Classifications

• • 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 [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/734—Complement-dependent cytotoxicity [CDC]
• • 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
  • C07K2317/75—Agonist effect on antigen
• • 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

Definitions

• the present invention relates generally to antibodies and antigen binding fragments thereof and more specifically to antibodies and antigen binding fragments having cytotoxicity against cancer cells and enhancing T-cell function.
• Cancer immunotherapy restores or boosts the natural defense against tumor by the human immune system.
• Such therapy usually targets specific biological molecules on cancer cells’ surface such as tumor-associated antigens (TAAs).
• TAAs tumor-associated antigens
• Anti-tumor activities are possibly achieved by directing the host immune system toward TAAs, which consequently establishes or induces adaptive immune responses against cancer cells.
• mAbs monoclonal antibodies
• CD137 (4-1BB/TNFRSF9) was identified in 1989 as an inducible gene that was expressed on antigen-primed T cells but not on resting ones. In addition, it is known to be expressed in dendritic (DCs), natural killer cells (NKs), activated CD4+ and CD8+ T lymphocytes, eosinophils, natural killer T cells (NKTs), and mast cells, but myeloid-derived suppressor cells (MDSCs) were investigated to not express this molecule on their surfaces.
• the anti-CD137 antibodies show great potential in anti-cancer activities due to its ability to activate cytotoxic T cells and to increase the production of interferon gamma (IFN-y).
• IFN-y interferon gamma
• rituximab was approved as the first monoclonal antibody (mAb) for the treatment of cancer in 1997, several mAbs have become standard of care for the treatment of both solid tumors and hematologic malignancies. Most of the approved mAbs (e.g., rituximab, trastuzumab, and cetuximab) target tumor-associated antigens on the surface of cancer cells and inhibit cell growth. Although several effective antibodies have emerged, long-term, durable responses remain elusive, and resistance and relapse remain major problems. Immunomodulatory antibodies have revolutionized cancer immunotherapy and helped gamer the breakthrough distinction.
• CD 137 is one of the TNF receptor family targets that have advanced into clinical trials.
• CD 137 regulates many immune cells, including CD4+ and CD8+ T cells, regulatory T cells (Treg), dendritic cells (DC), and natural killer (NK) cells.
• Treg regulatory T cells
• DC dendritic cells
• NK natural killer
• the present innovation is based on the discovery of anti-p95HER2 antibodies that show a superior activity to bind p95HER2-expressing cancer cells and of anti-CD73 antibodies that show a superior activity to neutralize the enzymatic activity of CD73 in the cell membranebound or soluble form.
• the present invention is further based on the discovery that bispecific antibodies targeting p95HER2 and CD 137 possess dual functions, antibody antibodydependent cell-mediated cytotoxicity (ADCC) and p95HER2-dependent T-cell activation, against p95HER-postive cancer cells, and that bispecific antibodies targeting CD73 and CD 137 possess extraordinary activity to boost T-cell effector function and inhibit tumor growth in vivo better than mono-treatment or combination treatment with each antibody.
• ADCC antibody antibodydependent cell-mediated cytotoxicity
• CD73 and CD 137 possess extraordinary activity to boost T-cell effector function and inhibit tumor growth in vivo better than mono-treatment or combination treatment with each antibody.
• the invention provides four antibodies or antigen binding fragments thereof: anti-p95HER2 antibody clone R4-3 (p95HER2 #R4-3), anti-p95HER2 antibody clone R4-5 (p95HER2 #R4-5), anti- p95HER2 antibody clone R4-11 (p95HER2 #R4- 11), anti- p95HER2 antibody clone R4-15 (p95HER2 #R4-15), anti- p95HER2 antibody clone R4-38 (p95HER2 #R4-38).
• the anti-p95HER2 antibodies provided herein include a heavy chain variable (VH) region including an amino acid sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO: 17, SEQ ID NO:25 and SEQ ID NO:33; and a light chain variable (VL) region including an amino acid sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO:2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO:26 and SEQ ID NO:34.
