WO2019179421A1 - Nouvelles molécules d'anticorps pd-1/ctla-4 bispécifiques - Google Patents

Nouvelles molécules d'anticorps pd-1/ctla-4 bispécifiques Download PDF

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WO2019179421A1
WO2019179421A1 PCT/CN2019/078664 CN2019078664W WO2019179421A1 WO 2019179421 A1 WO2019179421 A1 WO 2019179421A1 CN 2019078664 W CN2019078664 W CN 2019078664W WO 2019179421 A1 WO2019179421 A1 WO 2019179421A1
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ctla
bispecific antibody
sequence
binding
seq
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PCT/CN2019/078664
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Zhuozhi Wang
Jing Li
Yong Zheng
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Wuxi Biologics (Shanghai) Co., Ltd.
WuXi Biologics Ireland Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—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
    • C07K2317/70—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

  • the present disclosure generally relates to novel bispecific antibody molecules directed to human PD-1 and human CTLA-4.
  • Bispecific antibodies are growing to be the new category of therapeutic antibodies. They can bind two different targets or two different epitopes on a target, creating additive or synergistic effect superior to the effect of individual antibodies.
  • a lot of antibody engineering efforts have been put into designing new bispecific formats, such as DVD-Ig, CrossMab, BiTE etc. (Spiess et al. Molecular Immunology, 67 (2) , pp. 95–106 (2015) . ) .
  • these formats may potentially have various limitations in stability, solubility, short half-life, and immunogenicity.
  • PD-1 Programmed cell death 1
  • T cells that provide a major immune resistance mechanism by which tumor cells escaped immune surveillance.
  • the interaction of PD-1 expressed on activated T cells, and PD-L1 expressed on tumor cells negatively regulate immune response and damp anti-tumor immunity.
  • Cytotoxic T-lymphocyte-associated protein 4 is one of the validated targets of immune checkpoints. After T cell activation, CTLA-4 quickly expresses on those T cells, generally within one hour of antigen engagement with TCR. CTLA-4 can inhibit T cell signaling through competition with CD28. In addition to induced expression on activated T cells, CTLA-4 is constitutively expressed on the surface of regulatory T cells (Treg) , suggesting that CTLA-4 may be required for contact-mediated suppression and associated with Treg production of immunosuppressive cytokines such as transforming growth factor beta and iterleukin-10.
  • Treg regulatory T cells
  • an antibody means one antibody or more than one antibody.
  • the present disclosure provides novel bispecific PD-1/CTLA-4 antibody molecules, amino acid and nucleotide sequences thereof, and uses thereof.
  • the present disclosure provides herein a bispecific antibody molecule comprising a CTLA-4-binding domain and a PD-1 -binding domain, wherein:
  • CTLA-4-binding domain comprises:
  • CDR heavy chain complementarity determining region
  • the PD-1 binding domain comprises:
  • heavy chain CDR sequences selected from the group consisting of: the sequence of SEQ ID NOs: 1-3; and/or
  • the CTLA-4-binding domain comprises one independently selected from the group consisting of: a Fab and a single chain Fv antibody (scFv) ; and
  • the PD-1-binding domain comprises one independently selected from the group consisting of: a Fab and a scFv.
  • the CTLA-4-binding domain comprises a Fab.
  • the PD-1-binding domain comprises a Fab.
  • the CTLA-4-binding domain comprises a scFv.
  • the PD-1-binding domain comprises a scFv.
  • the CTLA-4-binding domain comprises a heavy chain variable region comprising 1, 2, or 3 CDR sequences selected from the sequence of SEQ ID NOs: 11-13 and/or a light chain variable region comprising 1, 2, or 3 CDR sequences selected from the sequence of SEQ ID NOs 14-16.
  • the CTLA-4-binding domain comprises a heavy chain variable region selected from the group consisting of the sequence of SEQ ID NOs: 17 and 21, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to CTLA-4.
  • the CTLA-4-binding domain comprises a light chain variable region selected from the group consisting of the sequence of SEQ ID NOs: 18 and 22, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to CTLA-4.
  • the CTLA-4-binding domain comprises:
  • the PD-1-binding domain comprises a heavy chain variable region comprising 1, 2, or 3 heavy chain CDR sequences selected from the group consisting of: the sequence of SEQ ID NOs: 1-3; and/or a light chain variable region comprising 1, 2, or 3 light chain CDR sequences selected from the group consisting of: the sequence of SEQ ID NOs: 4-6.
  • the PD-1-binding domain comprises a heavy chain variable region of the sequence of SEQ ID NO: 7 and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1.
  • the PD-1-binding domain comprises a light chain variable region of the sequence of SEQ ID NO: 8, and a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1.
  • the PD-1-binding domain comprises:
  • a heavy chain variable region comprising the sequence of SEQ ID NO: 7 and a light chain variable region comprising the sequence of SEQ ID NO: 8.
  • the CTLA-4-binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to CTLA-4, and/or the PD-1-binding domain further comprises one or more amino acid residue substitutions or modifications yet retains specific binding affinity to PD-1.
  • At least one of the substitutions or modifications is in one or more of the CDR sequences, and/or in one or more of the VH or VL sequences but not in any of the CDR sequences.
  • the bispecific antibody molecule further comprises an immunoglobulin (Ig) constant region, optionally a constant region of human Ig, or optionally a constant region of human IgG.
  • Ig immunoglobulin
  • the CTLA-4-binding domain is operably linked to the N terminus or the C terminus of the PD-1-binding domain.
  • the CTLA-4-binding domain comprises a Fab and the PD-1-binding domain comprises a scFv.
  • the PD-1-binding scFv comprises the sequence of SEQ ID NO: 25 or 29, and the CTLA-4-binding Fab comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 17 and a light chain variable region comprising the sequence of SEQ ID NO: 18.
  • the PD-1-binding scFv is operably linked to the C terminus of the heavy chain constant region following the CTLA-1-binding Fab.
  • the bispecific antibody comprises a heavy chain in the format of: VH (anti-CTLA-4) -CH1-Hinge-CH2-CH3-spacer-scFv (anti-PD1) or VH (anti-CTLA-4) -spacer-scFv (anti-PD1) -CH1-Hinge-CH2-CH3, which is associated with the light chain VL (anti-CTLA-4) -CL.
  • the bispecific antibody molecule comprising a heavy chain comprising the sequence of SEQ ID NO: 26 and a light chain comprising the sequence of SEQ ID NO: 27.
  • the PD-1-binding scFv is operably linked to the N terminus of the CTLA-4-binding Fab.
  • the bispecific antibody molecule comprises a heavy chain in the format of: scFv (anti-PD-1) -spacer-VH (anti-CTLA-4) -CH1-Hinge-CH2-CH3, associated with a light chain in the format of VL (anti-PD-1) -CL.
  • the bispecific antibody molecule comprising a heavy chain comprising the sequence of SEQ ID NO: 28 or 30 and a light chain comprising the sequence of SEQ ID NO: 27.
  • the CTLA-4-binding domain and/or the PD-1-binding domain is humanized.
  • the bispecific antibody molecule as provided herein is linked to one or more conjugate moieties.
  • the conjugate moiety comprises a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylators, a topoisomerase inhibitor, a tubulin-binders, or other anticancer drugs.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody molecule as provided herein, and a pharmaceutically acceptable carrier.
  • the present disclosure provides an isolated polynucleotide encoding the bispecific antibody molecule as provided herein.
  • the present disclosure provides a vector comprising the isolated polynucleotide as provided herein.
  • the present disclosure provides a host cell comprising the vector as provided herein.
  • the present disclosure provides a method of expressing the bispecific antibody molecule as provided herein, comprising culturing the host cell as provided herein under the condition at which the vector as provided herein is expressed.
  • the present disclosure provides a method of treating a disease or condition in a subject that would benefit from up-regulation of an immune response, comprising administering to the subject a therapeutically effective amount of the bispecific antibody molecule as provided herein or the pharmaceutical composition as provided herein.
  • the disease or condition that would benefit from up-regulation of an immune response is selected from the group consisting of cancer, a viral infection, a bacterial infection, a protozoan infection, a helminth infection, asthma associated with impaired airway tolerance, a neurological disease, multiple sclerosis, and an immunosuppressive disease.
  • the disease or condition is PD-1-related and/or CTLA-4-related.
  • the disease or condition is cancer, autoimmune disease, inflammatory disease, or infectious disease.
  • the cancer is lymphoma, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, uterine or endometrial cancer, rectal cancer, esophageal cancer, head and neck cancer, anal cancer, gastrointestinal cancer, intra-epithelial neoplasm, kidney or renal cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, pancreatic cancer, prostate cancer, sarcoma, skin cancer, squamous cell cancer, stomach cancer, testicular cancer, vulval cancer, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, penile carcinoma, solid tumors of childhood, tumor angiogenesis, spinal axis tumor, pituitary adenoma, or epidermoid cancer.
  • the disease or condition is an environmentally induced cancer induced by asbestos or hematologic malignancies, wherein said cancer is selected from multiple myeloma, B-cell lymphoma, Hodgkin lymphoma, primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia (CLL) , follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) , Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia (ALL) , mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combinations of said cancers.
  • B-cell lymphoma Hodg
  • the subject is human.
  • the administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.
  • the present disclosure provides a method of modulating CTLA-4 activity in a CTLA-4-expressing cell, comprising exposing the CTLA-4-expressing cell to the bispecific antibody molecule as provided herein.
  • the present disclosure provides use of the bispecific antibody molecule as provided herein in the manufacture of a medicament for treating a disease or condition that would benefit from up-regulation of an immune response.
  • the present disclosure provides use of the bispecific antibody molecule as provided herein in the manufacture of a medicament for treating a disease or condition that is PD-1 and/or CTLA-4-related.
  • Figure 1 shows the result of SDS-PAGE. The molecular weight estimated from each bands are similar to their theoretical molecular weight.
  • FIG. 1 shows the result of Size Exclusion Chromatography. The purity of are both above 95%.
  • Figure 3A shows the result of hCTLA-4-Binding ELISA. Both W3242-T4U3. G15-1 and W3242-T4U3. G17-4 bind to hCTLA-4 comparable as their parental antibody W3162-1.146.19-z12.
  • Figure 3B shows the result of hCTLA-4-Binding FACS. Both W3242-T4U3. G17-4 and W3242-T4U3. G15-1 can bind to hCTL-A4+ cells: W3242-T4U3. G15-1 with similar affinity and W3242-T4U3. G17-4 with reduced affinity.
  • Figure 3C shows the result of mCTLA-4-Binding ELISA. Both W3242-T4U3. G15-1 and W3242-T4U3. G17-4 can bind to mCTLA-4 with comparable affinity as their parental antibody.
  • Figure 3D shows the result of cyno CTLA-4 binding-ELISA. Both W3242-T4U3. G15-1 and W3242-T4U3. G17-4 bind to cyno CTLA-4.
  • Figure 4A shows the result of hPD-1-Binding ELISA.
  • the antibody W3242-T4U3.
  • G17-4 bind to hPD-1 comparable with parental antibody;
  • G15-1 binds to hPD-1 with 1.6x higher EC 50 than parental antibody.
  • Figure 4B shows the result of hPD-1-Binding FACS.
  • the antibody W3242-T4U3.
  • G17-4 bind to hPD-1 comparable with parental antibody; W3242-T4U3.
  • G15-1 binds to hPD-1 with 1.6x higher EC 50 than parental antibody.
  • Figure 4C shows the result of mPD-1-Binding ELISA.
  • the antibody W3242-T4U3.
  • G17-4 bind to hPD-1 comparable with parental antibody;
  • G15-1 binds to hPD-1 with 6x higher EC 50 than parental antibody.
  • Figure 4D shows the result of mPD-1-Binding FACS. Both W3242-T4U3. G17-4 and W3242-T4U3. G15-1 bind to mPD-1+ cells, comparable with their parental antibody.
  • Figure 5A shows the result of Human PD-1/CTLA-4 dual Binding ELISA.
  • G15-1 can bind to hPD-1 and hCTLA4 simultaneously: W3242-T4U3.
  • G15-1 has better dual binding activity than W3242-T4U3.
  • Figure 5B shows the result of Murine CTLA-4/PD-1 Dual Binding ELISA.
  • G15-1 can bind to murine PD-1 and CTLA4 simultaneously: W3242-T4U3.
  • G15-1 is better than W3242-T4U3.
  • Figure 6A shows the result of blocking CTLA-4 binding to CD80 on cell surface (FACS) .
  • FACS cell surface
  • Figure 6B shows the result of blocking CTLA-4 binding to CD86 on cell surface (FACS) .
  • FACS cell surface
  • Figure 7A shows the result of blocking soluble PD-L1 binding to hPD1 on cell surface (FACS) .
  • G15-1 can block PD-L1 binding to hPD1 by FACS.
  • Figure 7B shows the result of blocking soluble PD-L1 binding to mPD-1 on cell surface (FACS) .
  • FACS cell surface
  • Figure 8A shows the result of cytokine release of CD4+ T cells Stimulated with iDC (hMLR) .
  • G15-1 significantly enhanced IL-2 and IFN- ⁇ production in human allogeneic MLR in a dose-dependent manner.
  • Figure 8B shows the result of IFN-gamma release of Human PBMC Stimulated with SEB.
  • G15-1 enhance IFN-gamma production in human allogeneic MLR in a dose-dependent manner.
  • Figure 8C shows the result of IL-2 release of Human PBMC Stimulated with SEB. W3242-T4U3. G17-4 and W3242-T4U3. G15-1 enhance IL-2 production in human allogeneic MLR in a dose-dependent manner.
  • Figure 8D shows the result of Cytokine release of CD4+ T cells Stimulated with iDC in Treg MLR.
  • G15-1 significantly enhanced hIFN- ⁇ Secretion in Human Treg MLR.
  • Figure 8E shows the result of INF-gamma release of CD4+ T cells Stimulated with iDC in Treg MLR.
  • G15-1 significantly enhance IFN- ⁇ secretion in Human Treg MLR
  • Figure 9 shows that W3242-T4U3. G17-4 and W3242-T4U3. G15-1 bind to cyno PD-1 with EC 50 of 0.1214 and 0.0341 nM by ELISA, respectively.
  • Figure 10 shows that W3242-T4U3. G17-4 and W3242-T4U3. G15-1 block cyno CTLA-4 protein bind to hCD80+ cells with IC 50 of 3.889 and 38.73 nM by FACS, respectively.
  • Figure 11 shows that W3242-T4U3. G17-4 and W3242-T4U3. G15-1 block cyno CTLA-4 protein bind to hCD86+ cells with IC 50 of 79.1 nM by FACS.
  • Figure 12 shows that W3242-T4U3. G17-4 and W3242-T4U3. G15-1 block mouse CD80 binding to mouse CTLA-4 with IC 50 of 5.806 and 41.930 nM by ELISA.
  • Figure 13 shows that W3242-T4U3. G17-4 and W3242-T4U3. G15-1 are stable in human serum for at least 14 days, as measured by dual binding for PD-1 and CTLA-4 in ELISA.
