are replaced with corresponding amino acids derived from human immunoglobulins.
  • a humanized form of an antibody some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen.
  • humanized antibody retains an antigenic specificity similar to that of the original antibody.
  • a “chimeric antibody” refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.
  • a “hybrid” antibody refers to an antibody having heavy and light chains of different types, such as a mouse (parental) heavy chain and a humanized light chain, or vice versa.
  • Bispecific refers to a protein (e.g., an antibody) that binds to both PD-L1 and KDR.
  • a “bispecific antibody” is an artificial hybrid antibody having two different heavy/light chain pairs, giving rise to two antigen binding sites with specificity for different antigens.
  • Bispecific antibodies can be produced by a variety of methods including fusion of proteins or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann (1990) Clin. Exp. Immunol. 79:315-321; Kostelny et al. (1992) /. Immunol. 148, 1547-1553.
  • the bispecific molecules described herein can be prepared by conjugating the constituent binding specificities using other methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. A variety of coupling or cross-linking agents can be used for covalent conjugation.
  • Examples include protein A, carbodiimide, N-succinimidyl-S-acetyl- thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohexane-l-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al.
  • conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).
  • Antibodies can also be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly preferred
  • the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.
  • both binding portions can be encoded in the same vector and expressed and assembled in the same host cell.
  • a bispecific molecule described herein can be a single chain molecule comprising two single chain antibodies, or a complex having at least two antibody chains, or a combination thereof. Methods for preparing bispecific molecules can be found in, e.g. , U.S. Patent Number 5 ,260,203 ; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881,175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091 ,513; U.S.
  • ELISA immunosorbent assay
  • RIA radioimmunoassay
  • FACS analysis bioassay (e.g. , growth inhibition), or Western Blot assay known in the art or as described herein.
  • “Inhibiting a receptor” means diminishing and/or inactivating the ability of the receptor to transduce a signal, e.g., by diminishing and/or inactivating the intrinsic kinase activity of the receptor.
  • Identity refers to the number or percentage of identical positions shared by two amino acid or nucleic acid sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • peptide As used herein interchangeably to describe the arrangement of amino acid residues in a polymer.
  • a peptide, polypeptide, or protein can be composed of the standard 20 naturally occurring amino acid, in addition to rare amino acids and synthetic amino acid analogs. They can be any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation) .
  • Amino acid substitutions can be made, in some cases, by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target sit; or (c) the bulk of the side chain.
  • Naturally occurring residues can be divided into groups based on side-chain properties; (1) hydrophobic amino acids
  • substitutions include, without limitation, substitution of valine for alanine, lysine for arginine, glutamine for asparagine, glutamic acid for aspartic acid, serine for cysteine, asparagine for glutamine, aspartic acid for glutamic acid, proline for glycine, arginine for histidine, leucine for isoleucine, isoleucine for leucine, arginine for lysine, leucine for methionine, leucine for phenylalanine, glycine for proline, threonine for serine, serine for threonine, tyrosine for tryptophan, phenylalanine for tyrosine, and/or leucine for valine.
  • Methods and computer programs for determining sequence similarity are publically available, including, but not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12: 387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990), and the ALIGN program (version 2.0).
  • the well-known Smith Waterman algorithm may also be used to determine similarity.
  • the BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 at http://www.ncbi.nlm.nih.gov/blast/). In comparing sequences, these methods account for various substitutions, deletions, and other
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • Bispecific antibodies provided herein also include those for which binding characteristics have been improved by direct mutation, methods of affinity maturation, phage display, or chain shuffling. Affinity and specificity may be modified or improved by mutating CDRs and screening for antigen binding sites having the desired characteristics.
  • CDRs are mutated in a variety of ways. One way is to randomize individual residues or combinations of residues so that in a population of otherwise identical antigen binding sites, all twenty amino acids are found at particular positions. Alternatively, mutations are induced over a range of CDR residues by error prone PCR methods (see, e.g., Hawkins et al., /. Mol. Biol., 226: 889-896 (1992)).
  • phage display vectors containing heavy and light chain variable region genes may be propagated in mutator strains of E. coli (see, e.g., Low et al., /. Mol. Biol. , 250: 359-368 (1996)). These methods of mutagenesis are illustrative of the many methods known to one of skill in the art.
  • bispecific antibodies which comprise human constant domain sequences are preferred.
  • the bispecific antibodies may be, may comprise, or may combine members of any immunoglobulin class, such as IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof.
  • the antibody class may be selected to optimize effector functions (e.g., complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC)).
  • bispecific binding proteins involve the use of PD-L1 - and KDR-binding antibody fragments.
  • An Fv is the smallest fragment that contains a complete heavy and light chain variable domain, including all six hypervariable loops (CDRs).
  • variable domains are noncovalently associated.
  • the heavy and light chains may be connected into a single polypeptide chain (a "single-chain Fv” or "scFv") using a linker that allows the V H and V L domains to associate to form an antigen binding site.
  • the linker is (Gly-Gly-Gly-Gly-Serb. Since scFv fragments lack the constant domains of whole antibodies, they are considerably smaller than whole antibodies. scFv fragments are also free of normal heavy-chain constant domain interactions with other biological molecules which may be undesired in certain embodiments.
  • Fragments of an antibody containing VH, VL, and optionally CL, CHI, or other constant domains can also be used in the bispecific binding proteins.
  • Monovalent fragments of antibodies generated by papain digestion are referred to as Fab and lack the heavy chain hinge region.
  • Fragments generated by pepsin digestion, referred to as F(ab') 2 retain the heavy chain hinge and are divalent. Such fragments may also be recombinantly produced.
  • Many other useful antigen-binding antibody fragments are known in the art, and include, without limitation, diabodies, triabodies, single domain antibodies, and other monovalent and multivalent forms.
  • the PD-L1 binding region of the bispecific binding protein such as a bispecific antibody has an on rate constant (Kon) of at least about 10 2 M "1 s "1 ; at least about K ⁇ M ' V 1 ; at least about K ⁇ M ' V 1 ; at least about K ⁇ M ' V 1 ; or at least about lO'TVTV 1 , as measured by surface plasmon resonance.