• VH heavy chain variable
• VL light chain variable
• the antibody or antigen binding fragment has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:2 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:3, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:4, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO:5; and (b) VL CDR-L1, CDR- L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:6, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:7, and wherein CDR-L3 includes an amino acid sequence having at least 80%
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NOV and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO: 10 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO: 11, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO: 12, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO: 13; and (b) VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO: 14, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO: 15, and wherein CDR-L3 includes an amino acid sequence having at least 80% identity
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO: 17 and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO: 18 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO: 19, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:20, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO:21; and (b) VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:22, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:23, and wherein CDR-L3 includes an amino acid sequence having at
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:25 and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:26 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:27, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:28, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO:29; and (b) VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:30, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:31, and wherein CDR-L3 includes an amino acid sequence having
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:33 and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:34 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:35, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:36, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO:37; and (b) VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:38, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:39, and wherein CDR-L3 includes an amino acid sequence
• the antibody includes an Fc domain.
• the Fc domain is an IgG, IgE, IgM, IgD, IgA, or IgY domain.
• the IgG domain is an IgG 1 , IgG2, IgG3, or IgG4 domain.
• the IgGl domain includes an amino acid sequence of SEQ ID NO:41.
• the IgGl domain includes point mutations compared to a wild-type IgGl domain that modify or enhance antibody-dependent cellular cytotoxicity (ADCC) and/ or complement-dependent cytotoxicity (CDC). In many aspects, point mutations include S241D/A332L/I334E mutations.
• the invention provides a bispecific antibody including a first antigen binding region and a second antigen binding region, wherein the first antigen binding region binds to p95HER2.
• the bispecific antibody includes a VH region including complementarity-determining region (CDR)-Hl, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 19, CDR-H2 is set forth in SEQ ID NO:20, and CDR-H3 is set forth in SEQ ID NO:21 or sequences having 90% identity to SEQ ID NO: 19, 20 or 21 and the antigen binding specificity thereof; and a VL region including CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO:22, CDR-L2 is set forth in SEQ ID NO:23, and CDR-L3 is set forth in SEQ ID NO:24 or sequences having 90% identity to SEQ ID NO: 19, 20 or 21 and the antigen binding specificity thereof.
• CDR complementarity-determining region
• the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 17 and an antigen binding specificity of SEQ ID NOs: 19-21 and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 18 and an antigen binding specificity of SEQ ID NOs:22-24.
• the second antigen binding region binds to an immune checkpoint molecule, or an immune stimulatory molecule.
• the antibody includes a heavy chain sequence having the amino acid sequence of SEQ ID NO:46 or 47 or a sequence having 90% identity thereto and the binding specificity thereof.
• the bispecific antibody further includes a light chain having the amino acid sequence of SEQ ID NO: 18.
• the bispecific antibody includes an Fc domain.
• the Fc domain is an IgG domain, an IgE domain, an IgM domain, and IgD domain, an IgA domain, or an IgY domain.
• the Fc domain is an IgG domain.
• the IgG domain is an IgGl domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain.
• a scFv is linked to the C- terminus of the Fc domain.
• the bispecific antibody includes a linker between the Fab domain and the scFv domain.
• the Fab fragment is linked to the N- terminus of the Fc domain.
• the Fab includes a p95HER2 binding site
• the scFv includes a CD 137 binding site.
• the invention provides an antibody-drug conjugate including: a therapeutic agent, and any one of the antibodies or antigen binding fragments thereof described herein, or any one of the bispecific antibodies described herein.
• the therapeutic agent is covalently linked to the antibody, antigen binding fragment or bispecific antibody via a linker.
• the invention provides a pharmaceutical composition including any one of the antibodies or antigen binding fragments thereof described herein, any one of the bispecific antibodies described herein or any one of the antibody-drug conjugates described herein, and at least one pharmaceutical acceptable carrier.
• the pharmaceutically acceptable carrier is conjugated to the C- terminus of the C-terminus of the amino acid sequence of the antibody, antigen binding fragment, bispecific antibody, or antibody-drug conjugate.
• the invention provides a method of treating cancer including administering to a subject in need thereof an effective amount of any one of the antibodies or antigen binding fragments thereof described herein, any one of the bispecific antibodies described herein, or any one of the antibody-drug conjugates described herein, thereby treating cancer.
• the cancer is selected from prostate cancer, lung cancer, Non-Small Cell Lung Cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC), ovarian cancer, kidney cancer, urinary bladder cancer, uterine cancer, cervical cancer, ovarian cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, oral cavity cancer, pharynx cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, or leukemia.
• NSCLC Non-Small Cell Lung Cancer
• RRCC renal cell carcinoma
• the invention provides an isolated amino acid sequence as set forth in SEQ ID Nos: 1-42 or 46-47.