  • Figure 14 shows pharmacokinetics of W3242-T4U3. G17-4 and W3242-T4U3. G15-1, which have long half-life (168-199 hours) in vivo.
  • Figure 15 shows the tumor volume in the 3LL xenograft model overtime after treatment of W3242-T4U3. G17-4 and W3242-T4U3. G15-1.
  • antibody as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen.
  • a native intact antibody comprises two heavy (H) chains and two light (L) chains.
  • Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable region (V H ) and a first, second, and third constant region (C H1 , C H2 , C H3 , respectively) ;
  • mammalian light chains are classified as ⁇ or ⁇ , while each light chain consists of a variable region (V L ) and a constant region.
  • the antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding.
  • Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3) .
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding domains disclosed herein may be defined or identified by the conventions of Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273 (4) , 927 (1997) ; Chothia, C. et al., J Mol Biol. Dec 5; 186 (3) : 651-63 (1985) ; Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987) ; N.R.
  • the three CDRs are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen-binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively.
  • IgG1 gamma1 heavy chain
  • IgG2 gamma2 heavy chain
  • IgG3 gamma3 heavy chain
  • IgG4 gamma4 heavy chain
  • IgA1 alpha1 heavy chain
  • IgA2 alpha2 heavy chain
  • antibody molecule refers to an antigen-binding protein or polypeptide comprising at least one antibody fragment (such as CDR, and/or variable region sequence) .
  • An antibody molecule includes, for example, a monoclonal antibody, an antibody fragment or domain, a fusion protein comprising an antibody fragment or domain, a polypeptide complex comprising an antibody fragment or domain, and so on.
  • bivalent refers to an antibody or an antigen-binding domain having two antigen-binding sites; the term “monovalent” refers to an antibody or an antigen-binding domain having only one single antigen-binding site; and the term “multivalent” refers to an antibody or an antigen-binding domain having multiple antigen-binding sites.
  • the antibody or antigen-binding domain thereof is bivalent.
  • antigen-binding domain e.g. CTLA-4-binding domain or PD-1-binding domain
  • CTLA-4-binding domain or PD-1-binding domain refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure.
  • antigen-binding domain examples include, without limitation, a diabody, a Fab, a Fab', a F (ab') 2 , an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific antibody, a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody.
  • an antigen-binding domain is capable of binding to the same antigen to which the parent antibody binds.
  • an antigen-binding domain may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
  • Fab with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.
  • Fab' refers to a Fab fragment that includes a portion of the hinge region.
  • F (ab') 2 refers to a dimer of Fab’ .
  • a “fragment difficult (Fd) ” with regard to an antibody refers to the amino-terminal half of the heavy chain fragment that can be combined with the light chain to form a Fab.
  • Fd fragment may consists of the VH and CH1 domains
  • Fv with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen-binding site.
  • An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.
  • a number of Fv designs have been provided, including dsFvs, in which the association between the two domains is enhanced by an introduced disulphide bond; and scFvs can be formed using a peptide linker to bind the two domains together as a single polypeptide.
  • Fvs constructs containing a variable domain of a heavy or light immunoglobulin chain associated to the variable and constant domain of the corresponding immunoglobulin heavy or light chain have also been produced.
  • Fvs have also been multimerised to form diabodies and triabodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000) ) .
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85: 5879 (1988) ) .
  • ScFab refers to a fusion polypeptide with a Fd linked to a light chain via a polypeptide linker, resulting in the formation of a single chain Fab fragment (scFab) .
  • Fab-Fab refers to a fusion protein formed by fusing the Fd chain of a first Fab arm to the N-terminus of the Fd chain of a second Fab arm.
  • Fab-Fv refers to a fusion protein formed by fusing a heavy chain variable domain to the C-terminus of a Fd chain and a light chain variable domain to the C-terminus of a light chain.
  • a “Fab-dsFv” molecule can be formed by introducing an interdomain disulphide bond between the heavy chain variable domain and the heavy chain variable domain.
  • Single-chain Fv-Fc antibody or “scFv-Fc” refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.
  • Fc with regard to an antibody refers to that portion of the antibody consisting of the second and third constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via disulfide bonding.
  • the Fc portion of the antibody is responsible for various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) , and complement dependent cytotoxicity (CDC) , but does not function in antigen binding.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • chimeric means an antibody or antigen-binding domain, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species.
  • a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human animal, such as from mouse.
  • the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.
  • humanized means that the antibody or antigen-binding domain comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human.
  • operably link refers to a juxtaposition, with or without a spacer or a linker or an intervening sequence, of two or more biological sequences of interest in such a way that they are in a relationship permitting them to function in an intended manner.
  • polypeptide sequences When used with respect to polypeptides, it is intended to mean that the polypeptide sequences are linked in such a way that permits the linked product to have the intended biological function.
  • an antibody variable region may be operably linked to a constant region so as to provide for a stable product with antigen-binding activity.
  • an antigen-binding domain can be operably linked to another antigen-binding domain with an intervening sequence there between, and such intervening sequence can be a spacer or can comprise a much longer sequence such as a constant region of an antibody.
  • the term may also be used with respect to polynucleotides.
  • a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., promoter, enhancer, silencer sequence, etc. ) , it is intended to mean that the polynucleotide sequences are linked in such a way that permits regulated expression of the polypeptide from the polynucleotide.
  • fusion refers to combination of two or more amino acid sequences, for example by chemical bonding or recombinant means, into a single amino acid sequence which does not exist naturally.
  • a fusion amino acid sequence may be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by a method of introducing a construct containing the recombinant polynucleotides into a host cell.
  • an “antigen” as used herein refers to a compound, composition, peptide, polypeptide, protein or substance that can stimulate the production of antibodies or a T cell response in cell culture or in an animal, including compositions (such as one that includes a cancer-specific protein) that are added to a cell culture (such as a hybridoma) , or injected or absorbed into an animal.
  • An antigen reacts with the products of specific humoral or cellular immunity (such as an antibody) , including those induced by heterologous antigens.
  • CTLA-4 refers to the Cytotoxic T-lymphocyte-associated protein 4 derived from any vertebrate source, including mammals such as primates (e.g. humans, monkeys) and rodents (e.g., mice and rats) .
  • Exemplary sequence of human CTLA-4 includes Homo sapiens (human) CTLA-4 protein (NCBI Ref Seq No. AAL07473.1) .
  • Exemplary sequence of CTLA-4 includes Macaca fascicularis (monkey) CTLA-4 protein (NCBI Ref Seq No XP_005574071.1) .
  • CTLA-4 as used herein is intended to encompass any form of CTLA-4, for example, 1) native unprocessed CTLA-4 molecule, “full-length” CTLA-4 chain or naturally occurring variants of CTLA-4, including, for example, splice variants or allelic variants; 2) any form of CTLA-4 that results from processing in the cell; or 3) full length, a fragment (e.g., a truncated form, an extracellular/transmembrane domain) or a modified form (e.g. a mutated form, a glycosylated/PEGylated, a His-tag/immunofluorescence fused form) of CTLA-4 subunit generated through recombinant method.
  • a fragment e.g., a truncated form, an extracellular/transmembrane domain
  • a modified form e.g. a mutated form, a glycosylated/PEGylated, a His-tag/immun
  • anti-CTLA-4 antibody refers to an antibody or antigen-binding domain that is capable of specific binding CTLA-4 (e.g. human or monkey or mouse CTLA-4) .
  • PD-1 refers programmed cell death protein, which belongs to the superfamily of immunoglobulin and functions as co-inhibitory receptor to negatively regulate the immune system.
  • PD-1 is a member of the CD28/CTLA-4 family, and has two known ligands including PD-L1 and PD-L2.
  • Representative amino acid sequence of human PD-1 is disclosed under the NCBI accession number: NP_005009.2, and the representative nucleic acid sequence encoding the human PD-1 is shown under the NCBI accession number: NM_005018.2.
  • PD-L1 refers to programmed cell death ligand 1 (PD-L1, see, for example, Freeman et al. (2000) J. Exp. Med. 192: 1027) .
  • Representative amino acid sequence of human PD-L1 is disclosed under the NCBI accession number: NP_054862.1, and the representative nucleic acid sequence encoding the human PD-L1 is shown under the NCBI accession number: NM_014143.3.
  • PD-L1 is expressed in placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumor or cancer cells.
  • PD-L1 binds to its receptor PD-1 or B7-1, which is expressed on activated T cells, B cells and myeloid cells.
  • the binding of PD-L1 and its receptor induces signal transduction to suppress TCR-mediated activation of cytokine production and T cell proliferation.
  • PD-L1 plays a major role in suppressing immune system during particular events such as pregnancy, autoimmune diseases, tissue allografts, and is believed to allow tumor or cancer cells to circumvent the immunological checkpoint and evade the immune response.
  • Anti-PD-1 antibody refers to an antibody or antigen-binding domain that is capable of specific binding to PD-1 (e.g. human or monkey PD-1) with an affinity which is sufficient to provide for diagnostic and/or therapeutic use.
  • PD-1 e.g. human or monkey PD-1
  • the term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen.
  • the antibody molecules or antigen-binding domains provided herein specifically bind to human PD-1 and/or human CTLA-4 with a binding affinity (K D ) of ⁇ 10 -6 M (e.g., ⁇ 5x10 -7 M, ⁇ 2x10 -7 M, ⁇ 10 -7 M, ⁇ 5x10 -8 M, ⁇ 2x10 -8 M, ⁇ 10 -8 M, ⁇ 5x10 -9 M, ⁇ 4x10 -9 M, ⁇ 3x10 -9 M, ⁇ 2x10 -9 M, or ⁇ 10 -9 M) .
  • K D binding affinity
  • K D used herein refers to the ratio of the dissociation rate to the association rate (k off /k on ) , which may be determined by using any conventional method known in the art, including but are not limited to surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method.
  • the K D value can be appropriately determined by using flow cytometry.
  • the ability to “block binding” or “compete for the same epitope” as used herein refers to the ability of an antibody or antigen-binding domain to inhibit the binding interaction between two molecules (e.g. human CTLA-4 and an anti-CTLA-4 antibody, human PD-1 and an anti-PD-1 antibody) to any detectable degree.
  • an antibody or antigen-binding domain that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 85%, or greater than 90%.
  • epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids (also called linear or sequential epitope) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitope) .
  • Epitopes formed from contiguous amino acids are typically arranged linearly along the primary amino acid residues on the protein and the small segments of the contiguous amino acids can be digested from an antigen binding with major histocompatibility complex (MHC) molecules or retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5, about 7, or about 8-10 amino acids in a unique spatial conformation. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen.
  • an antibody or antigen-binding domain blocks binding of a reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding domain may be considered to bind the same/closely related epitope as the reference antibody.
  • the antibody names as used herein may include one or more suffix symbols which usually indicates the type of the antibody or particular modifications made to the antibody.
  • “uIgG4” means an antibody with human constant region of a IgG4 isotype
  • “z” means humanized antibody
  • SP means an antibody having S228P mutation in human IgG4.
  • a “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties.
  • conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g. Asp, Glu) , among amino acids with basic side chains (e.g. His, Lys, and Arg) , or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe) .
  • conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.
  • homolog and “homologous” as used herein are interchangeable and refer to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequences when optimally aligned.
  • Percent (%) sequence identity with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F.
  • effector functions refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C1 complex and Fc receptor.
  • exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell; and phagocytosis.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.
  • subject or “individual” or “animal” or “patient” as used herein refers to human or non-human animal, including a mammal or a primate, in need of diagnosis, prognosis, amelioration, prevention and/or treatment of a disease or disorder.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.
  • vector refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein.
  • a vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell.
  • vectors include plasmids, phagemids, cosmids, and artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • a vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication.
  • a vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.
  • a vector can be an expression vector or a cloning vector.
  • host cell refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.
  • CTLA-4-related disease or condition refers to any disease or condition caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of CTLA-4.
  • the CTLA-4 related condition is immune- related disorder, such as, for example, cancer, autoimmune disease, inflammatory disease or infectious disease, graft versus host disease (GVHD) , or transplant rejection.
  • GVHD graft versus host disease
  • a “PD-1-related” disease or condition as used herein refers to any condition that is caused by, exacerbated by, or otherwise linked to increased or decreased expression or activities of PD-1 (e.g. a human PD-1) .
  • Cancer refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis, and includes both solid tumors and non-solid cancers (hematologic malignancies) such as leukemia.
  • solid tumor refers to a solid mass of neoplastic and/or malignant cells.
  • cancer or tumors include hematological malignancies, oral carcinomas (for example of the lip, tongue or pharynx) , digestive organs (for example esophagus, stomach, small intestine, colon, large intestine, or rectum) , peritoneum, liver and biliary passages, pancreas, respiratory system such as larynx or lung (small cell and non-small cell) , bone, connective tissue, skin (e.g., melanoma) , breast, reproductive organs (fallopian tube, uterus, cervix, testicles, ovary, or prostate) , urinary tract (e.g., bladder or kidney) , brain and endocrine glands such as the thyroid.
  • oral carcinomas for example of the lip, tongue or pharynx
  • digestive organs for example esophagus, stomach, small intestine, colon, large intestine, or rectum
  • peritoneum liver and biliary passages
  • the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, renal cancer, bladder cancer, hepatocellular cancer, and colorectal cancer. In certain embodiments, the cancer is selected from a lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and B-cell lymphoma.
  • pharmaceutically acceptable indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
  • the present disclosure provides herein a bispecific antibody molecule.
  • the term “bispecific” as used herein means that, there are at least two antigen-binding domains (i.e. could be dual specific or multispecific) , each of which is capable of specifically binding to a different epitope.
  • the bispecific antibody molecule provided herein comprises a CTLA-4-binding domain and PD-1-binding domain, and the CTLA-4-binding domain comprises one independently selected from the group consisting of a Fab and a scFv; and the PD-1 binding domain comprises one independently selected from the group consisting of a Fab and a scFv.
  • the CTLA-4-binding domain comprises one or more (e.g. 1, 2, 3, 4, 5, or 6) CDR sequences of an anti-CTLA-4 antibody selected from the group consisting of: W3162-1.146.19, and W3162-1.146.19-z12.
  • W3162-1.146.19 refers to a rat monoclonal antibody having a heavy chain variable region of SEQ ID NO: 21, and a light chain variable region of SEQ ID NO: 22.
  • W3162-1.146.19-z12 refers to a humanized antibody based on W3162-1.146.19 that comprises a heavy chain variable region of SEQ ID NO: 17, and a kappa light chain variable region of SEQ ID NO: 18.
  • W3162-1.146.19-z12 has comparable affinity to the antigen as compared with its parent antibody W3162-1.146.19.