  • Kon on rate constant
  • the PD-L1 binding region has an on rate constant (Kon) between 10 2 M _1 s _1 and 10 3 M _1 s _1 ; between 10 3 M “1 s “1 and 10 4 M _1 s _1 ; between 10 4 M _1 s _1 and 10 5 M “1 s “1 ; or between 10 5 M “1 s “1 and 10 6 M _1 s _1 , as measured by surface plasmon resonance.
  • Kon on rate constant
  • the KDR binding region of the bispecific binding protein such as a bispecific antibody has an on rate constant (Kon) of at least about 10 2 M _1 s _1 ; at least about K ⁇ M ' V 1 ; at least about K ⁇ M ' V 1 ; at least about K ⁇ M ' V 1 ; or at least about lO'TVfV 1 , as measured by surface plasmon resonance.
  • Kon on rate constant
  • the KDR binding region has an on rate constant (Kon) between K ⁇ M ' 1 and K ⁇ M ' V 1 ; between K ⁇ M ' 1 and K ⁇ M ' V 1 ; between 10 4 M “1 s “1 and 10 5 M “1 s “1 ; or between 10 5 M “1 s “1 and 10 6 M “1 s “1 , as measured by surface plasmon resonance.
  • Kon on rate constant
  • the PD-L1 binding region of the bispecific binding protein such as a bispecific antibody has an off rate constant (Koff) of at most about lO ' V 1 ; at most about 10 " V 1 ; at most about lO ' V 1 ; or at most about 10 "6 s _1 , as measured by surface plasmon resonance.
  • the PD-L1 binding region has an off rate constant (Koff) of 10 "3 s _1 to 10 "4 s _1 ; of 10 "4 s _1 to lO ' V 1 ; or of 10 "5 s _1 to 10 "6 s _1 , as measured by surface plasmon resonance.
  • the KDR binding region of the bispecific binding protein such as a bispecific antibody has an off rate constant (Koff) of at most about 10 ⁇ 3 s _1 ; at most about 10 " V 1 ; at most about lO ' V 1 ; or at most about 10 "6 s _1 , as measured by surface plasmon resonance.
  • the KDR binding region has an off rate constant (Koff) of 10 " V 1 to 10 ⁇ 4 s _1 ; of 10 ⁇ 4 s _1 to lO ' V 1 ; or of 10 ⁇ 5 s _1 to 10 "6 s _1 , as measured by surface plasmon resonance.
  • the PD-L1 binding region of the bispecific binding protein such as a bispecific antibody has a dissociation constant (KD) of at most about 10 ⁇ 7 M; at most about 10 ⁇ 8 M; at most about 10 ⁇ 9 M; at most about 10 "10 M; at most about 10 "n M; at most about 10 " 12 M; or at most 10 ⁇ 13 M.
  • KD dissociation constant
  • the PD-L1 binding region of the bispecific binding protein such as a bispecific antibody has a dissociation constant (KD) to its targets of 10 "7 M to 10 "8 M; of 10 "8 M to 10 "9 M; of 10 "9 M to 10 10 M; of 10 10 M to 10 n M; of 10 n M to 10 " 12 M; or of 10 12 M to 10 "13 M.
  • KD dissociation constant
  • the KDR binding region of the bispecific binding protein such as a bispecific antibody has a dissociation constant (KD) of at most about 10 ⁇ 7 M; at most about 10 ⁇ 8 M; at most about 10 ⁇ 9 M; at most about 10 "10 M; at most about 10 "n M; at most about 10 " 12 M; or at most 10 "13 M.
  • KD dissociation constant
  • the KDR binding region of the bispecific binding protein such as a bispecific antibody has a dissociation constant (KD) to its targets of 10 "7 M to 10 "8 M; of 10 "8 M to 10 "9 M; of 10 "9 M to 10 “10 M; of 10 "10 M to 10 "n M; of 10 "n M to 10 " 12 M; or of 10 "12 M to 10 "13 M.
  • KD dissociation constant
  • the bispecific binding protein described herein may be a conjugate further comprising an imaging agent, a therapeutic agent, or a cytotoxic agent.
  • the imaging agent is a radio label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin.
  • the radio label is: 3 H, 14 C, 35 S, 90 Y, 99 Tc, in In, 125 I, 131 I, 177 Lu, or 153 Sm.
  • the therapeutic or cytotoxic agent is an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine (e.g., an immunostimulatory cytokine), an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin, or an apoptotic agent.
  • cytokine e.g., an immunostimulatory cytokine
  • an anti-angiogenic agent e.g., an anti-angiogenic agent
  • an anti-mitotic agent e.g., an anthracycline, toxin, or an apoptotic agent.
  • molecules that bind human PD-Ll to inhibit immunosuppression and which also bind to human KDR to inhibit angiogenesis combines the PD-Ll -binding domain of a first antibody that binds human PD-Ll with the KDR-binding domain of a second antibody that binds human KDR.
  • the PD-Ll -binding portion of the molecule may be an antigen-binding domain of an antibody such as a heavy and light chain variable domain pair. Suitable antibody heavy and light chain variable domains and antibodies that include them are provided herein.
  • the PD- Ll -binding portion of the bispecific binding proteins can be any agent that binds to PD-Ll and blocks immunosuppression. These include anti-PD-Ll antibodies and fragments, not limited to those antibodies disclosed herein, as well as peptides and proteins derived from human PDl, the natural ligand of human PD-Ll. As disclosed herein, the PD-Ll-binding region is linked to a region that binds human KDR.
  • hybrid molecules comprising a region that binds to human PD-Ll and blocks binding to human PDl, and a region that binds to human KDR and blocks binding to human KDR.
  • the PD-Ll- and KDR-binding regions may be joined by a linker as one polypeptide. Accordingly, provided is a human PD-Ll-binding region linked to a human KDR-binding region.
  • nucleic acid molecules that encode the antibodies or chains thereof described above.
  • the nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid is "isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York.
  • a nucleic acid of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences.
  • the nucleic acid is a cDNA molecule.
  • Nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from the library.
  • the nucleic acids and vectors containining the nucleic acids can be used to express antibodies or chains thereof described above.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector is a type of vector wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. , bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors
  • non-episomal mammalian vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • viral vectors e.g. , replication defective retroviruses, adenoviruses and adeno-associated viruses
  • recombinant host cell (or simply “host cell”), as used herein, is intended to refer to a cell that comprises a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. It is understood that the bispecific binding proteins, where used in a mammal for the purpose of prophylaxis or treatment, generally will be administered in the form of a composition additionally comprising at least one pharmaceutically acceptable carrier.
  • Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, sucrose, polysorbate, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the bispecific binding proteins.
  • a therapeutically effective amount of a bispecific binding protein such as a bispecific antibody is administered to a mammal in need thereof.
  • the term "administering” as used herein means delivering the bispecific binding protein such as a bispecific antibody to a mammal by any method that may achieve the result sought. Administration may be, for example, intravenously or intramuscularly. Although the bispecific binding proteins such as bispecific antibodies are particularly useful for administration to humans, they may be administered to other mammals as well.
  • the term “mammal” as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals. "Therapeutically effective amount” means an amount of bispecific binding protein such as a bispecific antibody that, when administered to a mammal, is effective in producing the desired therapeutic effect.
  • Bispecific binding proteins such as bispecific antibodies are useful for inhibiting tumors and other neoplastic diseases, as well as treating other pathologic conditions associated with immunosuppression.
  • Tumors that can be treated include primary tumors, metastatic tumors, and refractory tumors.
  • Refractory tumors include tumors that fail to respond or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone, or combinations thereof.
  • Refractory tumors also encompass tumors that appear to be inhibited by treatment with such agents, but recur up to five years, sometimes up to ten years or longer, after treatment is discontinued.
  • the bispecific binding proteins such as bispecific antibodies are effective for treating vascularized tumors and tumor that are not vascularized, or not yet substantially vascularized.
  • solid tumors which may be treated include breast carcinoma, lung carcinoma, colorectal carcinoma, pancreatic carcinoma, glioma, and lymphoma.
  • Some examples of such tumors include epidermoid tumors, squamous tumors, such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors, including small cell and non-small cell lung tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors.
  • Kaposi's sarcoma CNS neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas and cerebral metastases, melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma, glioblastoma, preferably glioblastoma multiforme, and leiomyosarcoma.
  • vascularized skin cancers for which the bispecific binding proteins such as a bispecific antibody are effective include squamous cell carcinoma, basal cell carcinoma and skin cancers that can be treated by suppressing the growth of malignant keratinocytes, such as human malignant
  • non-solid tumors include leukemia, multiple myeloma and lymphoma that are unresponsive to cytokines.
  • leukemias include acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), erythrocytic leukemia or monocytic leukemia.
  • lymphomas include Hodgkin's and non-Hodgkin's lymphoma.
  • the bispecific binding proteins such as a bispecific antibody are also used in the treatment of viral infections.
  • PD-1 expression on T cells correlates with viral load in HIV and HCV infected patients and PD-1 expression has been identified as a marker for exhausted virus-specific CD8 + T cells.
  • PD-1 + CD8 + T cells show impaired effector functions and PD-1 associated T cell exhaustion which can be restored by blocking the PD- 1/PD-Ll interaction. This results in recovery of virus-specific CD8 + T cell mediated immunity, indicating that interrupting PD-1 signaling using an antagonistic antibody restores T-cell effector functions.
  • Immunotherapy based on the blockade of PD-1/PD-L1 results in breakdown of T-cell tolerance not only to tumor antigens, but also provides a strategy to reactivate virus-specific effector T cells and eradicate pathogens in chronic viral infections. Accordingly, the antibodies and hybrid proteins of the invention are useful to treat chronic viral infections, including, without limitation, HCV and HIV, and lymphocytic
  • LCMV choriomeningitis virus
  • bispecific binding proteins such as bispecific antibodies can be advantageously administered with second agents to patients in need thereof.
  • a bispecific binding protein such as a bispecific antibody is administered to a subject with an anti-neoplastic agent.
  • the bispecific binding protein such as a bispecific antibody is administered to a subject with an angiogenesis inhibitor.
  • the bispecific binding protein such as a bispecific antibody is administered with an anti-inflammatory agent or an immunosuppressant.
  • Antineoplastic agents include cytotoxic chemotherapeutic agents, targeted small molecules and biological molecules, and radiation.
  • cytotoxic chemotherapeutic agents include cytotoxic chemotherapeutic agents, targeted small molecules and biological molecules, and radiation.
  • chemotherapeutic agents include cisplatin, dacarbazine (DTIC), dactinomycin, irinotecan, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprolide, megestrol, melphalan, mercaptopurine, plic
  • Targeted small molecules and biological molecules include, without limitation, inhibitors of components of signal transduction pathways, such as modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor-specific antigens.
  • growth factor receptors involved in tumorigenesis are the receptors for platelet-derived growth factor (PDGFR), insulin- like growth factor (IGFR), nerve growth factor (NGFR), and fibroblast growth factor (FGFR), and receptors of the epidermal growth factor receptor family, including EGFR (erbBl), HER2 (erbB2), erbB3, and erbB4.
  • EGFR antagonists include antibodies that bind to EGFR or to an EGFR ligand, and inhibits ligand binding and/or receptor activation.
  • the agent can block formation of receptor dimers or heterodimer with other EGFR family members.
  • Ligands for EGFR include, for example, EGF, TGF-a amphiregulin, heparin-binding EGF (HB-EGF) and betaregullulin.
  • An EGFR antagonist can bind externally to the extracellular portion of EGFR, which may or may not inhibit binding of the ligand, or internally to the tyrosine kinase domain.
  • EGFR antagonists further include agents that inhibit EGFR-dependent signal transduction, for example, by inhibiting the function of a component of the EGFR signal transduction pathway.
  • agents that inhibit EGFR-dependent signal transduction for example, by inhibiting the function of a component of the EGFR signal transduction pathway.
  • EGFR antagonists that bind EGFR include, without limitation, biological molecules, such as antibodies (and functional equivalents thereof) specific for EGFR, and small molecules, such as synthetic kinase inhibitors that act directly on the cytoplasmic domain of EGFR.
  • Small molecule and biological inhibitors include inhibitors of epidermal growth factor receptor (EGFR), including gefitinib, erlotinib, and cetuximab, inhibitors of HER2 (e.g., trastuzumab, trastuzumab emtansine (trastuzumab-DMl ; T-DM1) and pertuzumab), anti- VEGF antibodies and fragments (e.g., bevacizumab), antibodies that inhibit CD20 (e.g., rituximab, ibritumomab), anti-VEGFR antibodies (e.g., ramucirumab (IMC-1121B), IMC- 1C11, and CDP791), anti-PDGFR antibodies, and imatinib.