• the invention provides an isolated nucleic acid sequence encoding any one of the antibodies or antigen-binding fragments thereof, or the bispecific antibodies described herein. In another embodiment, the invention provides an isolated nucleic acid encoding any one of SEQ ID NOs: 1-42 or 46-47.
• the invention provides four antibodies or antigen binding fragments thereof: anti-CD73 antibody clone 3-4 (CD73 #3-4), anti-CD73 antibody clone 3-16 (CD73 #3-16), anti-CD73 antibody clone 3-1-28 (CD73 #3-1-28), and anti-CD73 antibody clone 3-1-37 (CD73 #3-1-37); and three affinity-maturated antibodies derived from anti-CD73 clone 3-1-28: anti-CD73 #3-1-28 (S51A Q54E), anti-CD73 #3-1-28 (S107D S109H), anti- CD73 #3-1-28 (S107D S109T A114S).
• the anti-CD73 antibodies provided herein include a heavy chain variable (VH) region including an amino acid sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO:49, SEQ ID NO:57, SEQ IDNO:65, and SEQ ID NO:73; and a light chain variable (VL) region including an amino acid sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:66 and SEQ ID NO:74, SEQ ID NO:81, SEQ ID NO:83 and SEQ ID NO:85.
• VH heavy chain variable
• VL light chain variable
• the antibody or antigen binding fragment has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:49 and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:50 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:51, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:52, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO: 53; and (b) VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:54, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:55, and wherein CDR-L3 includes an amino acid sequence having at
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:57 and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:58 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:59, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:60, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO:61; and (b) VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:62, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:63, and wherein CDR-L3 includes an amino acid sequence having
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:65 and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:66 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:67, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:68, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO: 69; and (b) VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:70, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:71, and wherein CDR-L3 includes an amino acid sequence
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:73 and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:74 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:75, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:76, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO: 77; and (b) VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:78, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:79, and wherein CDR-L3 includes an amino acid sequence
• the present invention provides three affinity- maturated antibodies derived from anti-CD73 clone 3-1-28: anti-CD73 #3-1-28 (S51A Q54E), anti-CD73 #3-1-28 (S107D S109H), anti-CD73 #3-1-28 (S107D S109T A114S).
• the anti-CD73 #3-1-28 (S51A Q54E) antibody or antigen binding fragment thereof having a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:81 includes VL CDR-L2 region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:82.
• the anti-CD73 #3-1-28 (S 107D S 109H) antibody or antigen binding fragment thereof having a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:83 includes VH CDR-H3 region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:84.
• the anti-CD73 #3-1-28 (S107D S109T Al 14S) antibody or antigen binding fragment thereof having a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:85 includes VH CDR-H3 region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:86.
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:65 and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:81 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:67, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:68, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO: 69; and (b) VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:70, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:82, and wherein CDR-L3 includes an amino acid sequence
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:65 and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:83 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:67, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:68, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO: 69; and (b) VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:70, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:84, and wherein CDR-L3 includes an amino acid sequence
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:65 and a VL region that includes an amino acid sequence having at least 80% identity to SEQ ID NO:85 includes (a) VH CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:67, wherein CDR-H2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:68, and wherein CDR-H3 includes an amino acid sequence having at least 80% identity to SEQ ID NO:69; and (b) VL CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 includes an amino acid sequence having at least 80% identity to SEQ ID NO:70, wherein CDR-L2 includes an amino acid sequence having at least 80% identity to SEQ ID NO:86, and wherein CDR-L3 includes an amino acid sequence
• antibodies or antigen binding fragments thereof provided herein include an Fc domain.
• the Fc domain is an IgG, IgE, IgM, IgD, IgA, or IgY domain.
• the IgG domain is an IgGl, IgG2, IgG3, or IgG4 domain.
• the IgGl domain includes an amino acid sequence of SEQ ID NO:87.
• IgGl includes point mutations compared to wild-type IgGl that modify or reduce antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). In many aspects, point mutations include K297A and K322A mutations.
• the invention provides a bispecific antibody including a first antigen binding region and a second antigen binding region, wherein the first antigen binding region binds to CD73.
• the bispecific antibody includes a VH region including complementarity-determining region (CDR)-Hl, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO:67, CDR-H2 is set forth in SEQ ID NO:68, and CDR-H3 is set forth in SEQ ID NO:69 or sequences having 90% identity to SEQ ID NO:67, 68 or 69 and the antigen binding specificity thereof; and a VL region including CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO:71, and CDR-L3 is set forth in SEQ ID NO:72 or sequences having 90% identity to SEQ ID NO:70, 71 or 72 and the antigen binding specificity thereof.