  • the CTLA-4-binding domain comprises heavy chain CDR1 comprising the sequence of SEQ ID NO: 11, heavy chain CDR2 comprising the sequence of SEQ ID NO: 12, and heavy chain CDR3 comprising the sequence of SEQ ID NO: 13, and/or light chain CDR1 comprising the sequence of SEQ ID NO: 14, light chain CDR2 comprising the sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the sequence of SEQ ID NO: 16.
  • Table 1 shows the CDR sequences of these 2 anti-CTLA-4 antibodies.
  • the heavy chain and light chain variable region sequences are also provided below in Table 2 and Table 3.
  • CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs provided herein for CTLA-4-binding domains, yet substantially retain the specific binding affinity to CTLA-4.
  • the CTLA-4-binding domains provided herein comprise SEQ ID NO: 13 (i.e. a heavy chain CDR3 sequence of anti-CTLA-4 W3162-1.146.19 and W3162-1.146.19-z12) .
  • Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with antigen and provide the most free energy to the affinity of antibody to antigen. It is also believed that the heavy chain CDR3 is by far the most diverse CDR of the antigen-binding site in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S. Nature. 302: 575-81) . The diversity in the heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM. Immunity. 13: 37-45) as well as desirable antigen-binding affinity (Schier R, etc. J Mol Biol. 263: 551-67) .
  • the CTLA-4-binding domains provided herein comprise any suitable framework region (FR) sequences, as long as the antigen-binding domains can specifically bind to CTLA-4.
  • the CDR sequences of W3162-1.146.19-z12 are obtained from mouse antibodies W3162-1.146.19, but they can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art such as recombinant techniques.
  • the CTLA-4-binding domains provided herein are humanized.
  • a humanized antigen-binding domain is desirable in its reduced immunogenicity in human.
  • a humanized antigen-binding domain is chimeric in its variable regions, as non-human CDR sequences are grafted to human or substantially human FR sequences.
  • Humanization of an antigen-binding domain can be essentially performed by substituting the non-human (such as murine) CDR genes for the corresponding human CDR genes in a human immunoglobulin gene (see, for example, Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239: 1534-1536) .
  • Suitable human heavy chain and light chain variable domains can be selected to achieve this purpose using methods known in the art.
  • “best-fit” approach can be used, where a non-human (e.g. rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as the human scaffold for grafting the non-human CDR sequences (see, for example, Sims et al, (1993) J. Immunol. 151: 2296; Chothia et al. (1987) J. Mot. Biol. 196: 901) .
  • a framework derived from the consensus sequence of all human antibodies may be used for the grafting of the non-human CDRs (see, for example, Carter et at. (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; Presta et al. (1993) J. Immunol., 151: 2623) .
  • the humanized antigen-binding domains provided herein are composed of substantially all human sequences except for the CDR sequences which are non-human.
  • the variable region FRs, and constant regions if present are entirely or substantially from human immunoglobulin sequences.
  • the human FR sequences and human constant region sequences may be derived different human immunoglobulin genes, for example, FR sequences derived from one human antibody and constant region from another human antibody.
  • the humanized antigen-binding domain comprise human FR1-4.
  • the humanized CTLA-4-binding domains provided herein comprise one or more FR sequences of W3162-1.146.19-z12.
  • the exemplary humanized anti-CTLA-4 antibody W3162-1.146.19-z12 retained the specific binding affinity to CTLA-4, and are at least comparable to, or even better than, the parent rat antibodies in that aspect.
  • the FR regions derived from human may comprise the same amino acid sequence as the human immunoglobulin from which it is derived.
  • one or more amino acid residues of the human FR are substituted with the corresponding residues from the parent non-human antibody. This may be desirable in certain embodiments to make the humanized antibody or its fragment closely approximate the non-human parent antibody structure.
  • the humanized CTLA-4-binding domain provided herein comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each of the human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all the FRs of a heavy or a light chain variable domain. In some embodiments, such change in amino acid residue could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains.
  • CTLA-4-binding domains provided herein comprise a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 17. In certain embodiments, CTLA-4-binding domains provided herein comprise a light chain variable domain sequence selected from the group consisting of SEQ ID NO: 22 and SEQ ID NO: 18.
  • the CTLA-4-binding domains provided herein comprise all or a portion of the heavy chain variable domain and/or all or a portion of the light chain variable domain.
  • the CTLA-4-binding domains provided herein are a single domain antibody which consists of all or a portion of the heavy chain variable domain provided herein. More information of such a single domain antibody is available in the art (see, e.g., U.S. Pat. No. 6,248,516) .
  • the PD-1-binding domain is capable of specifically binding to PD-1 (such as human PD-1) , and comprises one independently selected from the group consisting of: a Fab and a scFv.
  • the PD-1-binding domain comprises one or more (e.g. 1, 2, 3, 4, 5, or 6) CDR sequences of an anti-PD-1 antibody of W3052-2E5.
  • W3052-2E5 refers to a humanized monoclonal antibody having a heavy chain variable region of SEQ ID NO: 7, and a kappa light chain variable region of SEQ ID NO: 8.
  • the PD-1-binding domain comprises heavy chain CDR1 comprising the sequence of SEQ ID NO: 1, heavy chain CDR2 comprising the sequence of SEQ ID NO: 2, and heavy chain CDR3 comprising the sequence of SEQ ID NO: 3, and/or light chain CDR1 comprising the sequence of SEQ ID NO: 4, light chain CDR2 comprising the sequence of SEQ ID NO: 5, and light chain CDR3 comprising the sequence of SEQ ID NO: 6.
  • Table 4 shows the CDR sequences of the anti-PD-1 antibody.
  • the heavy chain and light chain variable region sequences are also provided below in Table 5 and Table 6.
  • CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs provided herein for PD-1-binding domains, yet substantially retain the specific binding affinity to PD-1 (e.g. human PD-1) .
  • PD-1 e.g. human PD-1
  • the PD-1-binding domains provided herein comprise SEQ ID NO: 3 (i.e. a heavy chain CDR3 sequence of anti-PD-1 antibody W3052-2E5) .
  • the PD-1-binding domains provided herein comprise suitable framework region (FR) sequences, as long as the antigen-binding domains can specifically bind to PD-1, respectively.
  • the CDR sequences of W3052-2E5 is obtained from rat antibodies, but they can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art such as recombinant techniques.
  • the PD-1-binding domains provided herein are humanized.
  • the exemplary humanized anti-PD-1 antibody W3052-2E5 retained the specific binding affinity to PD-1, and are at least comparable to, or even better than, the parent rat antibodies in that aspect.
  • the FR regions derived from human may comprise the same amino acid sequence as the human immunoglobulin from which it is derived.
  • one or more amino acid residues of the human FR are substituted with the corresponding residues from the parent non-human antibody. This may be desirable in certain embodiments to make the humanized antibody or its fragment closely approximate the non-human parent antibody structure.
  • the humanized PD-1 binding domain provided herein comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each of the human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all the FRs of a heavy or a light chain variable domain. In some embodiments, such change in amino acid residue could be present in heavy chain FR regions only, in light chain FR regions only, or in both chains.
  • the PD-1-binding domains provided herein comprise a heavy chain variable domain sequence comprising SEQ ID NO: 7. In certain embodiments, PD-1-binding domains provided herein comprise a light chain variable domain sequence comprising SEQ ID NO: 8.
  • the PD-1-binding domains provided herein comprise all or a portion of the heavy chain variable domain and/or all or a portion of the light chain variable domain.
  • the PD-1-binding domains provided herein are a single domain antibody which consists of all or a portion of the heavy chain variable domain provided herein. More information of such a single domain antibody is available in the art (see, e.g., U.S. Pat. No. 6,248,516) .
  • the bispecific antibody molecule provided herein comprises an CTLA-4-binding domain comprising one or more (e.g. 1, 2, 3, 4, 5, or 6) CDR sequences of SEQ ID NO: 11-16 (i.e. derived from W3162-1.146.19 and W3162-1.146.19-z12) , and a PD-1-binding domain comprising one or more (e.g. 1, 2, 3, 4, 5, or 6) CDR sequences of SEQ ID NO: 1-6 (i.e.
  • CTLA-4-binding domain comprises one independently selected from the group consisting of: a Fab and a scFv
  • PD-1-binding domain comprises one independently selected from the group consisting of: a Fab and a scFv.
  • the bispecific antibody molecule provided herein comprises a CTLA-4-binding domain comprising:
  • heavy chain CDR1 comprising the sequence of SEQ ID NO: 11
  • heavy chain CDR2 comprising the sequence of SEQ ID NO: 12
  • heavy chain CDR3 comprising the sequence of SEQ ID NO: 13
  • light chain CDR1 comprising the sequence of SEQ ID NO: 14
  • light chain CDR2 comprising the sequence of SEQ ID NO: 15
  • light chain CDR3 comprising the sequence of SEQ ID NO: 16;
  • a PD-1-binding domain comprising:
  • heavy chain CDR1 comprising the sequence of SEQ ID NO: 1
  • heavy chain CDR2 comprising the sequence of SEQ ID NO: 2
  • heavy chain CDR3 comprising the sequence of SEQ ID NO: 3
  • light chain CDR1 comprising the sequence of SEQ ID NO: 4
  • light chain CDR2 comprising the sequence of SEQ ID NO: 5
  • light chain CDR3 comprising the sequence of SEQ ID NO: 6;
  • the CTLA-4-binding domain comprises one independently selected from the group consisting of: a Fab and a scFv
  • the PD-1-binding domain comprises one independently selected from the group consisting of: a Fab and a scFv.
  • the CTLA-4-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 17, 21, or a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to CTLA-4 (e.g. human CTLA-4) , and/or a light chain variable region comprising the sequence of SEQ ID NO: 18, 22, or a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to CTLA-4 (e.g. human CTLA-4) .
  • the PD-1 binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 7, or a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1 (e.g. human PD-1) , and/or a light chain variable region comprising the sequence of SEQ ID NO: 8 or a homologous sequence thereof having at least 80%sequence identity yet retaining specific binding affinity to PD-1 (e.g. human PD-1) .
  • the CTLA-4-binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 17 and a light chain variable region comprising the sequence of SEQ ID NO: 18 (derived from W3162-1.146.19-z12)
  • the PD-1 binding domain comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 7 and and a light chain variable region comprising the sequence of SEQ ID NO: 8 (W3052-2E5) .
  • CTLA-4-binding domains and/or the PD-1-binding domains provided herein comprise one independently selected from the group consisting: a Fab and a scFv.
  • Various techniques can be used for the production of such antigen-binding domains.
  • Illustrative methods include, enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) ; and Brennan et al., Science, 229: 81 (1985) ) , recombinant expression by host cells such as E. Coli (e.g. for Fab, Fv and ScFv antibody fragments) , and screening from a phage display library as discussed above (e.g. for ScFv) .
  • Other techniques for the production of antibody fragments will be apparent to a skilled practitioner.
  • the CTLA-4-binding domain and/or the PD-1-binding domain is or comprises a scFv.
  • Generation of scFv is described in, for example, WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458.
  • scFv may be fused to an effector protein at either the amino or the carboxyl terminus to provide for a fusion protein (see, for example, Antibody Engineering, ed. Borrebaeck) .
  • An scFv can comprise from a VH linked directly or via a peptide linker to a VL.
  • the VH can be at the N-terminus and the VL can be at the C terminus of the scFv. In certain embodiments, the VL can be at the N-terminus and the VH can be at the C terminus of the scFv.
  • the PD-1-binding domain comprises a scFv comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 7 (W3052-2E5 VH) linked to a light chain variable region (VL) comprising the sequence of SEQ ID NO: 8 (W3052-2E5 VL) via a peptide linker.
  • the CTLA-4-binding domain comprises a scFv comprising a VH comprising the sequence of SEQ ID NO: 17 and a VL comprising the sequence of SEQ ID NO: 18 (derived from W3162-1.146.19-z12) via a peptide linker.
  • the peptide linker can comprise a single or repeated sequences composed of threonine/serine and glycine, such as TGGGG (SEQ ID NO: 35) , GGGGS (SEQ ID NO: 31) , GGGGSGGGGS (SEQ ID NO: 32) , GGGGSGGGGSGGGGS (SEQ ID NO: 33) or SGGGG (SEQ ID NO: 36) or its tandem repeats (e.g. 2, 3, 4, or more repeats) .
  • the peptide linker comprises GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 34) .
  • the PD-1-binding domain comprises a scFv comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 7 (W3052-2E5 VH) linked to the N terminus of a light chain variable region (VL) comprising the sequence of SEQ ID NO: 8 (W3052-2E5 VL) via a peptide linker.
  • the peptide linker comprises or is SEQ ID NO: 33.
  • the PD-1-binding domain comprises a scFv comprising SEQ ID NO: 25.
  • the PD-1-binding domain comprises a scFv comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 7 (W3052-2E5 VH) linked to the C terminus of a light chain variable region (VL) comprising the sequence of SEQ ID NO: 8 (W3052-2E5 VL) via a peptide linker.
  • the peptide linker comprises or is SEQ ID NO: 33.
  • the PD-1-binding domain comprises a scFv comprising SEQ ID NO: 29.
  • the CTLA-4-binding domain and/or the PD-1-binding domain is a Fab.
  • the PD-1-binding Fab comprises a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8 (derived from W3052-2E5) .
  • the CTLA-4-binding Fab a heavy chain variable region comprising SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 18 (derived from W3162-1.146.19-z12) .
  • the heavy chain variable region and the light chain variable region can be disulfidely bonded.
  • disulfidely bonded refers to linkage via one or more disulfide bond (optionally in addition to another bond) .
  • a disulfide bond can be formed between, for example, one cysteine residue of an antibody heavy chain and another cysteine residue of the light chain.
  • the CTLA-4-binding and/or the PD-1-binding domains are multivalent, such as bivalent, trivalent, tetravalent.
  • valent refers to the presence of a specified number of antigen binding sites in a given molecule.
  • bivalent tetravalent
  • hexavalent denote the presence of two binding site, four binding sites, and six binding sites, respectively, in an antigen-binding molecule.
  • a bivalent molecule can be monospecific if the two binding sites are both for specific binding of the same antigen or the same epitope.
  • a trivalent molecule can be bispecific, for example, when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope) .
  • the CTLA-4-binding and/or the PD-1-binding domains in the bispecific antibody molecule provided herein can be bivalent, trivalent, or tetravalent, with at least two binding sites specific for the same antigen or epitope. This, in certain embodiments, provides for stronger binding to the antigen or the epitope than a monovalent counterpart.
  • the first valent of binding site and the second valent of binding site are structurally identical (i.e.
  • CTLA-4-binding and/or the PD-1-binding domains comprises two or more antigen binding sites (e.g. scFv or Fab) operably linked together, with or without a spacer.
  • antigen binding sites e.g. scFv or Fab
  • the CTLA-4-binding domain is operably linked to the N terminus or the C terminus of the PD-1-binding domain. In certain embodiments, the PD-1-binding domain is operably linked to the N terminus or the C terminus of the CTLA-4-binding domain.
  • the operable linkage can be direct chemical bond linkage or linkage via a spacer or via an intervening sequence.