  • EGFR epidermal growth factor receptor
  • HER2 e.g., trastuzumab, trastuzumab emtansine (trastuzumab-DMl ; T
  • Small molecule kinase inhibitors can be specific for a particular tyrosine kinase or be inhibitors of two or more kinases.
  • the compound N-(3,4-dichloro-2-fluorophenyl)-7-( ⁇ [(3aR,6aS)-2- methyloctahydrocyclopenta[c] pyrrol-5 -yljmethyl ⁇ oxy)-6-(methyloxy)quinazolin-4-amine also known as XL647, EXEL-7647 and KD-019
  • RTKs receptor tyrosine kinases
  • EGFR epigallocate
  • EphB4 KDR
  • Flt4 Flt4
  • ErbB2 ErbB2
  • treatment of a subject in need comprises administration of a rho
  • Dasatinib (BMS-354825; Bristol-Myers Squibb, New York) is another orally bioavailable, ATP-site competitive Src inhibitor. Dasatanib also targets Bcr-Abl (FDA- approved for use in patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL)) as well as c-Kit, PDGFR, c-FMS, EphA2, and SFKs.
  • Bcr-Abl FDA- approved for use in patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL)
  • CML chronic myelogenous leukemia
  • ALL Philadelphia chromosome positive
  • c-Kit c-Kit
  • PDGFR chronic myelogenous leukemia
  • c-FMS chronic lymphoblastic leukemia
  • EphA2 Eph
  • a bispecific binding protein such as a bispecific antibody is used in combination with an anti- viral agent to treat a chronic virus infection.
  • an anti- viral agent for HCV, the following agents can be used.
  • HCV protease inhibitors include, without limitation, boceprevir, telaprevir (VX-950), ITMN-191 , SCH-900518, TMC-435, BI-201335, MK-7009, VX-500, VX-813, BMS790052, BMS650032, and VBY376.
  • HCV nonstructural protein 4B (NS4B) inhibitors include, but are not limited to, clemizole, and other NS4B-RNA binding inhibitors, including but not limited to benzimidazole RBIs (B-RBIs) and indazole RBIs (I- RBIs).
  • HCV nonstructural protein 5A (NS5A) inhibitors include, but are not limited to, BMS-790052, A-689, A-831, EDP239, GS5885, and PP1461.
  • HCV polymerase (NS5B) inhibitors include, but are not limited to nucleoside analogs (e.g. , valopicitabine, R1479, R1626, R7128), nucleotide analogs (e.g.
  • ribavirin or a ribavirin analog such as Taribavirin (viramidine; ICN 3142), Mizoribine, Merimepodib (VX- 497), Mycophenolate mofetil, and Mycophenolate can be used.
  • the first and second agents can be administered sequentially or simultaneously.
  • Each agent can be administered in single or multiple doses, and the doses can be administered on any schedule, including, without limitation, twice daily, daily, weekly, every two weeks, and monthly.
  • the invention also includes adjunctive administration of the bispecific antibodies.
  • Adjunctive administration means that a second agent is administered to a patient in addition to a first agent that is already being administered to treat a disease or disease symptom.
  • adjunctive administration includes administering a second agent to a patient in which administration of the first agent did not treat, or did not sufficiently treat, the disease or disease symptom.
  • adjunctive administration includes administration of the second agent to a patient whose disease has been effectively treated by administration of the first agent.
  • a dose of a bispecific binding protein such as a bispecific antibody is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, or once every two weeks.
  • two, three or four doses of a bispecific binding protein such as a bispecific antibody is administered to a subject every day, every couple of days, every third day, once a week or once every two weeks.
  • a dose(s) of a bispecific binding protein such as a bispecific antibody is administered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days.
  • a dose of a bispecific binding protein such as a bispecific antibody is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.
  • Methods of administration include but are not limited to parenteral, intradermal, intravitrial, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, transmucosal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
  • parenteral intradermal, intravitrial, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, transmucosal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
  • administration is left to the discretion of the practitioner. In most instances, administration will result in the release of a bispecific binding protein such as a bispecific antibody into the bloodstream.
  • intravitrial administration of a bispecific binding protein such as a bispecific antibody is preferred.
  • a bispecific binding protein such as a bispecific antibody
  • administration may selectively target a local tissue without substantial release of a bispecific binding protein such as a bispecific antibody into the bloodstream.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant.
  • a bispecific binding protein such as a bispecific antibody is formulated as a suppository, with traditional binders and vehicles such as triglycerides.
  • a bispecific binding protein such as a bispecific antibody is delivered in a vesicle, in particular a liposome (See Langer, 1990, Science 249:1527 - 1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Bacterial infection, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353 - 365 (1989); Lopez Berestein, ibid., pp. 317 - 327; see generally ibid.).
  • a bispecific binding protein such as a bispecific antibody is delivered in a controlled release system (See, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 - 138 (1984)). Examples of controlled-release systems are discussed in the review by Langer, 1990, Science 249:1527 - 1533 may be used.
  • a pump may be used (See Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574).
  • polymeric materials can be used (See Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; See also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71 :105).
  • administration schedules are provided for illustrative purposes only and should not be considered limiting. A person of ordinary skill in the art will readily understand what doses and dosing schedules are suitable.
  • PCT/US2015/011657; and PCT/US2015/054569 are incorporated herein by reference, in their entireties.
  • This example descibes transient expression of bi-specific antibody against VEGFR2 and PDL1 in HEK293 and purification by Protein A; aggregation analysis (by SEC-UPLC), serum stability test and thermal stability test (Tm measurement by using and DSC).
  • Antibody R3B1 (against VEGFR2) were isolated by panning on the immobilized VEGFR2 directly from Dyax Fab 310 phagemid library.
  • a light chain shuffling library was built by paring the light chain pool with the heavy chain of R3B1.
  • B1C4 which can bind to both human and murine VEGFR2, was isolated.
  • a further modification in its light chain CDR3 and heavy chain CDR1 and CDR2 was done by soft mutation method, in which only 10% of residues were mutated to the other 19 amino acids (excluding cysteine).
  • B1C4A7 a higher affinity and more stable antibody, was identified by screening the soft-mutated library in the very stringent conditions.