• CDR complementarity-determining region
• the bispecific antibody includes a VH region including complementarity-determining region (CDR)-H 1 , CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 67, CDR-H2 is set forth in SEQ ID NO:68, and CDR-H3 is set forth in SEQ ID NO:69 or sequences having 90% identity to SEQ ID NO:67, 68 or 69 and the antigen binding specificity thereof; and a VL region including CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO:70, CDR-L2 is set forth in SEQ ID NO: 82, and CDR-L3 is set forth in SEQ ID NO:72 or sequences having 90% identity to SEQ ID NO:70, 82 or 72 and the antigen binding specificity thereof.
• CDR complementarity-determining region
• the bispecific antibody includes a VH region including complementarity-determining region (CDR)-H1, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 67, CDR-H2 is set forth in SEQ ID NO:68, and CDR-H3 is set forth in SEQ ID NO:69 or sequences having 90% identity to SEQ ID NO:67, 68 or 69 and the antigen binding specificity thereof; and a VL region including CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO:70, CDR-L2 is set forth in SEQ ID NO:84, and CDR-L3 is set forth in SEQ ID NO:72 or sequences having 90% identity to SEQ ID NO:70, 84 or 72 and the antigen binding specificity thereof.
• CDR complementarity-determining region
• the bispecific antibody includes a VH region including complementarity-determining region (CDR)-Hl, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO:67, CDR-H2 is set forth in SEQ ID NO:68, and CDR-H3 is set forth in SEQ ID NO:69 or sequences having 90% identity to SEQ ID NO:67, 68 or 69 and the antigen binding specificity thereof; and a VL region including CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO:70, CDR-L2 is set forth in SEQ ID NO:86, and CDR-L3 is set forth in SEQ ID NO:72 or sequences having 90% identity to SEQ ID NO:70, 86 or 72 and the antigen binding specificity thereof.
• CDR complementarity-determining region
• the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO:65 and an antigen binding specificity of SEQ ID NOs:67-69 and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO:66 and an antigen binding specificity of SEQ ID NOs:70-72.
• the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO:65 and an antigen binding specificity of SEQ ID NOs:67-69 and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO:81 and an antigen binding specificity of SEQ ID NOs:70, 82 and 72.
• the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 65 and an antigen binding specificity of SEQ ID NOs:67-69 and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO:82 and an antigen binding specificity of SEQ ID NOs:70, 84 and 72.
• the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO:65 and an antigen binding specificity of SEQ ID NOs:67-69 and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO:85 and an antigen binding specificity of SEQ ID NOs:70, 86 and 72.
• the second antigen binding region binds to an immune checkpoint molecule, or an immune stimulatory molecule.
• the antibody includes a heavy chain sequence having the amino acid sequence of SEQ ID NO:88, 89, 90 or 91 or a sequence having 90% identity thereto and the binding specificity thereof.
• the bispecific antibody further includes a light chain having the amino acid sequence of SEQ ID NO:66.
• the invention provides an antibody-drug conjugate including: a therapeutic agent, and any one of the antibodies or antigen binding fragments thereof described herein, or any one of the bispecific antibodies described herein.
• the invention provides a pharmaceutical composition including any one of the antibodies or antigen binding fragments thereof described herein, any one of the bispecific antibodies described herein, or anyone of the antibody-drug conjugates described herein, and at least one pharmaceutically acceptable carrier.
• the invention provides a method of treating cancer including administering to a subject in need thereof an effective amount of any one of the antibodies or antigen binding fragments thereof described herein, any one of the bispecific antibodies described herein, or any one of the antibody-drug conjugates described herein, thereby treating cancer.
• the invention provides an isolated amino acid sequence as set forth in SEQ ID NOs:49-86 or 88-91.
• the invention provides an isolated nucleic acid sequence encoding any one of the antibodies or antigen-binding fragments thereof, or the bispecific antibodies described herein. In another embodiment, the invention provides an isolated nucleic acid encoding any one of SEQ ID NOs:49-86 or 88-91.
• FIGURES 1A-1C show the screening for phage clones targeted to p95HER2 by flow cytometer.
• the phage clones that recognize p95HER2 were enriched after four rounds of panning. Positive phage binders were selected by staining p95HER2-HEK293 cells with phage-containing supernatant.