  • spacer refers to an artificial amino acid sequence having 1, 2, 3, 4 or 5 amino acid residues, or a length of between 5 and 15, 20, 30, 50 or more amino acid residues, joined by peptide bonds and are used to link one or more binding domains, such as a scFv and a Fab or an IgG.
  • the spacer can be identical to or different from the peptide linker in the scFv.
  • the spacer comprises 1, 2, 3, 4 or more sequential or tandem repeats of SEQ ID NOs: 31, 32, 33, 35 and 36.
  • the spacer comprises GGGGS (SEQ ID NO: 31) . In certain embodiments, the spacer comprises GGGGSGGGGS (SEQ ID NO: 32) , GGGGSGGGGSGGGGS (SEQ ID NO: 33) , GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 34) .
  • the intervening sequence as used herein can be any amino acid sequence located between the CTLA-4-binding domain and the PD-1-binding domain, as long as both the CTLA-4-binding domain and the PD-1-binding domain are capable of binding to its respective antigen. In an illustrative example, the intervening sequence can comprise a heavy chain constant region, or a light chain constant region.
  • the PD-1-binding domain comprises a scFv and the CTLA-4-binding domain comprises a Fab or an IgG.
  • the PD-1-binding domain scFv can be operably linked to the N terminus or the C-terminus of the heavy chain of the CTLA-4-binding Fab or IgG (e.g. the C-terminus of the heavy chain constant region following the PD-1-binding Fab) , or to the N terminus or the C-terminus of the light chain of the CTLA-4-binding Fab or IgG, or any combination thereof, and vice versa.
  • the bispecific antibody molecule can comprise a heavy chain in the format of: VH (anti-CTLA-4) -CH1-Hinge-CH2-CH3-scFv (anti-PD-1) or scFv (anti-PD-1) -VH (anti-CTLA-4) -CH1-Hinge-CH2-CH3, and a light chain in the format of: VL (anti-CTLA-4) -CL.
  • VH anti-CTLA-4 and VL (anti-CTLA-4) refer respectively to the heavy and light chain variable domain of the anti-CTLA-4 antibody provided herein
  • scFv anti-PD-1 refers to an scFv derived from the anti-PD-1 antibody provided herein
  • CL refers to the light chain constant region
  • CH1-Hinge-CH2-CH3 are collectively heavy chain constant region.
  • the bispecific antibody molecule can comprise a light chain in the format of: VL (anti-CTLA-4) -CL-spacer-scFv (anti-PD-1) , or scFv (anti-PD-1) -spacer-VL (anti-CTLA-4) -CL, a heavy chain VH (anti-CTLA-4) -CH1-Hinge-CH2-CH3, by the same token.
  • the CTLA-4-binding domain comprises a scFv and the PD-1-binding domain comprises a Fab or an IgG.
  • the CTLA-4-binding domain scFv can be linked to the N terminus or the C-terminus of the heavy chain of the anti-PD-1 Fab or IgG (e.g. the C-terminus of the heavy chain constant region following the PD-1-binding Fab) , or to the N terminus or the C-terminus of the light chain of the anti-PD-1 Fab or IgG, or any combination thereof, and vice versa.
  • the bispecific antibody molecule can comprise a heavy chain in the format of: VH (anti-PD-1) -CH1-Hinge-CH2-CH3-scFv (anti-CTLA-4) or scFv (anti-CTLA-4) -VH (anti-PD-1) -CH1-Hinge-CH2-CH3, and a light chain VL (anti-PD-1) -CL.
  • the bispecific antibody molecule can comprise a light chain in the format of: scFv (anti-CTLA-4) -VL (anti-PD-1) -CL or VL (anti-PD-1) -CL-scFv (anti-CTLA-4) , and a heavy chain VH (anti-PD-1) -CH1-Hinge-CH2-CH3, by the same token.
  • the CTLA-4-binding domain may be monovalent (i.e. one scFv or Fab) or multivalent (e.g. more than one scFv or Fab) .
  • the PD-1-binding domain may be monovalent or multivalent.
  • the CTLA-4-binding domain may be monovalent (i.e. one scFv or Fab) or multivalent (e.g. more than one scFv or Fab) , and/or the PD-1-binding domain may be monovalent or multivalent.
  • the bispecific antibody molecule comprise a heavy chain in the format of: VH (anti-CTLA-4) -CH1-Hinge-CH2-CH3-spacer-scFv (anti-PD-1) , and a light chain in the format of VL (anti-CTLA-4) -CL, wherein the VH (anti-CTLA-4) comprises a sequence of SEQ ID NO: 17, the VL (anti-CTLA-4) comprises an amino acid sequence of SEQ ID NO: 18, and the scFv (anti-PD-1) comprises the sequence of SEQ ID NO: 25.
  • the spacer comprises the sequence of SEQ ID NO: 34.
  • the heavy chain constant region is of human IgG4 isotype, and optionally contains mutations of S228P and/or L235E.
  • the heavy chain constant region comprises the sequence of SEQ ID NO: 23.
  • the light chain constant region comprises the sequence of SEQ ID NO: 24.
  • the bispecific antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 26 and a light chain comprising the amino acid sequence of SEQ ID NO: 27. This antibody is also called W3242-T4U3. G15-1 in the present disclosure.
  • the bispecific antibody molecule comprises a heavy chain in the format of: scFv (anti-PD-1) -spacer-VH (anti-CTLA-4) -CH1-Hinge-CH2-CH3, and a light chain in the format of VL (anti-CTLA-4) -CL, wherein the VH (anti-CTLA-4) comprises a sequence of SEQ ID NO: 17, the VL (anti-CTLA-4) comprises an amino acid sequence of SEQ ID NO: 18, and the scFv (anti-PD-1) comprises the sequence of SEQ ID NO: 25 or 29.
  • the spacer comprises the sequence of SEQ ID NO: 32? .
  • the heavy chain constant region is of human IgG4 isotype, and optionally contains mutations of S228P and/or L235E.
  • the heavy chain constant region comprises the sequence of SEQ ID NO: 23.
  • the light chain constant region comprises the sequence of SEQ ID NO: 24.
  • the bispecific antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 28 and a light chain comprising the amino acid sequence of SEQ ID NO: 27. This antibody is also called W3242-T4U3. G17-1 in the present disclosure.
  • the bispecific antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 30 and a light chain comprising the amino acid sequence of SEQ ID NO: 27.
  • This antibody is also called W3242-T4U3. G17-4 in the present disclosure.
  • Table 7 and 8 show the combination of heavy chain and light chain sequences of the bispecific antibody molecules of W3242-T4U3. G15-1, W3242-T4U3. G17-1 and W3242-T4U3. G17-4.
  • CL refers to light chain constant region
  • CH refers to heavy chain constant region
  • VL refers to light chain variable region
  • VH refers to heavy chain variable region
  • Anti-PD-1 refers to anti-PD-1 antibody, in particular, the sequence provided in the table is the sequence of anti-PD-1 antibody W3052-2E5;
  • Anti-CTLA-4 refers to anti-CTLA-4 antibody, in particular, the sequence provided in the table is the sequence derived from anti-CTLA-4 antibody W3162-1.146.19-z12.
  • the bispecific antibody molecules provided herein may further comprise an immunoglobulin constant region.
  • an immunoglobulin constant region comprises a heavy chain and/or a light chain constant region.
  • the heavy chain constant region comprises CH1, hinge, and/or CH2-CH3 regions.
  • the heavy chain constant region comprises an Fc region.
  • the light chain constant region comprises C ⁇ or C ⁇ .
  • the bispecific antibody molecules provided herein can have a constant region of an immunoglobulin (Ig) , optionally a human Ig, optionally a human IgG.
  • the constant region can be in any suitable isotype.
  • the bispecific antibody molecules provided herein comprises a constant region of IgG1 isotype, which could induce ADCC or CDC, or a constant region of IgG4 or IgG2 isotype, which has reduced or depleted effector function..
  • the bispecific antibody molecules provided herein have reduced or depleted effector function. In some embodiments, the bispecific antibody molecules provided herein have a constant region of IgG4 isotype, which has reduced or depleted effector function. Effector functions such as ADCC and CDC can lead to cytotoxicity to cells expressing PD-1. Many cells such as T cells normally express PD-1. In order to avoid potential unwanted toxicity to those normal cells, certain embodiments of the antibodies and antigen-binding fragments provided herein can possess reduced or even depleted effector functions.
  • Assays are known to evaluate ADCC or CDC activities, for example, Fc receptor binding assay, C1q binding assay, and cell lysis assay, and can be readily selected by people in the art.
  • the bispecific antibody molecules provided herein have reduced side effects.
  • the bispecific antibody molecules provided herein can comprise at least one fully human antigen-binding domain and Fc region and therefore reduced immunogenicity than a humanized antibody counterpart.
  • the bispecific antibody molecules provided herein are capable of specifically binding to both human PD-1 and human CTLA-4.
  • the bispecific antibody molecules provided herein retain the specific binding affinity to both PD-1 and CTLA-4, in certain embodiments are at least comparable to, or even better than, the parent antibodies in that aspect.
  • the bispecific antibody molecules provided herein have a specific binding affinity to CTLA-4 which is sufficient to provide for diagnostic and/or therapeutic use.
  • Binding of bispecific antibody molecules can be represented by “half maximal effective concentration” (EC 50 ) value, which refers to the concentration of an antibody where 50%of its maximal effect (e.g., binding or inhibition etc. ) is observed.
  • the EC 50 value can be measured by methods known in the art, for example, sandwich assay such as ELISA, Western Blot, flow cytometry assay, and other binding assay.
  • Binding affinity of the antigen-binding domains provided herein can also be represented by K D value, which represents the ratio of dissociation rate to association rate (k off /k on ) when the binding between the antigen and antigen-binding molecule reaches equilibrium.
  • the antigen-binding affinity e.g. K D
  • K D can be appropriately determined using suitable methods known in the art, including, for example, flow cytometry assay.
  • the bispecific antibody molecules provided herein specifically bind to human CTLA-4 protein at an EC 50 (i.e. 50%binding concentration) of no more than: 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, or 0.2 nM by ELISA; or specifically bind to cell surface human CTLA-4 at an EC 50 of no more than: 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.8 nM, 0.6 nM, or 0.5 nM by FACS.
  • an EC 50 i.e. 50%binding concentration
  • the bispecific antibody molecules provided herein cross-react with Cynomolgus monkey CTLA-4 and murine CTLA-4.
  • the bispecific antibody molecules provided herein specifically bind to mouse CTLA-4 at an EC 50 of no more than: 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM by ELISA.
  • the bispecific antibody molecules provided herein specifically bind to Cynomolgus monkey CTLA-4 at an EC 50 of no more than: 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM by ELISA.
  • the bispecific antibody molecules provided herein specifically bind to human PD-1 protein at an EC 50 of no more than: 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM by ELISA; or specifically bind to cell surface human PD-1 at an EC 50 of no more than: 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, or 0.6 nM by FACS.
  • the bispecific antibody molecules provided herein cross-react with murine PD-1 and Cynomolgus monkey PD-1.
  • the bispecific antibody molecules provided herein specifically bind to murine PD-1 protein at an EC 50 of no more than: 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, 0.9 nM, 0.7 nM, 0.5 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.08 nM, or 0.05 nM by ELISA; or specifically bind to cell surface murine PD-1 at an EC 50 of no more than: 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 8 nM, 7 nM, 6 nM, 5 nM, or 4 nM by FACS.
  • the bispecific antibody molecules provided herein specifically bind to Cynomolgus monkey PD-1 at an EC 50 of no more than: 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, 0.2 nM, or 0.1 nM by ELISA;
  • the bispecific antibody molecules provided herein are capable of binding to both human PD-1 and human CTLA-4 at an EC 50 of no more than: 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.03 nM, or 0.02 nM by ELISA.
  • the bispecific antibody molecules provided herein are capable of binding to both murine PD-1 and murine CTLA-4 at an EC 50 of no more than: 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, or 0.07 nM by ELISA.
  • the bispecific antibody molecules provided herein are capable of specifically binding to human PD-1 with a binding affinity (K D ) of no more than: 20x10 -8 M, 15x10 -8 M, 12x10 -8 M, 10x10 -8 M, 9x10 -8 M, 8x10 -8 M, 7x10 -8 M, 6x10 -8 M, 5x10 -8 M, 4x10 -8 M, 3x10 -8 M, 2x10 -8 M, or 1x10 -8 M as measured by surface plasmon resonance (SPR) .
  • K D binding affinity
  • the bispecific antibody molecules provided herein are capable of specifically binding to murine PD-1 with a binding affinity (K D ) of no more than: 60x10 -9 M, 50x10 -9 M, 40x10 -9 M, 30x10 -9 M, 20x10 -9 M, 10x10 -9 M, 9x10 -9 M, 8x10 -9 M, 7x10 -9 M, 6x10 -9 M, 5x10 -9 M, 4x10 -9 M, or 3x10 -9 M as measured by SPR.
  • K D binding affinity
  • the bispecific antibody molecules provided herein are capable of specifically binding to Cynomolgus monkey PD-1 with a binding affinity (K D ) of no more than: 20x10 -8 M, 10x10 -8 M, 9x10 -8 M, 8x10 -8 M, 7x10 -8 M, 6x10 -8 M, 5x10 -8 M, 4x10 -8 M, 3x10 -8 M, 2x10 -8 M, 1x10 -8 M, 0.9x10 -8 M, or 0.8x10 -8 M as measured by SPR.
  • K D binding affinity
  • the bispecific antibody molecules provided herein are capable of specifically binding to human CTLA-4 with a binding affinity (K D ) of no more than: 40x10 -9 M, 30x10 -9 M, 20x10 -9 M, 10x10 -9 M, 9x10 -9 M, 8x10 -9 M, 7x10 -9 M, 6x10 -9 M, 5x10 -9 M, 4x10 -9 M, 3x10 -9 M, 2x10 -9 M, 1x10 -9 M, 0.8x10 -9 M, 0.6x10 -9 M, or 0.5x10 -9 M as measured by SPR.
  • K D binding affinity
  • the bispecific antibody molecules provided herein are capable of specifically binding to murine CTLA-4 with a binding affinity (K D ) of no more than: 50x10 -9 M, 40x10 -9 M, 30x10 -9 M, 20x10 -9 M, 10x10 -9 M, 9x10 -9 M, 8x10 -9 M, 7x10 -9 M, 5x10 -9 M, 2x10 -9 M, 1x10 -9 M, 0.9x10 -9 M, 0.8x10 -9 M, 0.5x10 -9 M, or 0.3x10 -9 M as measured by SPR.
  • K D binding affinity
  • the bispecific antibody molecules provided herein are capable of specifically binding to Cynomolgus monkey CTLA-4 with a binding affinity (K D ) of no more than: 50x10 -8 M, 40x10 -8 M, 30x10 -8 M, 20x10 -8 M, 10x10 -8 M, 9x10 -8 M, 8x10 -8 M, 7x10 -8 M, 6x10 -8 M, 5x10 -8 M, 4x10 -8 M, 3x10 -8 M, 2x10 -8 M, or 1x10 -8 M as measured by SPR.