  • Anti-PDLl antibodies tccR3D7 and tccR3Al l were also identified by panning on the PDL1 from Dyax Fab 310 phagemid library.
  • D7A8 an antibody with higher affinity and lower oxidization sensitivity, was isolated from the tccR3D7_HCDRl library, in which three amino acids of methionine locating in tccR3D7 heavy chain CDR1 were mutated randomly.
  • anti-PDLl antibody D7A8 (or Al l) was reformatted to the single- chain variable fragment (scFv) in the orientation of VH-(SG 4 )5-VL first; then the scFv was appended to the c-terminal of conventional antibody BlAl or B1C4A7 (against VEGFR2) connected by a linker ( Figure 1).
  • leucines in the BlAl or B1C4A7 position 234 and 235 (IgGl-CH2 domain) were mutated to alanine.
  • bi-specific antibody (A1-A8) and (A7-A8) was inserted to mammalian expression vector pBhl and transiently expressed in HEK293 cell line. The supernatant was harvested and loaded to Protein A column to purify bi-specific antibodies.
  • bi-specific antibody A1-A8 and (A7-A8) were analyzed by using SDS-PAGE, SEC-UPLC and DSC.
  • the serum stability of these two bi-specific antibodies was examined by incubating the BsAb in mouse serum following Western blotting.
  • Mammalian expression vectors carried bi-specific antibodies were transfected to the free style HEK293 cells by using Polyjet transfection reagent according to the manufacture instruction.
  • the 6-day culture was harvested and filtered supernatant was loaded to the affinity column protein A connected to the AKTAxpress protein purification system.
  • the purified antibody was buffer exchanged to PBS and filtered.
  • Antibody concentration was obtained by measuring absorbance at 280 nm in Nanodrop photometer and calculated by using the theoretical coefficient.
  • Antibody samples were prepared in reduced and non-reduced conditions by adding loading dye with or without reducing agent.
  • the sample tubes were heated at 100°C for 10 min and the reduced and non-reduced samples, along with a molecular weight standard, were loaded onto a 4-12% NUPAGE® NOVEX bis-tris gel.
  • the gels were run at a 200 v constant voltage for about 50 minutes.
  • the gel was then stained gel with protein dye and destained in QH20 until the protein band was observed. A picture of the gel is shown in ( Figure 19)
  • Mammalian expression vectors carried bi-specific antibodies were transfected to the free style HEK293 cells.
  • Bi-specific antibodies were purified by Protein A and buffer exchanged to PBS. The antibody concentration was measured by Nanodrop photometer at 280 nm and calculated by using the theoretical coefficient. The results are shown in the table below.
  • bi-specific antibody A1-A8 and A7-A8 could be expressed transiently in HEK-293 cells.
  • Over 10 mg of BsAb (A1-A8) and BsAb (A7-A8) were recovered from 1L supernatant harvested in day 6 after Protein A purification and buffer exchange.
  • Tml of BsAb (A7-A8)-LALA was about ⁇ 60°C measured by DSC scanning, which was lower than the lowest Tml of the parental antibody B1C4A7 and D7A8-LALA ( ⁇ 69°C for both).
  • the lowest Tml of BsAb (A7-A8) was contributed by scFv D7A8 as reported that scFv antibody fragment was not as stable as IgG.
  • Further engineering of scFv such as using a longer linker between heavy and light chain variable domain of D7A8, or adding a disulfide bond between two domains, may improve its thermo stability.
  • This example descibes in vitro characterization of bi-specific antibody A7-A8 and comparison of the potency with its parental antibodies B1C4A7 (against VEGFR2) and dsD7A8-LALA (against PDLl).
  • bi-specific antibody A7-A8 obtained by transiently expressed in HEK293 and purified by Protein A, had less than 1 % higher molecular weight aggregation detected by SEC-UPLC analysis.
  • BsAb (A7-A8) was also stable in 37° C mouse serum up to 5 days.
  • Bi-specific antibody A7-A8 which can cross react with human and mouse species, alone with its parental antibody B1C4A7 (against VEGFR2) and dsD7A8-LALA (against PDLl), was studies the binding ability to soluble and cell expressed VEGFR2 and PDLl (both human and mouse species), inhibiting the ligands (VEGF and PDLl) binding to the respective receptors (VEGFR2 and PD1), blocking VEGFR2/downstream molecules phosphorylation and stimulating cytokine IL2 and INFy secretion.
  • VEGFR2 and PDLl both human and mouse species
  • VEGFR2-Fc human and mouse
  • PDLl-Fc human and mouse
  • Immulon 2HB plates 1 ⁇ g/ml at 4°C overnight.
  • the plates were blocked with 3% PBSM.
  • Serially diluted bi-specific and mono-specific antibodies were added to the blocked plates and incubated at RT for about 1 hour.
  • HRP conjugated anti-human IgG Fab specific (HRPanti-hFab) antibody was added (1/5000 diluted in 3% PBSM) after washing the plates. The plate was washed again, and then TMB reaction buffer was added to develop the color.
  • the plates were read at OD 450 in a plate reader after stopping the reaction with IN H2SO4.
  • VEGF human and mouse
  • PDL1 human and mouse
  • the binding affinities were evaluated using surface plasmon resonance (SPR) in a Biacore T200 instrument (GE Healthcare).
  • SPR surface plasmon resonance
  • a CM5 chip was equilibrated in running buffer HBSEP (running buffer) at 10 ⁇ /ml.
  • Two flow cells of a CM5 chip were activated with 1 :1 NHS/EDC injection for five minutes.
  • the second flow cell was immobilized with 5 ⁇ g/ml of hVEGFR2 diluted in 10 mM sodium acetate buffer pH 5 to reach 40-100 RU of immobilized protein.
  • the surfaces were subsequently blocked with a 5 -minute injection of ethanolamine.
  • Human PDLl-Fc was labeled with a biotin labeling kit from Thermo-Fisher according to the manufacturer's instructions.
  • Human VEGFR2 was coated to the Immulon 2HB plate at ⁇ g/ml at 4°C overnight.
  • the plate was blocked with 3% PBSM.
  • 1 nM of bi-specific antibody or mono-specific antibodies were mixed with biotin labeled hPDLl in a PBSM blocked round 96-well plate and incubated at RT for 1 hour.
  • the mixtures were transferred to the PBSM blocked hVEGFR2 coated plate and incubated at room temperature for an additional 1 hour.