• FIGURE 1A illustrates phage clones with specific binding to p95HER2-HEK293 cells were determined by flow cytometer.
• FIGURE IB illustrates clones (as numbered 1 to 40) specifically bound to p95HER2-HEK293 cells with AMFI higher than 800 were selected for sequencing. Negative control (NC), stained with secondary antibody alone.
• NC Negative control
• FIGURE 1C illustrates phage clones with distinct sequences (marked in bold with grey background) were selected for conversion to monoclonal antibodies. Clones were named as R4-X, where X is the number in FIGURE 1A.
• FIGURE 2 shows the purity of Protein A-purified anti- p95HER2 antibody leads by SDS- PAGE. The purity and integrity of one-step Protein A-purified anti- p95HER2 antibody leads was analyzed by SDS-PAGE under non-reducing and reducing conditions.
• FIGURES 3A-3B show the binding activity of purified anti-p95HER2 antibody leads by flow cytometer.
• FIGURE 3A is a graph illustrating that P95HER2-expressing HEK293 cells were surface stained with anti-p95HER2 antibody (30, 3, 0.3 nM) on ice for Ice then incubated with A488-conjugated anti-human IgG secondary antibody on ice for additional Bit.
• FIGURE 3B is a graph illustrating that P95HER2-expressing NCI-H292 stable cells were surface stained with selected anti-p95HER2 antibodies and the analyzed by flow cytometer. Binding curve and EC50 were shown.
• FIGURES 4A-4B show the binding epitope mapping of anti-p95HER2 antibody leads.
• FIGURE 4A illustrates that anti-p95HER2 #R4-3, R4-5, R4-11, R4-15 and R4-38 antibodies showed widely identical responses against peptides with the consensus motif KFPDE at moderate to high spot intensities and high signal-to-noise ratios. Even though the intensity levels did not vary remarkably, antibody R4-3 showed a slightly weaker and antibody R4-38 the strongest response of these antibodies.
• FIGURE 4B is a heat map showing anti- p95HER2 antibodies binding to linear 5, 10 and 15 amino acid peptides with peptide-peptide overlaps. Binding intensity was shown for each peptide, corrected with its own background value.
• FIGURE 5 shows the symmetric format of an anti- p95HER2-CD137 bispecific antibody (bsAb).
• FIGURES 6A-6B show the protein aggregation and integrity of anti-p95HER2- CD137 bsAb by SEC-HPLC and pCE-SDS-PAGE, respectively.
• FIGURE 6A shows the analysis of protein aggregation by SEC-HPLC.
• FIGURE 6B shows the analysis of protein integrity by pCE-SDS-PAGE under non-reducing and reducing conditions, and the data were summarized in the table below.
• FIGURES 7A-7B show the binding affinity of anti-p95HER2-CD137 bsAb against p95HER2 and CD 137 antigen by biolayer interferometry.
• FIGURE 7A illustrates binding kinetics against human p95HER2 antigen.
• FIGURE 7B illustrates binding kinetics against human CD 137 antigen.
• FIGURE 8 shows the simultaneous binding to p95HER2 and CD 137 antigens by anti-p95HER2-CD137 bsAb.
• FIGURES 9A-9B show that anti-p95HER2-CD137 bsAb induces IFN-y production of T-cells upon coculturing with p95HER2-expressing NCI-H292 cells.
• FIGURE 9A illustrates the expression level of p95HER2 on p95HER2-expressing NCI-H292 cells.
• FIGURE 9B illustrates IFN-y production of T cells upon coculturing with p95HER2-expressin NCI-H292 lung cancer cells.
• FIGURES 10A-10B show the antibody-dependent cell-mediated cytotoxicity of anti-p95HER2-CD137 bsAb p95HER2-expressing NCI-H292 cells.
• FIGURE 10A illustrates the expression level of CD 137 on CD137-expressing HEK293 cells (left). ADCC of anti- p95HER2-CD137 bsAbs with normal or ADCC-enhancing IgGl format (right).
• FIGURE 10B illustrates the expression level of p95HER2 on p95HER2-expressing NCI-H292 cells (left).
• Anti-p95HER2-CD137 bsAb with normal IgGl format induced a dose-dependent ADCC as well as #R4-11 IgGl monoclonal antibody (right).
• FIGURES 11A-11C show the anti-tumor activity of anti-p95HER2-CD137 bsAb by MDA-MB-231 breast cancer model in vivo.
• FIGURE 11B illustrates tumor volume.