  • K D binding affinity
  • the bispecific antibody molecules provided herein are capable of blocking human CTLA-4 binding to human CD80 on cell surface with at an IC 50 (i.e. 50%inhibiting concentration) of no more than: 1000 nM, 800 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, or 8 nM by FACS.
  • IC 50 i.e. 50%inhibiting concentration
  • the bispecific antibody molecules provided herein are capable of blocking human CTLA-4 binding to human CD86 on cell surface with at an IC 50 of no more than: 1000 nM, 800 nM, 500 nM, 400 nM, 300 nM, 200 nM, 180 nM, 150 nM, 140 nM, 130 nM, 125 nM, 120 nM, 100 nM, 80 nM, 60 nM, 40 nM, 20 nM, 10 nM, or 5 nM by FACS.
  • the bispecific antibody molecules provided herein are capable of blocking Cynomolgus monkey CTLA-4 binding to human CD80 on cell surface with at an IC 50 of no more than: 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, or 4 nM by FACS.
  • the bispecific antibody molecules provided herein are capable of blocking Cynomolgus monkey CTLA-4 binding to human CD86 on cell surface with at an IC 50 of no more than: 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 85 nM, or 80 nM by FACS.
  • the bispecific antibody molecules provided herein are capable of blocking murine CTLA-4 binding to murine CD80 on cell surface with at an IC 50 of no more than: 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 45 nM, 42 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, or 5 nM by FACS.
  • the bispecific antibody molecules provided herein are capable of blocking human PD-L1 binding to human PD-1 on cell surface with at an IC 50 of no more than: 20 nM, 15 nM, 12 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, or 0.8 nM by FACS.
  • the bispecific antibody molecules provided herein are capable of blocking human PD-L1 binding to murine PD-1 on cell surface with at an IC 50 of no more than: 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 18 nM, 16 nM, or 15 nM by FACS.
  • the bispecific antibody molecules provided herein block binding of human PD-1 to its ligand and thereby providing biological activity including, for example, inducing cytokine production from the activated T cells (such as CD4+ T cells and CD8+ T cells) , inducing proliferation of activated T cells (such as CD4+ T cells and CD8+ T cells) , and reversing T reg’s suppressive function.
  • cytokines include IL-2 and IFN ⁇ .
  • IL-2 refers to interleukin 2, a type of cytokine signaling molecule in the immune system that regulates the activities of white blood cells (e.g. leukocytes) .
  • Interferon gamma is a cytokine that is produced by natural killer (NK) , NK T cells, CD4+ and CD8+T cells, which is a critical activator of macrophages and inducer of major histocompatibility complex (MHC) molecule expression.
  • NK natural killer
  • MHC major histocompatibility complex
  • the cytokine production can be determined using methods known in the art, for example, by ELISA. Methods can also be used to detect proliferation of T cells, including [ 3 H] thymidine incorporation assay.
  • the bispecific antibody molecules provided herein are capable of specifically enhancing IL-2 and/or IFN- ⁇ production in human PBMCs stimulated with SEB in a dose-dependent manner, as measured by human allogeneic mixed lymphocyte reaction (MLR) .
  • MLR human allogeneic mixed lymphocyte reaction
  • the bispecific antibody molecules provided herein are capable of specifically enhancing IL-2 and/or IFN- ⁇ production in CD4+ T cells stimulated with iDC in a dose-dependent manner, as measured by human allogeneic mixed lymphocyte reaction (MLR) .
  • MLR human allogeneic mixed lymphocyte reaction
  • the bispecific antibody molecules provided herein are capable of specifically enhancing IFN- ⁇ production in CD4+ T cells stimulated with iDC, as measured by human Treg mixed lymphocyte reaction (MLR) .
  • MLR human Treg mixed lymphocyte reaction
  • the bispecific antibody molecules provided herein are capable of simultaneous stimulating cells from both the innate and the adaptive immune system.
  • the antigen-binding domains and bispecific antibody molecules provided herein also encompass various variants thereof.
  • the variants comprise one or more modifications or substitutions in one or more CDR sequences as provided in Table 1, or Table 4, one or more variable region sequences (but not in any of the CDR sequences) provided in Table 2, or Table 5, and/or the constant region (e.g. Fc region) .
  • Such variants retain specific binding affinity to CTLA-4 and/or PD-1 of their parent antibodies, but have one or more desirable properties conferred by the modification (s) or substitution (s) .
  • the variants may have improved antigen-binding affinity, improved productivity, improved stability, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function (s) , improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility to conjugation (e.g. one or more introduced cysteine residues) .
  • the parent antibody sequence may be screened to identify suitable or preferred residues to be modified or substituted, using methods known in the art, for example “alanine scanning mutagenesis” (see, for example, Cunningham and Wells (1989) Science, 244: 1081-1085) .
  • target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • substitution at a particular amino acid location demonstrates an interested functional change, then the position can be identified as a potential residue for modification or substitution.
  • the potential residues may be further assessed by substituting with a different type of residue (e.g. cysteine residue, positively charged residue, etc. ) .
  • the CTLA-4-binding domains and/or the PD-1 binding domains provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences, and/or one or more FR sequences, and/or one or more variable region sequences.
  • a variant comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR sequences and/or FR sequences and/or one or more variable region sequences in total.
  • the CTLA-4-binding domains comprise 1, 2, 3, 4, 5, or 6 CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NOs: 11-16, and in the meantime retain the binding affinity to CTLA-4 at a level similar to or even higher than its parent antibody.
  • the anti-CTLA-4-binding domains comprise one or more variable region sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NOs: 17-18 and 21-22, and in the meantime retain the binding affinity to CTLA-4 at a level similar to or even higher than its parent antibody.
  • a total of 1 to 10 amino acids have been substituted, inserted, or deleted in a variable region sequence comprising SEQ ID NOs: 17-18 and 21-22.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs) .
  • the PD-1-binding domains comprise 1, 2, or 3 CDR sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NOs: 1-6, and in the meantime retain the binding affinity to PD-1 at a level similar to or even higher than its parent antibody.
  • the PD-1-binding domains comprise one or more variable region sequences having at least 80% (e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NOs: 7-8, and in the meantime retain the binding affinity to PD-1 at a level similar to or even higher than its parent antibody.
  • a total of 1 to 10 amino acids have been substituted, inserted, or deleted in a variable region sequence comprising SEQ ID NOs: 7-8.
  • the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs) .
  • the antigen-binding domains and bispecific antibody molecules provided herein also encompass a glycosylation variant, which can be obtained to either increase or decrease the extent of glycosylation of the bispecific antibody molecules.
  • the antigen-binding domains and bispecific antibody molecules provided herein may comprise one or more amino acid residues with a side chain to which a carbohydrate moiety (e.g. an oligosaccharide structure) can be attached.
  • Glycosylation of antibodies is typically either N-linked or O-linked.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline.
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence in the is substituted. A new glycosylation site can be created in a similar way by introducing such a tripeptide sequence or serine or threonine residue.
  • the antigen-binding domains and bispecific antibody molecules also encompass a cysteine-engineered variant, which comprises one or more introduced free cysteine amino acid residues.
  • a free cysteine residue is one which is not part of a disulfide bridge.
  • a cysteine-engineered variant is useful for conjugation with for example, a cytotoxic and/or imaging compound, a label, or a radioisoptype among others, at the site of the engineered cysteine, through for example a maleimide or haloacetyl.
  • Methods for engineering antibody polypeptides to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.
  • the antigen-binding domains and bispecific antibody molecules provided herein also encompass an Fc variant, which comprises one or more amino acid residue modifications or substitutions at its Fc region and/or hinge region.
  • the antigen-binding domains and bispecific antibody molecules comprise one or more amino acid substitution (s) that improves pH-dependent binding to neonatal Fc receptor (FcRn) .
  • FcRn neonatal Fc receptor
  • Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell.
  • Methods of engineering an antibody molecule to improve binding affinity with FcRn are well-known in the art, see, for example, Vaughn, D. et al, Structure, 6 (1) : 63-73, 1998; Kontermann, R.
  • the antigen-binding domains and bispecific antibody molecules comprise one or more amino acid substitution (s) that alters the antibody-dependent cellular cytotoxicity (ADCC) .
  • Certain amino acid residues at the Fc region e.g. at the CH2 domain
  • carbohydrate structures on the antibody can be changed to alter (e.g. enhance, decrease, or deplete) ADCC activity.
  • the antigen-binding domains and bispecific antibody molecules comprise a human IgG4 constant region in which the 228 th amino acid residue is altered, for example from Ser228Pro (S228P, which may prevent or reduce strand exchange) , and/or the 235 th amino acid residue is altered, for example from Leu235Glu (L235E, which may alter Fc receptor interactions.
  • S228P Ser228Pro
  • L235E Leu235Glu
  • the antigen-binding domains and bispecific antibody molecules comprise one or more amino acid substitution (s) that alters Complement Dependent Cytotoxicity (CDC) , for example, by improving or diminishing C1q binding and/or CDC (see, for example, WO99/51642; Duncan & Winter Nature 322: 738-40 (1988) ; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821) ; and WO94/29351 concerning other examples of Fc region variants.
  • CDC Complement Dependent Cytotoxicity
  • the antigen-binding domains and bispecific antibody molecules comprise one or more amino acid substitution (s) in the interface of the Fc region to facilitate and/or promote heterodimerization.
  • modifications comprise introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance can be positioned in the cavity so as to promote interaction of the first and second Fc polypeptides to form a heterodimer or a complex.
  • the bispecific antibody molecules further comprise a conjugate moiety.
  • the conjugate moiety can be linked to the bispecific antibody molecules.
  • a conjugate moiety is a non-proteinaceous moiety that can be attached to the bispecific antibody molecules. It is contemplated that a variety of conjugate moieties may be linked to the bispecific antibody molecules provided herein (see, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) . These conjugate moieties may be linked to the bispecific antibody molecules by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.
  • the bispecific antibody molecules disclosed herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugates.
  • a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate.
  • the bispecific antibody molecules may be linked to a conjugate moiety indirectly, or through another conjugate moieties.
  • the bispecific antibody molecules may be conjugated to biotin, then indirectly conjugated to a second conjugate moiety that is conjugated to avidin.
  • the conjugate moieties can be a clearance-modifying agent, a toxin (e.g., a chemotherapeutic agent) , a detectable label (e.g., a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label) , or purification moiety.
  • a “toxin” can be any agent that is detrimental to cells or that can damage or kill cells.
  • toxin include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine) , alkylating agents (e.g.,
  • detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or ⁇ -D-galactosidase) , radioisotopes (e.g.
  • the conjugate moiety can be a clearance-modifying agent which helps increase half-life of the antibody.
  • Illustrative example include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules.
  • the conjugate moiety can be a purification moiety such as a magnetic bead.
  • the bispecific antibody molecule provided herein is used for a base for a conjugate.
  • the present disclosure provides isolated polynucleotides that encode the bispecific antibody molecules provided herein.
  • nucleic acid or “polynucleotide” as used herein refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses polynucleotides containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) , alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19: 5081 (1991) ; Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985) ; and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994) ) .
  • the isolated polynucleotides comprise one or more nucleotide sequences as shown in SEQ ID NOs: 9, 10, 19 and 20, and/or a homologous sequence thereof having at least 80% (e.g. at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and/or a variant thereof having only degenerate substitutions, and encodes the variable region of the exemplary antibodies provided herein.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) .
  • the encoding DNA may also be obtained by synthetic methods.
  • the isolated polynucleotide that encodes the bispecific antibody molecule can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art.
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g. SV40, CMV, EF-1 ⁇ ) , and a transcription termination sequence.
  • the present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the bispecific antibody molecules, at least one promoter (e.g., SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence, and at least one selection marker.
  • a promoter e.g., SV40, CMV, EF-1 ⁇
  • vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40) , lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBA
  • RTM. pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos etc.
  • Vectors comprising the polynucleotide sequence encoding the bispecific antibody molecule can be introduced to a host cell for cloning or gene expression.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the vectors provided.
  • Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms.
  • Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12, 424) , K. bulgaricus (ATCC 16, 045) , K. wickeramii (ATCC 24, 178) , K.
  • waltii ATCC 56, 500
  • K. drosophilarum ATCC 36, 906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402, 226)
  • Pichia pastoris EP 183, 070
  • Candida Trichoderma reesia
  • Neurospora crassa Neurospora crassa
  • Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated bispecific antibody molecules are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruiffly) , and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • vertebrate cells have been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: 59 (1977) ) ; baby hamster kidney cells (BHK, ATCC CCL 10) ; Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.
  • mice sertoli cells TM4, Mather, Biol. Reprod. 23: 243-251 (1980) ) ; monkey kidney cells (CV1 ATCC CCL 70) ; African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2) ; canine kidney cells (MDCK, ATCC CCL 34) ; buffalo rat liver cells (BRL 3A, ATCC CRL 1442) ; human lung cells (W138, ATCC CCL 75) ; human liver cells (Hep G2, HB 8065) ; mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982) ) ; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) .
  • the host cell is 2
  • Host cells are transformed with the above-described expression or cloning vectors for production of the bispecific antibody molecules and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the bispecific antibody molecules may be produced by homologous recombination known in the art.
  • the host cells used to produce the bispecific antibody molecule provided herein may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma) , Minimal Essential Medium (MEM) , (Sigma) , RPMI-1640 (Sigma) , and Dulbecco's Modified Eagle's Medium (DMEM) , Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleotides (such as adenosine and thymidine) , antibiotics (such as GENTAMYCIN TM drug) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the bispecific antibody molecules can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the bispecific antibody molecules thereof prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • Protein A immobilized on a solid phase is used for immunoaffinity purification of the bispecific antibody molecules.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the bispecific antibody molecules.
  • Protein A can be used to purify antibodies that are based on human gamma1, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) .
  • Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. 5: 1567 1575 (1986) ) .
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the bispecific antibody molecule comprises a CH3 domain
  • the Bakerbond ABX TM resin J. T. Baker, Phillipsburg, N. J. ) is useful for purification.
  • the mixture comprising the antibody molecule of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt) .
  • compositions comprising the bispecific antibody molecule and one or more pharmaceutically acceptable carriers.
  • Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
  • Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins.
  • Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate.
  • compositions that comprise one or more bispecific antibody molecules as disclosed herein and one or more antioxidants such as methionine. Further provided are methods for preventing oxidation of, extending the shelf-life of, and/or improving the efficacy of a bispecific antibody molecule as provided herein by mixing the bispecific antibody molecule with one or more antioxidants such as methionine.
  • pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (
  • Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
  • compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the pharmaceutical compositions are formulated into an injectable composition.
  • the injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion.
  • Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
  • a sterile, lyophilized powder is prepared by dissolving a bispecific antibody molecule as disclosed herein in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
  • the solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH.
  • the resulting solution will be apportioned into vials for lyophilization.