  • HRP- strep was added (1/5000 diluted in 3% PBSM) after washing the plate.
  • the plate was washed again and TMB reaction buffer was added to develop the color.
  • the plate was read at OD450 in a plate reader after stopping the reaction with IN H2SO4.
  • PAE-KDR cells in DMEM with glutamine, supplemented with 10% heat-inactive FBS
  • EOMA cells in DMEM with glutamine, supplemented with 10% heat-inactive FBS
  • MDA-MB-231 cells in RPMI1640 with glutamine, supplemented with 10% heat-inactive FBS
  • B16-F10 cells in RPMI1640 with glutamine, supplemented with 10% heat- inactive FBS were grown until 90% confluent. Cells were harvested, washed first, and then resuspended at 5xl0 5 cells/ml in ice cold FACS Buffer (PBS, 1% BSA).
  • Stripping buffer 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulphate and 62.5 mM Tris-HCl pH 6.7
  • Loading buffer 4 ml 100% glycerol, 2.4 ml of pH6.8 1 M Tris/HCl, 0.8 g SDS, 4 mg bromophenol blue, 0.5 ml 2-mercaptoethanol and 3.1 ml ddfhO.
  • PAE/KDR (0.4xl0 6 ) cells in 1 ml of 10% FBS/DMEM or EOMA (lxlO 6 ) cells in 1.5 ml of 10% FBS DMEM culture medium was added into 12 well-plates, incubated for 4 hours at 37°C, 5% CO2 until the cells attached. The medium was removed and the serum free DMEM was added to starve the cells overnight. Next day serially diluted antibodies were added to the cells and incubated for 20 min at 37°C, 5% CO2 first, and then added VEGF (final concentration 30 ng/ml and 50 ng/ml for PAE/KDR and EOMA, respectively, into cells; the cell mixture was incubated for an additional 10 minutes at 37°C, 5% CO2.
  • the cells were washed with serum free culture medium once. Then, a 100 ⁇ lyses buffer was added and the cells were scrapped from dish to 1.5 ml Eppendorf tubes and incubated for about 2 hours on ice. The lazed samples were centrifuged for 10 min at 10,000 rpm 4°C and the supernatant were collected. The concentration of protein was measured by Bio-Rad protein assay kit. Then, 30 ⁇ g protein was mixed with 8 ⁇ load buffer and boiled for 5 min. The mixture was loaded onto a 4-12% NUPAGE gel and the gel was run to separate the proteins. The proteins and markers were transferred together onto a PVDF membrane overnight.
  • the membrane was blocked with 3% PBS milk, then the anti-phospho-VEGFR2 (1/1000) or antiphospho-MAPK was added to the membrane.
  • the membrane was incubated at 4°C overnight. After washing membrane twice with 0.1% T-PBS, 1 :1500 of an anti-rabbit antibody conjugated HRP was added and incubated with the membrane at RT for 1 hour.
  • the membrane was washed with PBST three times before ECL was added and picture taken with ImageQuant LAS 4000 (for the phosphor-protein).
  • the membrane was washed twice with PBST and then incubated with a rabbit anti-KDR antibody or anti-MAPK antibody (1 to 400 dilutions, in 10 ml 3% PBSM).
  • the membrane was incubated with rabbit antibodies at 4°C overnight.
  • the membrane was washed again and am HRP-anti-rabbit antibody (1 to 1500 dilutions) was added to the membrane. After an incubation at RT for 1 hour, the membrane was washed three times before ECL was added and luminescent pictures were taken in ImageQuant LA4000 (for the total protein).
  • PBMCs were isolated from LeukoPak (an enriched leukapheresis containing highly concentrated blood cells including monocytes, lymphocytes, platelets, plasma, as well as red cells) using Histopaque-1077 per manufacturer's instructions.
  • PBMCs were cultured at 2 x 10 4 cells per well in 96 well plate containing IMDM (supplemented with 2 mM Glutamine, 25 mM HEPES, 3.024 g/L Sodium Bicarbonate) and 10% FBS and activated by 0.01 ⁇ g/mL SEB for 5 to 7 days in the presence of serially diluted bi-specific antibody or its parental antibodies.
  • IMDM supplied with 2 mM Glutamine, 25 mM HEPES, 3.024 g/L Sodium Bicarbonate
  • the EC50 was 0.128 nM to hVEGFR2, 0.598 Nm to mVEGFR2, 0.079 nM to hPDLl and 0.137 nM to mPDLl respectively; the relevant EC50 for B1C4A7 to hVEGFR2 and mVEGFR2 was 0.107 nM and 0.466 nM respectively; for dsD7A8 to hPDLl and mPDLl was 0.121 nM and 0.172 nM respectively.
  • the serially diluted BsAb (A7-A8)- LALA alone with its parental mono-specific antibody B1C4A7 (against VEGFR2) and dsD7A8-LALA (against PDLl), were incubated with a fixed amount of either human or mouse VEGFR2-Fc (0.5 ⁇ g/ml final) in solution at RT for 1 hour, after which the mixtures were transferred to 96-well plates coated with either human VEGF or mouse VEGF and incubated for an additional 1 hour. The amount of h/mVEGFR2 that bound to the immobilized h/mVEGF was quantified by incubation of the plates with HRP-anti-human IgG Fc specific antibody.
  • the serially diluted BsAb (A7-A8)-LALA alone with its parental mono-specific antibody B1C4A7 (against VEGFR2) and dsD7A8- LALA (against PDLl), were incubated with a fixed amount of Biotin labeled either human or mouse PDLl-Fc (0.5 ⁇ g/ml final) in solution at RT for 1 h, after which the mixtures were transferred to 96-well plates coated with either human PDLl or mouse PDLl and incubated for an additional hour. The amount of h/mPDL that bound to the immobilized h/mPDl was quantified by incubation of the plates with HRP-strep.
  • BsAb (A7-A8) could also strongly block ligand VEGF or PDLl binding to their respective receptor VEGFR2 and PD1.
  • IC50 for hVEGF-hVEGFR2 interaction was 1.29 nM for BsAb (A7-A8)-LALA, versus 1.72 nM for B1C4A7.
  • IC50 for mVEGF-mVEGFR2 interaction was 0.801 nM for BsAb (A7-A8)-LALA, versus 0.973 nM for B1C4A7.