  • Each vial can contain a single dosage or multiple dosages of the bispecific antibody molecule or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
  • Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
  • methods are provided to treat a condition in a subject that would benefit from up-regulation of immune response, comprising administering a therapeutically effective amount of the bispecific antibody molecule as provided herein to a subject in need thereof.
  • the disease or condition that would benefit from up-regulation of an immune response is selected from the group consisting of cancer, a viral infection, a bacterial infection, a protozoan infection, a helminth infection, asthma associated with impaired airway tolerance, a neurological disease, multiple sclerosis, and an immunosuppressive disease.
  • Therapeutic methods comprising: administering a therapeutically effective amount of the bispecific antibody molecule as provided herein to a subject in need thereof, thereby treating or preventing a PD-1 related and/or a CTLA-4-related condition or a disorder.
  • PD-1-related conditions and disorders can be immune related disease or disorder, tumors and cancers, autoimmune diseases, or infectious disease.
  • the PD-1-related conditions and disorders include tumors and cancers, for example, non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL) , primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-
  • the PD-1-related conditions and disorders include autoimmune diseases.
  • Autoimmune diseases include, but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component) , alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diabetes, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED) , autoimmune lymphoproliferative syndrome (ALPS) , autoimmune thrombocytopenic purpura (ATP) , Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS) , chronic inflammatory demyelinating polyneuropathy (CIPD) , cicatricial pemphigold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos'disease, dermatomyo
  • the PD-1-related conditions and disorders include infectious disease.
  • infectious disease include, for example, chronic viral infection, for example, fungus infection, parasite/protozoan infection or chronic viral infection, for example, malaria, coccidioiodmycosis immitis, histoplasmosis, onychomycosis, aspergilosis, blastomycosis, candidiasis albicans, paracoccidioiomycosis, microsporidiosis, Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fa
  • the subject has been identified as being likely to respond to a PD-1 antagonist.
  • the presence or level of PD-L1 on an interested biological sample can be indicative of whether the subject from whom the biological sample is derived could likely respond to a PD-1 antagonist.
  • Various methods can be used to determine the presence or level of PD-L1 in a test biological sample from the subject.
  • the test biological sample can be exposed to anti-PD-L1 antibody or antigen-binding fragment thereof, which binds to and detects the expressed PD-L1 protein.
  • PD-L1 can also be detected at nucleic acid expression level, using methods such as Polymerase Chain Reaction (qPCR) , reverse transcriptase PCR, microarray, Serial analysis of gene expression (SAGE) , Fluorescence in situ hybridization (FISH) , and the like.
  • the test sample is derived from a cancer cell or tissue, or tumor infiltrating immune cells.
  • presence or upregulated level of the PD-L1 in the test biological sample indicates likelihood of responsiveness.
  • the term “upregulated” as used herein, refers to an overall increase of no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%or greater, in the protein level of PD-L1 in the test sample, as compared to the PD-L1 protein level in a reference sample as detected using the same antibody.
  • the reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from whom the test sample is obtained.
  • the reference sample can be a non-diseased sample adjacent to or in the neighborhood of the test sample (e.g. tumor) .
  • the subject is resistant or has developed resistance to PD-1 antagonist therapy or PD-L1 inhibitor therapy.
  • the subject can be one who progressed (e.g., experienced tumor growth) during therapy with a PD-1 inhibitor (e.g., an antibody molecule as described herein) and/or a PD-L1 inhibitor (e.g., antibody molecule) .
  • a PD-1 inhibitor e.g., an antibody molecule as described herein
  • a PD-L1 inhibitor e.g., antibody molecule
  • the present disclosure also provides therapeutic methods comprising: administering a therapeutically effective amount of the bispecific antibody molecule as provided herein to a subject in need thereof, thereby treating or preventing a CTLA-4-related condition or a disorder.
  • the CTLA-4-related condition or a disorder is cancer, autoimmune disease, inflammatory disease, infectious disease, graft versus host disease (GVHD) , or transplant rejection.
  • GVHD graft versus host disease
  • cancer examples include but are not limited to, lymphoma, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, uterine or endometrial cancer, rectal cancer, esophageal cancer, head and neck cancer, anal cancer, gastrointestinal cancer, intra-epithelial neoplasm, kidney or renal cancer, leukemia, liver cancer, lung cancer (e.g.
  • non-small cell lung cancer and small cell lung cancer melanoma, myeloma, pancreatic cancer, prostate cancer, sarcoma, skin cancer, squamous cell cancer, stomach cancer, testicular cancer, vulval cancer, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, penile carcinoma, solid tumors of childhood, tumor angiogenesis, spinal axis tumor, pituitary adenoma, or epidermoid cancer.
  • autoimmune diseases include, but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component) , alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED) , autoimmune lymphoproliferative syndrome (ALPS) , autoimmune thrombocytopenic purpura (ATP) , Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS) , chronic inflammatory demyelinating polyneuropathy (CIPD) , cicatricial pemphigold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos'disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobul
  • Inflammatory disorders include, for example, chronic and acute inflammatory disorders.
  • inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy and ventilator induced lung injury.
  • infectious disease examples include, but are not limited to, fungus infection, parasite/protozoan infection or chronic viral infection, for example, malaria, coccidioiodmycosis immitis, histoplasmosis, onychomycosis, aspergilosis, blastomycosis, candidiasis albicans, paracoccidioiomycosis, microsporidiosis, Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis,
  • an bispecific antibody molecule as provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.
  • the bispecific antibody molecule as provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg. In certain of these embodiments, the bispecific antibody molecule is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 3 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. In certain embodiments, the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) .
  • a single dose may be administered, or several divided doses may be administered over time.
  • the bispecific antibody molecule disclosed herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
  • parenteral e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection
  • non-parenteral e.g., oral, intranasal, intraocular, sublingual, rectal, or topical routes.
  • the bispecific antibody molecules disclosed herein may be administered alone or in combination with one or more additional therapeutic means or agents.
  • the bispecific antibody molecules disclosed herein may be administered in combination with another therapeutic agent, for example, a chemotherapeutic agent or an anti-cancer drug.
  • an bispecific antibody molecule as disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments the bispecific antibody molecule and the additional therapeutic agent (s) may be administered as part of the same pharmaceutical composition.
  • a bispecific antibody molecule administered “in combination” with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent.
  • a bispecific antibody molecule administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the bispecific antibody molecule and second agent are administered via different routes.
  • additional therapeutic agents administered in combination with the bispecific antibody molecule disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians'Desk Reference 2003 (Physicians'Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002) ) or protocols well known in the art.
  • the present disclosure further provides methods of using the bispecific antibody molecule thereof.
  • the present disclosure also provides use of the bispecific antibody molecule provided herein in the manufacture of a medicament for treating a PD-1 and/or CTLA-4 related disease or condition in a subject.
  • the bispecific antibodies provided herein are advantageous over existing therapies in many aspects.
  • the bispecific antibodies provided herein can block both PD1 and CTLA4 pathways, and they particularly inhibit Treg function.
  • the bispecific antibodies provided herein are superior to monospecific anti-PD1 antibodies, or monospecific anti-CTLA4 antibodies, or combination of monospecific anti-PD1 antibodies and monospecific anti-CTLA4 antibodies.
  • the bispecific antibodies provided herein are also advantageous in that they are cross-reactive to human, monkey and murine PD1 and CTLA4.
  • the bispecific antibodies may be used to treat the patients who are resistant to or relapse from anti-PD1 or anti-CTLA4 monotherapy.
  • the bispecific antibodies may also increase the response rate comparing with anti-PD1 or anti-CTLA4 alone.
  • the bispecific antibodies may also reduce the toxicity of anti-CTLA4 or anti-PD1 by using lower therapeutic dose.
  • EXAMPLE 1 Generation and characterization of PD-1 humanized monoclonal antibody of W3052-2E5
  • the humanized monoclonal PD-1 antibody W3052-2E5 was generated as described in WO2018053709A1. Generally, SD rats were immunized with human PD-1 extracellular domain (ECD) protein and the B lymphocytes isolated from lymph node of the immunized SD rats were combined with myeloma cells to obtain a hybridoma, which was isolated, selected and sub-cloned. The total RNA of the hybridoma was extracted and the cDNA was synthesized and amplified. The framework region of the rat VH and VL genes were replaced with human frameworks by CDR-grafting technique and were cloned into expression vectors to create corresponding clones of humanized antibodies. The monoclonal antibody W3052-2E5 was obtained after affinity maturation by point mutation (s) in the CDR and/or framework regions.
  • ECD extracellular domain
  • the humanized W3052-2E5 antibody has a heavy chain variable region of SEQ ID NO: 7, a kappa light chain variable region of SEQ ID NO: 8, and a human IgG4 constant region.
  • W3052-2E5 binds to human PD-1 transfected CHO-S cells with EC 50 of 2.20 nM, to mouse PD-1 transfected 293F cells with EC 50 of 12.9 nM and to activated cynomolgus PBMC in a dose dependent way, as determined by flow cytometry.
  • W3052-2E5 binds to human PD-1 with EC 50 of 0.18 nM, to mouse PD-1 with EC 50 of 0.37 nM and to cynomolgus PD-1 with EC50 of 0.25 nM by ELISA.
  • W3052-2E5 binds specifically to human PD-1, but not to CD28 and CD47, as measured by flow cytometry.
  • W3052-2E5 blocks human PD-L1 binding to PD-1 transfected CHO-S cells with IC 50 of 2.14 nM, blocks mouse PD-L1 binding to PD-1 transfected 293F cells with IC50 of 13 nM, and blocks human PD-L2 binding to PD-1 in a dose-dependent manner, as determined by ELISA.
  • the binding affinity of W3052-2E5 to human PD-1 by SPR assay was 6.13 nM (KD value) .
  • the binding affinity of W3052-2E5 to mouse PD-1 by SPR assay was 3.99 nM (KD value) .
  • the binding affinity of W3052-2E5 to human PD-1 by FACS assay was 0.23 nM (KD value) .
  • the binding affinity of W3052-2E5 to mouse PD-1 by FACS assay was 29 nM (KD value) .
  • W3052-2E5 increased IL-2 secretion, IFN- ⁇ secretion in a dose-dependent manner, as measured by ELISA in both human and mouse T cell function assays.
  • W3052-2E5 increased and CD4 + T cells proliferation in a dose-dependent manner, as measured by 3H-thymidine incorporation assay in both human and mouse T cell function assays.
  • Tests in W3052-2E5 on cell proliferation and cytokine production by autologous antigen specific immune response showed that W3052-2E5 can enhance the function of human CD4 + T cell by increase IFN- ⁇ secretion and CD4 + T cells proliferation in a dose-dependent manner.
  • W3052-2E5 also can reverse the suppressive function of Tregs by restoring the IFN- ⁇ secretion and the T-cell proliferation.
  • W3052-2E5 does not mediated ADCC or CDC activity on activated CD4 + T cells.
  • Anti-CLTA-4 monoclonal rat antibody W3162-1.146.19 was obtained by hybridoma approach. Its rat/human chimeiric antibody W3162-1.146.19. xAb, and the humanized anti-CTLA-4 antibody W3162-1.146.19-z12 was also generated as described in WO2018209701A1.
  • Reference benchmark antibody W316-BMK1 was generated based on the variable region sequence of Ipilimumab (Yervoy) as disclosed in patent document US20150283234.
  • variable region was synthesized in Sangon Biotech (Shanghai, China) , and then cloned into modified pcDNA3.4 expression vectors with constant region of human IgG1, or human IgG4.
  • xAb promoted IL-2 secretion in a dose dependent manner, comparable with WBP316-BMK1.
  • W3162-1.146.19-z12 bound to human CTLA-4 with EC 50 of 0.03 nM and 0.04 nM, respectively, slightly higher than EC 50 of WBP316-BMK1 0.01 nM.
  • W3162-1.146.19-z12 s also bound to monkey CTLA-4 with EC50 of 0.05 nM, and to murine CTLA-4 with EC50 0.19 nM.
  • WBP316-BMK1 did not bind to murine CTLA-4.
  • W3162-1.146.19-z12 bound to human CTLA-4 on cell surface with EC 50 of 1.58 nM, whereas WBP316-BMK1 had slightly different EC 50 of 0.83 nM.
  • W3162-1.146.19-z12 has a binding affinity of KD value at 1.92 nM to cynomolgus monkey CTLA-4-ECD.
  • W3162-1.146.19-z12 In an FACS binding affinity assay to cell surface CTLA-4, W3162-1.146.19-z12 had high affinity of KD value at 5.05 nM, whereas the affinity of WBP316-BMK1 is 0.97 nM.
  • W3162-1.146.19-z12 had similar effect as WBP316-BMK1 in blocking ligands binding with coated human CTLA-4, with IC 50 of 0.87 nM, and 0.40 nM for human CD80, and 0.71nM, and 0.42 nM for human CD86, respectively.
  • W3162-1.146.19-z12 could more effectively block CTLA-4/ligand binding than WBP316-BMK1.
  • Ipilimumab only blocked 32%CTLA-4 binding to human CD80 and 40%of CTLA-4 binding to human CD86.
  • antibody W3162-1.146.19-z12 blocked 71%of CTLA-4 binding on human CD80 and 73%of CTLA-4 binding on human CD86.
  • IC50 of WBP316-BMK1 and W3162-1.146.19-z12 directing against human CD80 were 3.23 and 6.60 nM, respectively.
  • IC50 of WBP316-BMK1, W3162-1.146.19-z12 directing against human CD86 were 2.52 and 5.15 nM, respectively.
  • both W3162-1.146.19-z12 and WBP316-BMK1 could enhance IL-2 release from the PBMCs in a dose-dependent manner.
  • W3162-1.146.19-z12 was stable in tested low and high temperature conditions (4°C or 37°C for 20 hours, or 45°C or 50°C for 2 hours) .
  • W3162-1.146.19-z12 did not have non-specific binding to other antigens (Factor VIII, FGFR-ECD, PD-1, CTLA-4.ECD, VEGF, HER3.
  • ECD, 4-1BB ECD, CD22. ECD, CD3e. ECD. ) .
  • the epitopes of WBP316-BMK1 and W3162-1.146.19-z12 overlap to each other, except a few residues such as N145 and P138.
  • the N-glycosylation site on N145 of CTLA-4 could be the epitope of W3162-1.146.19-z12, but not for WBP316-BMK1.
  • the N145 residue is conserved in CTLA-4 of cynomoguls monkey and mouse.
  • Mouse cancer cell line CT26 was used to establish xenograft mouse model to test the tumor inhibition effects of W3162-1.146.19-z12. Each mouse was inoculated subcutaneously at the right axillaries with tumor cells. When the average tumor volume reaches 60-80 mm 3 .
  • the anti-CTLA-4 antibody W3162-1.146.19-z12 and isotype control were used for treatment with intravenously injected into mice twice a week. When average tumor volume reached approximately 70 mm3, W3162-1.146.19-z12 (1 mg/kg, 3 mg/kg, 10 mg/kg) and control antibodies (10 mg/kg) were injected twice a week for two weeks.
  • W3162-1.146.19-z12 significantly inhibited tumor growth in a dose-dependent manner. At 1 mg/kg dose, W3162-1.146.19-z12 inhibited tumor growth, compared with control group. At 3 mg/kg dose, W3162-1.146.19-z12 inhibit tumor volume to 160 mm 3 at day 19, whereas 10 mg/kg W3162-1.146.19-z12 induced tumor regression at the end of the study period.
  • DNA sequences encoding the extracellular domain sequence of human PD-1 (Uniport No.: Q15116) were synthesized in Sangon Biotech (Shanghai, China) , and then subcloned into modified pcDNA3.3 expression vectors with 6xhis in C-terminal. Protein of human, cyno and mouse CTLA4 and mouse and cyno PD1 were purchased from Sino Biological.
  • Expi293 cells (Invitrogen-A14527) were transfected with the purified expression vector pcDNA3.3. Cells were cultured for 5 days and supernatant was collected for protein purification using Ni-NTA column (GE Healthcare, 175248) . The obtained human PD-1 was QC’ed by SDS-PAGE and SEC, and then stored at -80 °C.
  • DNA sequence encoding the variable region of anti-CTLA4 antibody Ipilimumab (as disclosed in patent document US20150283234) , anti-PD1 antibody nivolumab (as disclosed as clone 5C4 in the U.S. patent US9084776B2) ) was synthesized in Sangon Biothech (Shanghai, China) , and then subcloned into modified pcDNA3.4 expression vectors with constant region of human IgG1 or human IgG4 (S228P) .
  • the obtained anti-CTLA4 benchmark antibody was named as WBP316-BMK1
  • the obtained anti-PD-1 benchmark antibody was named as WBP305-BMK1.
  • Anti-PD-1 antibody W3052-2E5 antibody was generated in house after immunizing rats with human PD-1 and mouse PD-1 protein and was converted to IgG4 (S228P) format.
  • Anti-CTLA4 W3162-1.146.19-z12 antibody was generated in house after immunizing rats with human CTLA-4 and mouse CTLA-4 protein and was converted to IgG4 (S228P) format.
  • the plasmid containing VH and VL gene were co-transfected into Expi293 cells. Cells were cultured for 5 days and supernatant was collected for protein purification using Protein A column (GE Healthcare, 175438) or Protein G column (GE Healthcare, 170618) . The obtained antibodies were tested by SDS-PAGE and SEC, and then stored at -80 °C.
  • CHO-S Thermo Fisher, Cat. A1155701
  • 293F cells Thermo Fisher, Cat. R79007
  • mouse PD-1 high expression stable cell line WBP305.293F. mPro1. B4
  • G15 format bispecific antibodies DNA sequence encoding W3162-1.146.19-z12 variable light chain on the N-terminal constant region of light chain was cloned into modified pcDNA3.3 expression vector. DNA sequence encoding scFv (VH- (G4S) 3 -VL) of W3052-2E5 on the C-terminal of W3162-1.146.19-z12 constant region of human IgG4 (S228P) heavy chain was cloned into modified pcDNA3.3 expression vector. The obtained bispecific antibody was named “W3242-T4U3. G15-1” .
  • G17 format bispecific antibodies DNA sequence encoding W3162-1.146.19-z12 variable light chain on the N-terminal constant region of light chain was cloned into modified pcDNA3.3 expression vector. DNA sequence encoding scFv (VH- (G4S) 3 -VL, ) of W3052-2E5 on the N-terminal of W3162-1.146.19-z12 variable region of human IgG4 (S228P) heavy chain was cloned into modified pcDNA3.3 expression vector. The obtained bispecific antibody was named “W3242-T4U3. G17-1” .
  • Heavy chain and light chain expression plasmids were co-transfected into ExpiCHO cells using ExpiCHO expression system kit (ThermoFisher-A29133) according to the manufacturer’s instructions. 10 days after transfection, the supernatants were collected and used for protein purification using Protein A column (GE Healthcare-17543802) and further size exclusion chromatography (GE Healthcare-17104301) . Antibody concentration was measured by Nano Drop. The purity of proteins was evaluated by SDS-PAGE and HPLC-SEC. Two Bispecific antibodies, i.e. W3242-T4U3. G17-4 and W3242-T4U3. G15-1 were obtained after expression and purification.
  • Heavy chain and light chain expression plasmids were co-transfected into Expi293 cells (ThermoFisher-A14527) according to the manufacturer’s instructions. Five days after transfection, the supernatants were harvested and used for protein purification using Protein A column (GE Healthcare-17543802) and further size exclusion chromatography (GE Healthcare-17104301) . Antibody concentration was measured by Nano Drop. The endotoxin level was confirmed by using endotoxin detection kit (GenScript-L00350) , and the endotoxin level of two Bispecific antibodies was both less than 10 EU/mg. The purity of proteins was evaluated by SDS-PAGE and HPLC-SEC. Two BsAbs, i.e. W3242-T4U3. G17-4 and W3242-T4U3. G15-1 were obtained after expression and purification.
  • Figure 1 showed the result of the SDS-PAGE.
  • the molecular weight estimated from each bands are similar to their theoretical molecular weight.
  • the SEC result was shown in Figure 2.
  • the purity of all are above 95%.
  • a DSF assay was performed using 7500 Fast Real-Time PCR system (Applied Biosystems) . Briefly, 19 ⁇ L of bispecific antibody solution was mixed with 1 ⁇ l of 62.5x SYPRO Orange solution (TheromFisher-S6650) and added to a 96 well plate. The plate was heated from 26 °C to 95 °C at a rate of 2 °C/min and the resulting fluorescence data was collected. The data was analyzed automatically by its operation software and Th was calculated by taking the maximal value of negative derivative of the resulting fluorescence data with respect to temperature. Ton can be roughly determined as the temperature of negative derivative plot beginning to decrease from a pre-transition baseline. The DSF result is shown in Table 9.
  • hCTLA-4-Binding ELISA The result of hCTLA-4-Binding ELISA was shown in Figure 3A. Both W3242-T4U3. G17-4 and W3242-T4U3. G15-1 bind to hCTLA-4 comparable as their parental Ab.
  • the result of hCTLA-4-Binding FACS was shown in Figure 3B. Both W3242-T4U3. G17-4 and W3242-T4U3. G15-1 can bind to hCTLA-4+ cells: W3242-T4U3. G15-1 with similar binding to their parental Ab and W3242-T4U3. G17-4 with slightly reduced binding.
  • the result of mCTLA-4-Binding ELISA was shown in Figure 3C. Both W3242-T4U3.
  • G15-1 can bind to mCTLA-4 with comparable affinity as their parental Ab.
  • the result of cynoCTLA4 binding-ELISA was shown in Figure 3D. Both W3242-T4U3.
  • G15-1 bind to cyno CTLA-4.
  • hPD-1-Binding ELISA The result of hPD-1-Binding ELISA was shown in Figure 4A.
  • G17-4 bind to hPD-1 comparable with parental antibody; W3242-T4U3.
  • G15-1 binds to hPD-1 with 1.6x higher EC 50 than parental antibody.
  • engineered human PD-1 expressing cells W305-CHO-S.hPro1. C6 were seeded at 1 ⁇ 10 5 cells/well in U-bottom 96-well plates (COSTAR 3799) . 3.16-Fold titrated antibodies with 1%BSA DPBS (Corning, 21-031-CVR) from 20 ⁇ g/ml to 0.0002 ⁇ g/ml were added to the cells. Plates were incubated at 4 °C for 1 hour. After wash, 100 ⁇ L 1: 125 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4 °C for 1 hour. The binding of the antibodies onto the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.
  • MFI mean fluorescence intensity
  • hPD-1-Binding FACS The result of hPD-1-Binding FACS was shown in Figure 4B.
  • G17-4 binds to hPD-1 comparable with parental antibody; W3242-T4U3.
  • G15-1 binds to hPD-1 with 1.6x higher EC 50 than parental antibody.
  • engineered mouse PD-1 expressing cells W305-293F. mPro1. B4 were seeded at 1 ⁇ 10 5 cells/well in U-bottom 96-well plates (COSTAR 3799) .
  • 4.0-Fold titrated antibodies with 1%BSA DPBS from 80 ⁇ g/ml to 0.000076 ⁇ g/ml were added to the cells. Plates were incubated at 4 °C for 1 hour. After wash, 100 ⁇ L 1: 150 diluted FITC-labeled goat anti-human antibody (Jackson 109-095-008) was added to each well and the plates were incubated at 4 °C for 1 hour. The binding of the antibodies onto the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.
  • MFI mean fluorescence intensity
  • the bispecific antibody’s binding to cyno PD-1 was measured using an ELISA method. As shown in Figure 9, the antibodies bind to cyno PD-1 with EC 50 of 1.12 and 0.03 nM, respectively.
  • ELISA assay was developed as below.
  • a 96-well ELISA plate (Nunc MaxiSorp, ThermoFisher) was coated overnight at 4 °C with 0.5 ⁇ g/ml antigen-1 (hPD1-ECD, W305-hPro1. ECD. mFc (in house) ) in carbonate-bicarbonate buffer.
  • Streptavidin-HRP (Lifetechnologies, #SNN1004) (1: 20000 diluted) is added and incubated on the plates for 1 hour at room temperature. After washing six times with 300 ⁇ L per well of PBS containing 0.5% (v/v) Tween 20, 100 ⁇ L tetramethylbenzidine (TMB) Substrate (in house) is added for the detection pre well. The reaction is stopped after approximate 5 minutes through the addition of 100 ⁇ L per well of 2 M HCl. The absorbance of the wells is measured at 450 nm with a multiwall plate reader ( M5 e ) ..
  • an ELISA assay was developed as below.
  • a 96-well ELISA plate (Nunc MaxiSorp, ThermoFisher) was coated overnight at 4 °C with 1.0 ⁇ g/ml antigen-1 (mCTLA-4-ECD, W316-mPro1. ECD. hFc (in house) ) in carbonate-bicarbonate buffer.
  • Murine CTLA-4/PD-1 Dual Binding ELISA was shown in Figure 5B.
  • G17-4 can bind to murine PD-1 and CTLA4 simultaneously: W3242-T4U3.
  • G15-1 is better than W3242-T4U3.
  • Biacore T200, Series S Sensor Chip CM5, Amine Coupling Kit, and 10x HBS-EP were purchased from GE Healthcare.
  • Goat anti-human IgG Fc antibody was purchased from Jackson ImmunoResearch Lab (catalog number 109-005-098) .
  • the activation buffer was prepared by mixing 400 mM EDC and 100 mM NHS immediately prior to injection.
  • the CM5 sensor chip was activated for 420 s with the activation buffer.
  • 30 ⁇ g/mL of goat anti-human IgG Fc antibody in 10 mM NaAc (pH 4.5) was then injected to Fc1-Fc4 channels for 200s at a flow rate of 5 ⁇ L/min.
  • the chip was deactivated by 1 M ethanolamine-HCl (GE) . Then the antibodies were captured on the chip. Briefly, 4 ⁇ g/mL antibodies in running buffer (HBS-EP+) was injected individually to Fc3 channel for 30 s at a flow rate of 10 ⁇ L/min. Eight different concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625 nM) of analyte ECD of CTLA4 or PD-1 and blank running buffer were injected orderly to Fc1-Fc4 channels at a flow rate of 30 ⁇ L/min for an association phase of 120 s, followed by 2400 s dissociation phase. Regeneration buffer (10 mM Glycine pH 1.5) was injected at 10 ⁇ L/min for 30 s following every dissociation phase.
  • Table 11 is the summary of High affinity on human, cyno and murine PD-1.
  • Table 12 is the summary of High affinity on human, cyno and murine CTLA-4.
  • Human CD80-or CD86-expressing CHO-K1 cells were added to each well of a 96-well plate (COSTAR 3799) at 1 x 10 5 per well and centrifuged at 1500 rpm for 4 minutes at 4°C before removing the supernatant. Serial dilutions of test antibodies, positive and negative controls were mixed with biotinylated human CTLA4. ECD. hFc (in house) . Due to different density of ligands on cell surface, 0.066 and 0.037 ⁇ g/mL of hCTLA-4. ECD. hFc-Biotin was used for human CD80 and 86 cells, respectively.
  • FIG. 6A The result of blocking CTLA-4 binding to CD80 on cell surface (FACS) was shown in Figure 6A. Both W3242-T4U3. G15-1 and W3242-T4U3. G17-4 can block CD80 binding to hCTLA4 by FACS.
  • FIG. 6B The result of blocking CTLA-4 binding to CD86 on cell surface (FACS) was shown in Figure 6B. Both W3242-T4U3. G15-1 and W3242-T4U3. G17-4 can block CD86 binding to hCTLA4 by FACS.
  • Human CD80 expressing CHO-K1 cells were added to each well of a 96-well plate (COSTAR 3799) at 1 x 10 5 per well and centrifuged at 1500 rpm for 4 minutes at 4°C before removing the supernatant. Serial dilutions of test antibodies, positive and negative controls were mixed with cynoCTLA-4. ECD. His. A concentration of 0.1 ⁇ g/mL of cynoCTLA-4.ECD. His was used for human CD80 cells. Then the mixtures of antibody and cynoCTLA-4 protein were added to the cells and incubated for 1 hour at 4 °C. The cells were washed two times with 200 ⁇ l FACS washing buffer (DPBS containing 1%BSA) .
  • FACS washing buffer DPBS containing 1%BSA
  • Biotinylated anti-His mAb (GenScript A00613) 1 to 600 diluted in FACS buffer was added to the cells and incubated at 4 °C for 1 hour. After washing two times, Streptavidin PE (eBioscience 12-4317-87) 1 to 333 diluted in FACS buffer was added to the cells and incubated at 4 °C for 1 hour. Additional washing steps were performed two times with 200 ⁇ L FACS washing buffer followed by centrifugation at 1500 rpm for 4 minutes at 4 °C. Finally, the cells were resuspended in 60 ⁇ L FACS washing buffer and fluorescence values were measured by flow cytometry and analyzed by FlowJo.
  • the bispecific antibodies can block cyno CTLA4 binding to hCD80 and hCD86, as shown in Figure 10 and 11.
  • the two bispecific antibodies block cyno CTLA4 binding to hCD80+ cells, with IC50 of 3.889 and 38.73 nM, respectively.
  • the bispecific antibodies can block cyno CTLA4 binding to hCD86+ cells.
  • the bispecific antibodies can also block murine CD80 binding to murine CTLA-4, with IC 50 of 5.806 and 41.930 nM, as shown in Figure 12.
  • engineered human PD-1 expressing cells W305-CHO-S. hPro1.
  • C6 in house
  • 3.16-Fold titrated antibodies from 80 ⁇ g/ml to 0.0008 ⁇ g/ml coupled with 5ug/ml in house human PD-L1 protein W315-hPro1.