  • IC50 for hPDLl-hPDl interaction was 2.51 nM for BsAb (A7-A8)-LALA, versus 3.04 nM for dsD7A8-LALA.
  • IC50 for the mPDLl-mPDl interaction was 5.59 nM for BsAb (A7-A8)-LALA, versus 2.80 nM for dsD7A8-LALA.
  • BsAb (A7-A8)-LALA also could bind to cell expressed human VEGFR2, mouse VEGFR2, human PDL1 and mouse PDL1.
  • the EC50 values of BsAb (A7-A8)-LALA to KDR/PAE, MDA-MB-231 and B16-F10 were 0.533 nM, 0.223 nM and 0.122 nM respectively; the relevant EC50 for B1C4A7 to KDR/PAE was 0.522 nM, for dsD7A8 to MDA-MB-231 and B16-F10 the values were 0.159 nM and 0.042 nM
  • the EC50 and IC50 for bi-specific antibody A7-A8 and its parental antibody B1C4A7 and dsD7A8-LALA are listed in the table below.
  • the very close EC50 and IC50 between BsAb (A7-A8)-LALA and its relevant parental antibody either B1A1 or dsD7A8-LALA indicates that BsAb (A7-A8)-LALA is as potent as either B1A1 or dsD7A8-LALA.
  • hVEGFR-Fc or hPDLl-Fc was immobilized at pH 5 onto a Series S CM5 sensor chip using standard amine coupling chemistry.
  • Antibodies were injected at 30 ⁇ /min at concentrations ranging from 1.5 to 100 nM over the immobilized surface using IX HBSEP as the running buffer.
  • the contact time (association phase) was 3 minutes.
  • the dissociation time was 6-10 minutes.
  • Regeneration was performed after each binding cycle with an injection of 20 mM HCL for 30 seconds at 30 ul/min flow rate.
  • Sensorgrams were obtained at each concentration and the derived curves were fit to a 1:1 Langmuir binding model using Biaevaluation software. The results are shown in Figure 26.
  • the on-rate (ka), off-rate (kd) and the calculated KD is list on the top of each graph.
  • BsAb (A1-A8)-LALA associated to either VEGFR2 or PDLl fast and dissociated from either VEGFR2 or PDLl slowly.
  • the very slow off rate from VEGFR2 and PDLl was out of the machine measurement range.
  • the KD values of BsAb (A7-A8) were about 47 pM and 1.2 pM for the hVEGFR2 and hPDLl respectively, comparable to 52 pM for B1C4A7 and 7.7 pM for dsD7A8-LALA.
  • Scheme 1 shown in Figure 27 was used for the cross binding ELISA.
  • Complexes 2 and 3 can be found on the immobilized hVEGFR2 surface, but only complex 3 can be detected by strep-HRP.
  • Complex 1, which can be found in the solution, will be washing out during the processing.
  • bi-specific antibody A7-A8 could bind to VEGFR2 and PDL1 simultaneously examining by cross binding ELISA. This result indicates that the
  • the first target bound bi-specific antibody can bind to second target.
  • Serum starved cells were incubated with various amounts of antibodies at room temperature for 30 min, followed by stimulation with 30 ng/ml of hVEGF (for KDR-PAE) or 50 ng/ml of mVEGF (for EOMA), for additional 15 min.
  • the cells were lysed and protein concentration was measured.
  • 30 ⁇ g of proteins were resolved with SDS PAGE and subjected to immunoblotting analysis using anti-phosphotyrosine antibodies to the receptor and MAPK. The signals were detected using enhanced chemoilluminescense. The results are shown in Figure 29.
  • BsAb (A7-A8)-LALA The potency of BsAb (A7-A8)-LALA was examined by testing its ability to block phosphorylation of human VEGFR2 or mouse VEGFR2 and downstream molecule MAPK.
  • Figure 29 shows that the phosphor protein bands of human VEGFR2, mVEGFR2 and MAPK are much weaker in the present of 100 or 10 nM of BsAb (A7-A8)-LALA and B1C4A7.
  • the difference of band density between treated with BsAb (A7-A8) and B1C4A7 was so minor and could not be differentiated by eye. Cytokine secretion assay
  • BsAb (A7-A8)-LALA retains the potency of its parental antibody B1C4A7 (against VEGFR2) and dsD7A8-LALA (against PDL1).
  • This example descibes in vitro characterization of bi-specific antibody A1-A8 and comparison of the potency with its parental antibodies Bl Al (against VEGFR2) and dsD7A8-LALA (against PDL1).
  • bi-specific antibody A1-A8 obtained by transient expression in HEK293 and purified by Protein A, had less than 1 % higher molecular weight aggregation detected by SEC-UPLC analysis.
  • BsAb (A1-A8) was also very stable in mouse serum after incubating at 37 °C for up to 5 days.
  • Bi-specific antibody A1-A8 which can cross react with human and mouse species, alone with its parental antibody B1A1 (against VEGFR2) and dsD7A8-LALA (against PDL1), was studied for its binding ability to soluble and cell expressed VEGFR2 and PDL1 , inhibiting the ligands (VEGF and PDL1), binding to the receptors (VEGFR2 and PD1), blocking VEGFR2/downstream molecule phosphorylation and stimulating cytokine IL2 and INFy secretion.
  • BsAb (A1-A8)-LALA can strongly bind to the recombinant human VEGFR2, human PDL1 and mouse PDL1.
  • the EC50 is 0.128 nM for hVEGFR2, 0.088 nM for hPDLl and 0.160 nM for mPDLl , respectively; the relevant EC50 for BlAl to hVEGFR2 is 0.127 nM, for dsD7A8 to hPDLl and mPDLl is 0.121 nM and 0.172 nM, respectively.
  • BsAb (A1-A8) could strongly block ligand VEGF or PDL1 binding to their respective receptor, VEGFR2 and PD1.
  • IC50 for hVEGF-hVEGFR2 interaction was 1.19 nM for BsAb (A1-A8)-LALA versus 1.60 nM for BlAl.
  • IC50 for hPDLl-hPDl interaction was 2.51 nM for BsAb (A1-A8)-LALA versus 3.04 nM for dsD7A8-LALA.
  • IC50 for the mPDLl-mPDl interaction was 3.20 nM for BsAb (A1-A8)-
  • the binding assay was carried out in the manner described above. The results are shown in Figure 33.