  • ECD. mFc were added to the cells. Plates were incubated at 4 °C for 1 hour. After wash, the binding of W315-hPro1.
  • mFc to cell expressive human PD-1 was detected by FITC-labeled goat anti-mouse antibody (abcam 98716 1: 125) .
  • the competition binding of antibodies to the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.
  • mFc to cell expressive human PD-1 was detected by FITC-labeled goat anti-mouse antibody (abcam 98716 1: 125) .
  • the competition binding of antibodies to the cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.
  • MLR Mixed lymphocyte reaction
  • PBMCs Human peripheral blood mononuclear cells
  • Ficoll-Paque STEMCELL-07861 PLUS gradient centrifugation. Isolated PBMCs were cultured in complete RPMI-1640 (containing 10%FBS and 1%PS) supplemented with 100 U recombinant human IL-2 (SL PHARM) .
  • Human monocytes were isolated using Human Monocyte Enrichment Kit (Miltenyi Biotec-130-050-201) according to the manufacturer’s instructions. Cell concentration was adjusted in complete RPMI-1640 medium (Gibco-22400089) supplemented with 800 U/mL recombinant human GM-CSF (R&D 215-GM) and 50 ng/mL rhIL-4 (R&D, AG1815401) . Cell suspension was seeded at a concentration of 2 ⁇ 10 6 cells/mL, 2.5 mL/well in 6-well plate. Cells were cultured for 5 to 7 days to differentiate into immature dendritic cells (iDCs) . Cytokines were replenished every 2-3 days by replacing half of the media with fresh media supplemented with cytokines.
  • iDCs immature dendritic cells
  • Human CD4+ T cells were isolated using Human CD4+ T cell Enrichment kit (STEMCELL-19052) according to the manufacturer’s protocol.
  • CD4+ T cells DCs and various concentrations of antibodies (2-fold, 2.5-fold and 10-fold serially diluted from 335 nM to 0.067 nM) were added to 96-well round bottom plates in complete RPMI-1640 medium. The plates were incubated at 37°C in a 5%CO2 incubator. IL-2 and IFN- ⁇ in the supernatant were quantified on day 3 and day 5, respectively (data not shown) .
  • PBMCs various concentrations of antibodies (2-fold, 2.5-fold and 10-fold serially diluted from 335 nM to 0.067 nM) and SEB (Staphylococcal enterotoxin B) at the concentration of 10 ng/mL were added to 96-well round bottom plates in complete RPMI-1640 medium. The plates were incubated at 37°C, 5%CO2. IL-2 and IFN- ⁇ quantitation were determined on day 3 and day 5 respectively.
  • SEB Staphylococcal enterotoxin B
  • Human IFN- ⁇ were measured by ELISA using matched antibody pairs. Recombinant human IFN- ⁇ (Peprotech, 300-02) was used as standards. The plates were pre-coated with capture antibody specific for human IFN- ⁇ (Lifetechnologies, M700A; M7001B) . After blocking, standards or samples were pipetted into each well and incubated for 2 hours at ambient temperature. Following removal of the unbound substances, the biotin-conjugated detecting antibody specific for IFN- ⁇ was added to the wells and incubated for 1 hour. The HRP-conjugated streptavidin was then added to the wells for 30 minutes at ambient temperature. The color was developed by dispensing 100 ⁇ L of TMB substrate, and then stopped by 100 ⁇ L of 2N HCl. The absorbance was read at 450 nm using a microplate spectrophotometer ( M5e) .
  • human IL-2 test plates were pre-coated with 1.0 ⁇ g/ml human IL-2 antibody (R&D System MAB602) at 4 °C for 16-20 hours. After 1 hour blocking with 2%BSA (BovoGen) in DBPS, the supernatants containing IL-2 were added to the plates and incubated at room temperature for 2 hours. After washing three times with PBST (containing 0.05%Tween 20) , biotinylated human IL-2 antibody (R&D system, BAF202) was diluted and added at concentration of 0.5 ⁇ g/mL. The plates were incubated at room temperature for 1 hour.
  • PBST containing 0.05%Tween 20
  • the bound biotinylated antibody was detected by 1: 20000 diluted streptavidin conjugated HRP (Invitrogen, SNN1004) . After 1 hour incubation, the color was developed by dispensing 100 ⁇ L of TMB substrate, and then stopped by 100 ⁇ L of 2M HCl. The absorbance at 450 nm and 540 nm was measured using a microplate spectrophotometer.
  • Human CD4+CD25+ Treg cells were separated from fresh hPBMC by isolation Kit (Miltenyi 130-093-631) and amplified for 2 weeks.
  • Human CD4+ T cells separated from another donor by Human CD4+ T cell Enrichment kit (STEMCELL-19052) were mixed with Treg , iDC and test antibodies (10-fold dilution, from 335 nM to 3.35 nM) .
  • the amount of Treg, CD4+ T and iDC cells were 1E5, 1E5 and 1E4 pre well and incubated in 96-well plates. The plates were kept at 37°C in a 5%CO2 incubator for 5 days. IFN- ⁇ in the supernatant was quantified by ELISA and T cell proliferation was measured by 3H-thymidine incorporation (Perkin Elmer, NET027001MC) .
  • Both FACS and ELISA assays were used to test whether the antibodies binding to other targets.
  • the testing antibodies, isotype control antibodies were tested binding to different proteins including Factor VIII, FGFR-ECD, PD-1, CTLA-4.
  • E and XAg were undisclosed proteins.
  • Several 96-well plates (Nunc-Immuno Plate, Thermo Scientific) was coated with the individual antigens (2 ⁇ g/mL) at 4 °C overnight.
  • PE conjugated goat anti-human IgG Fc fragment (Jackson, Catalog number 109-115-098) was diluted to final concentration 5 ⁇ g/ml in PBS with 1%BSA, then added to re-suspend cells and incubated at 4 °C in the dark for 30 min. Additional washing steps were performed twice with 180 ⁇ L PBS containing 1%BSA followed by centrifugation at 1500 rpm for 4 minutes at 4°C. Finally, the cells were re-suspended in 100 ⁇ L PBS containing 1%BSA and fluorescence values were measured by flow cytometry (BD Canto II) and analyzed by FlowJo. Results are shown in Table 35.
  • Table 34 is the summary of non-specific binding (ELISA) .
  • Table 35 is the summary of non-specific binding (FACS) .
  • Human ICOS, BTLA, CD28 and PD-1 were purchased from Sino Biological.
  • HRP-conjugated goat anti-human IgG Fc was purchased from Bethyl (Cat: A80-304P) .
  • a 96-well plate was coated with human CTLA-4.
  • ECD-6xHis or other antigens (1.0 ⁇ g/mL) at 4 °C for 16-20 hours.
  • After 1 hour blocking with 2%BSA in DBPS, testing antibodies, as well as positive and negative control antibodies were added to the plates and incubated at room temperature for 1 hour.
  • the binding of the antibodies to the plates were detected by HRP-conjugated goat anti-human IgG antibody (1: 5000 dilution) with 1 hr incubation.
  • the color was developed by dispensing 100 ⁇ L of TMB substrate for 8 mins, and then stopped by 100 ⁇ L of 2N HCl.
  • the absorbance at 450 nM was measured using a microplate spectrophot
  • Non-tissue culture treated flat-bottom 96-well plates were pre-coated with 1.0 ⁇ g/ml in house made human CD28 ECD. mFc (20368) , human CTLA4 ECD. his, human ICOS ECD. mFc (20374) and human PD-1 protein overnight at 4°C.
  • 100 ⁇ L 10-fold titrated antibodies from 20 nM to 0.02 pM were pipetted into each well and incubated for 1 hour at ambient temperature.
  • HRP-labeled goat anti-human IgG was added to the wells and incubated for 1 hour. The color was developed by dispensing 100 ⁇ L TMB substrate, and then stopped by 100 ⁇ L 2N HCl. The absorbance was read at 450 nm using a Microplate Spectrophotometer.
  • G17-4 bind to human CTLA-4 and human PD-1, but not to human CD28 and human ICOS (data not shown) .
  • Antibodies were incubated in freshly isolated human serum (serum content > 90%) at 37°C. On indicated time points, an aliquot of serum treated sample were removed from the incubator and snap frozen in liquid N2, and then stored at -80°C until ready for test. The samples were quickly thawed immediately prior to the stability test. Briefly, plates were pre-coated with 0.5 ⁇ g/mL of W316-hPro1. ECD. hFc (in house) at 4°C overnight. After 1-hour blocking, testing antibodies were added to the plates at various concentrations (4-fold serially diluted from 5.0 nM to 0.0003 nM) . The plates were incubated at ambient temperature for 1 hour.
  • TMB tetramethylbenzidine
  • the antibodies were incubated with 37 C human serum for 14 days. At different time points, the antibodies were taken and their dual binding activity to PD1 and CTLA4 was measured. As shown in Figure 13, these antibodies’ binding to both targets did not change over the 14 day period, demonstrating that W3242-T4U3. G17-4 W3242-T4U3. G15-1 are stable in 37 °C human serum for at least two weeks.
  • C57BL/6 mice with age of 6-8 weeks were injected with 13.3 mg/kg of antibodies. At different time point, blood samples were taken. The antibody concentration in the blood was measure using human Fc-binding ELISA. The data was analyzed and the pharmacokinetic parameters were calculated.
  • mice The pharmacokinetics of the two antibodies were tested in mice. As shown in Figure 14, they had long half-life (168-199 hours) in vivo.
  • mice Female C57BL/6 mice with age of 6-8 weeks and weight approximately 17-19g were used for this study. A total of 60 mice were purchased from Shanghai Sino-British SIPPR/BK Laboratory Animal Co., LTD.
  • the 3LL tumor cells were maintained in vitro as a monolayer culture in RPMI-1640 medium (GIBCO, 22400089) supplemented with 10%fetal bovine serum (GIBCO, 10100147) , 100 U/ml penicillin and 100 ⁇ g/ml streptomycin at 37°C in an atmosphere of 5%CO2 in air.
  • the tumor cells were routinely sub-cultured twice weekly by trypsin-EDTA treatment.
  • the cells growing in an exponential growth phase were harvested and counted for tumor inoculation.
  • Each mouse were inoculated subcutaneously at the right axillaries. Cells of 2 x 10 6 in 0.1 ml of PBS were inoculated into each mouse for tumor development. The animals were random grouped when the average tumor volume reaches 60-80 mm 3 , then treatment was started for the efficacy study.
  • mice were assigned into groups using randomized block design based upon their tumor volumes. This ensured that all the groups were comparable at the baseline.
  • mice The tumor inoculated mice were treated with the W3242-T4U3. G15-1 and W3242-T4U3. G17-4 (intraperitoneal injection of 13mg/kg, twice per week for 3 weeks) .
  • the monoclonal antibodies anti-PD-1 antibody W3052-2E5 and anti-CTLA-4 antibody W3162-1.146.19-z12 were treated with same time schedule at same molar concentration 10 mg/kg.
  • 3LL xenograft model was used to test the anti-tumor efficacy of these bispecific antibodies. As shown in Figure 15, both bispecific antibodies significantly inhibited tumor growth. Moreover, the W3242-T4U3. G15-1 was more efficacious than monotherapy.

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Abstract

L'invention concerne des molécules d'anticorps bispécifiques anti-CTLA-4/PD-1, des polynucléotides isolés codant pour celles-ci, des compositions pharmaceutiques les comprenant, et leurs utilisations.
PCT/CN2019/078664 2018-03-19 2019-03-19 Nouvelles molécules d'anticorps pd-1/ctla-4 bispécifiques WO2019179421A1 (fr)

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WO2022218383A1 (fr) * 2021-04-14 2022-10-20 康方药业有限公司 Utilisation d'anticorps dans un traitement anti-tumoral

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WO2015048312A1 (fr) * 2013-09-26 2015-04-02 Costim Pharmaceuticals Inc. Méthodes de traitement de cancers hématologiques
CN104974253A (zh) * 2014-04-01 2015-10-14 上海中信国健药业股份有限公司 抗ctla-4/pd-1双特异性抗体、其制备方法及应用
CN104987421A (zh) * 2015-05-13 2015-10-21 北京比洋生物技术有限公司 抗ctla-4和pd-1的双重可变结构域免疫球蛋白
WO2016061142A1 (fr) * 2014-10-14 2016-04-21 Novartis Ag Molécules d'anticorps de pd-l1 et leurs utilisations
CN105754990A (zh) * 2016-01-29 2016-07-13 深圳精准医疗科技有限公司 一种pd-1/ctla-4双特异性抗体的制备方法及其应用
EP3091031A1 (fr) * 2015-05-04 2016-11-09 Affimed GmbH Combinaison d'un anticorps bispécifique avec une molécule modulatrice immunitaire pour le traitement d'une tumeur
WO2017106061A1 (fr) * 2015-12-14 2017-06-22 Macrogenics, Inc. Molécules bispécifiques présentant une immunoréactivité par rapport à pd-1 et à ctla-4 et leurs procédés d'utilisation
WO2018036473A1 (fr) * 2016-08-23 2018-03-01 中山康方生物医药有限公司 Anticorps bifonctionnel anti-ctla4 et anti-pd -1, composition pharmaceutique et utilisation associées

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Publication number Priority date Publication date Assignee Title
WO2014209804A1 (fr) * 2013-06-24 2014-12-31 Biomed Valley Discoveries, Inc. Anticorps bispécifiques
WO2015048312A1 (fr) * 2013-09-26 2015-04-02 Costim Pharmaceuticals Inc. Méthodes de traitement de cancers hématologiques
CN104974253A (zh) * 2014-04-01 2015-10-14 上海中信国健药业股份有限公司 抗ctla-4/pd-1双特异性抗体、其制备方法及应用
WO2016061142A1 (fr) * 2014-10-14 2016-04-21 Novartis Ag Molécules d'anticorps de pd-l1 et leurs utilisations
EP3091031A1 (fr) * 2015-05-04 2016-11-09 Affimed GmbH Combinaison d'un anticorps bispécifique avec une molécule modulatrice immunitaire pour le traitement d'une tumeur
CN104987421A (zh) * 2015-05-13 2015-10-21 北京比洋生物技术有限公司 抗ctla-4和pd-1的双重可变结构域免疫球蛋白
WO2017106061A1 (fr) * 2015-12-14 2017-06-22 Macrogenics, Inc. Molécules bispécifiques présentant une immunoréactivité par rapport à pd-1 et à ctla-4 et leurs procédés d'utilisation
CN105754990A (zh) * 2016-01-29 2016-07-13 深圳精准医疗科技有限公司 一种pd-1/ctla-4双特异性抗体的制备方法及其应用
WO2018036473A1 (fr) * 2016-08-23 2018-03-01 中山康方生物医药有限公司 Anticorps bifonctionnel anti-ctla4 et anti-pd -1, composition pharmaceutique et utilisation associées

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022218383A1 (fr) * 2021-04-14 2022-10-20 康方药业有限公司 Utilisation d'anticorps dans un traitement anti-tumoral

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