  • the results indicate that BsAb (A1-A8)-LALA also could bind to cell expressed human VEGFR2, human PDLl and mouse PDLl .
  • the EC50 values of BsAb (A1-A8)- LALA to KDR/PAE, MDA-MB-231 and B16-F10 were 0.779 nM, 0.402 nM and 0.113 nM respectively; the relevant EC50 values for BlAl to KDR/PAE is 0.177 nM, for dsD7A8 to MDA-MB-231 and B16-F10 were 0.159 nM and 0.042 nM, respectively. (Figure 33).
  • the EC50 and IC50 for bi-specific antibody A1-A8 and its parental antibody BlAl and dsD7A8-LALA are listed in the table below.
  • the very close EC50 and IC50 between BsAb (A1-A8)-LALA and its relevant parental antibody either BlAl or dsD7A8-LALA indicates that BsAb (A1-A8)-LALA is as potent as either BlAl or dsD7A8-LALA.
  • the kinetic assay was carried out in the manner described above. The results are shown in Figure 34.
  • the on-rate (ka), off-rate (kd) and the calculated KD is list on the top of each graph.
  • KD values of BsAb (A1-A8) were about 200 pM and 3.2 pM for the hVEGFR2 and hPDLl, respectively, comparable to 164 pM for B1A1 and 7.7 pM for dsD7A8-LALA.
  • the binding assay was carried out in the manner described above. The results are shown in Figure 35. The data points are the means + S.D. of six determinations.
  • bi-specific antibody A18 could bind to VEGFR2 and PDL1 simultaneously examined by cross binding ELISA ( Figure 36). This result indicates that the conformation of bi-specific antibody did not change after binding to the first target; therefore, the first target bound bi-specific antibody could bind to the second target.
  • BsAb (A1-A8)-LALA The potency of BsAb (A1-A8)-LALA was examined by testing its ability to block phosphorylation of human VEGFR2 and downstream molecule MAPK.
  • Figure 36 shows that in the present of 1.5 ⁇ (7.5 nM) of BsAb (A1-A8)-LALA and BsAb (A7-A8)-LALA, the phosphor protein bands for both VEGFR2 and MAPK were much weaker than the bands without antibody treatment.
  • the concentration for the parental antibody was 1.5 ⁇ g/ml (10 nM); therefore, more mono-specific antibody was used than bi-specific antibodies in this experiment.
  • the cytokine secretion assay was carried out in the manner described above.
  • the serially diluted BsAb (A1-A8)-LALA and BsAb (A7-A8) were added to PBMC (from donor BG) which was activated by 0.01 ⁇ g/mL SEB.
  • PBMC from donor BG
  • BsAb A7-A8
  • supernatants were collected for the measurements of IL-2 and IFNy by using Duoset kit per manufacturer's instructions.
  • the data points were the means + S.D. of duplicate determination. The results are shown in Figure 37.
  • mice were allocated randomly into 5 experimental groups according to their tumor sizes. Each group consisted of 13 mice. The mice were treated with antibodies as shown in the table below. Each dose was adjusted to 200 ⁇ with PBS and injected to the mice by i.p. twice per week up to three weeks. Body weights and tumor volumes were measured twice weekly. The results are shown in the table below.
  • B1C4A7, D7A8, their combination, and BsAb (A7-A8) reduced volumes of the tumor. It was found that combination of the two antibodies (B1C4A7 and D7A8) and BsAb (A7-A8) were more potent than two individual antibodies alone in reducing tumor volume in this CT26 model. Since all antibodies were fully human antibodies, immunogenicity was developed for the treatment longer than 2 weeks. Results were therefore reported up to 3 weeks after the treatment. The results also indicate that no significant decrease in body weight was found in any of the treatment groups (data not shown).
  • mice were inoculated subcutaneously at the right lower flank with MC38 murine colon carcinoma cells (1 x 10 6 cells/mouse) for tumor development.
  • MC38 murine colon carcinoma cells (1 x 10 6 cells/mouse) for tumor development.
  • the mice were allocated randomly into 9 experimental groups according to their tumor sizes. Each group consisted of 13 mice.
  • the mice were treated with antibodies as shown in Figures 39A and 39B.
  • Each dose was adjusted to 200 ⁇ with PBS and injected to animals by i.p. twice per week up to three weeks. Body weights and tumor volumes were measured twice weekly. The results are shown in Figures 39A and 39B.
  • B1C4A7 antibody against VEGFR2 only showed a mediate potency.
  • bi-specific antibodies as depicted in Figure 1A (i.e., HC-C terminal fusion) were generated and examined.
  • different orientations of bispecific antibody against VEGFR2 and PDL1 were generated using anti- PDLl antibody All or D7A8 and anti-VEGFR2 antibody B1A1 or B1C4A7.
  • These bi-specific antibodies, related components, and binding specificities are listed in the table below.
  • the bi-specific antibodies the following two linkers were used:
  • mutations were introduced into the scFv to add a disulfide bond between two variable domains. Mutations were also introduced to the CH2 domain to diminish the effector function (LALA version IgG) where the LL residues (bold in, e.g., SEQ ID NOs: 28, 30, and 32) in the CH2 were changed to AA.
  • LALA version IgG effector function
  • BsAb (D7A8-B1A1) variants for BsAb (D7A8-BlAl)_30cc, monomers accounted for more than 97.3%, while for D7A8 IgG, BsAb (D7A8-B1A1) and BsAb (D7A8-B1A1)_30, monomers accounted for more than 99%, 86.6%, and 90.2%, respectively. Binding of these bi-specific antibodies to PDLl and VEGR2 were analyzed in the same manner described above. The results are shown in Figure 42. It was found these bi- specific antibodies to PDLl and VEGR2 as potent as the parental antibodies.

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Abstract

L'invention concerne des protéines de liaison bispécifiques tels que des anticorps bispécifiques qui se lient à la fois au PD-L1 humain et au KDR humain. Les protéines de liaison bispécifiques sont utiles pour le traitement de maladies et d'états caractérisés par l'immunosuppression et/ou une angiogenèse excessive.
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CN110498857B (zh) * 2019-08-09 2022-09-09 安徽瀚海博兴生物技术有限公司 一种抗vegf-抗pd1双特异性抗体
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