• FIGURE 11C illustrates tumor mass.
• FIGURE 12 shows the pharmacokinetic profiles of anti-p95HER2-CD137 bsAb in vivo.
• FIGURES 13A-13C shows the screening for phage clones targeted to CD73 by ELISA.
• FIGURE 13A shows ELISA results of phage clones isolated from third-round MDA- MB-231 cell-based panning.
• FIGURE 13B shows ELISA results of phage clones isolated from third-round CD73 -overexpressing Expi293 cell-based panning in the third round.
• FIGURE 13C shows a list of clone name numbered in FIGURE 13A and 13B.
• FIGURE 14 shows the purity of Protein A-purified anti-CD73 antibody leads by SDS- PAGE.
• FIGURE 15 shows the binding of anti-CD73 antibody to CD73 -expressing MDA- MB-231 breast cancer cells.
• FIGURE 16 shows the blockade of CD73 enzymatic activity by anti-CD73 antibody.
• FIGURES 17A-17C show the screening of affinity-maturated clones derived from anti-CD73 clone 3-1-28 antibody.
• FIGURE 17A illustrates CD73 binding activities of phage clones.
• FIGURE 17B illustrates binding activities of antibodies converted from phage clones numbered in FIGURE 17A.
• FIGURE 17C illustrates binding kinetics of antibodies selected in FIGURE 17B.
• FIGURES 18A-18B show the blockade of membrane -bound and soluble CD73 enzymatic activity by affinity-maturated anti-CD73#3-l-28 antibodies.
• FIGURE 18A shows membrane -bound CD73 expressed by MDA-MB-231 cells.
• FIGURE 18B shows soluble CD73 secreted by MDA-MB-231 cells.
• FIGURE 19 shows the symmetric format of an anti-CD73-CD137 bispecific antibody (bsAb).
• FIGURES 20A-20B show the protein aggregation and integrity of anti-CD73- CD137 bsAb by SEC-HPLC and pCE-SDS-PAGE, respectively.
• FIGURE 20A illustrates the analysis of protein aggregation by SEC-HPLC.
• FIGURE 20B illustrates Analysis of protein integrity by pCE-SDS-PAGE under non-reducing and reducing conditions.
• FIGURES 21A-21B show the binding affinity of anti-CD73-CD137 bsAb against CD73 and CD 137 antigen, respectively measured by biolayer interferometry.
• FIGURE 21A shows binding kinetics against human CD73 antigen.
• FIGURE 21B shows binding kinetics against human CD 137 antigen.
• FIGURE 22 shows the simultaneous binding of CD73 and CD 137 antigens by anti- CD73-CD 137 bsAb.
• FIGURE 23 shows the blockade of CD73 enzymatic activity by anti-CD73-CD 137 bsAb.
• Anti-CD73-CD137 bsAb showed a comparable efficacy to inhibit CD73 enzymatic activity to parental anti-CD73 antibody.
• FIGURE 24 shows the rescue of AMP-suppressed T-cell activation by anti-CD73-
• CD 137 bsAb CD 137 bsAb.
• FIGURES 25A-25C show that anti-CD73-CD137 bsAb induces IFN-y production of T-cells upon coculturing with CD73 -expressing cancer cells.
• FIGURE 25A illustrates the expression level of CD73 on MDA-MB-231 and NCI-H292 cells.
• FIGURE 25B illustrates IFN-y production of T-cells upon coculturing with CD73-expressing MDA-MB-231, breast cancer cells.
• FIGURE 25C illustrates IFN-y production of T-cells upon coculturing with CD73-expressing NCI-H292, lung cancer cells.
• FIGURES 26A-26E show the anti-tumor activity of anti-CD73-CD137 bsAb by MDA-MB-231 breast cancer model in vivo.
• FIGURE 26A illustrates tumor volumes.
• FIGURE 26B illustrates tumor mass.
• FIGURE 26C illustrates percent of alive huCD45+ cells.
• FIGURE 26D illustrates percent of CD8+ T cells among CD3+ cells.
• FIGURE 26E illustrates percent of CD8+ T cells among CD4+ cells.
• FIGURE 27 shows the pharmacokinetic profiles of anti-CD73-CD137 bsAb in vivo.
• the present innovation is based on the discovery of anti-p95HER2 antibodies that show a superior activity to bind p95HER2-expressing cancer cells and of anti-CD73 antibodies that show a superior activity to neutralize the enzymatic activity of CD73 in the cell membranebound or soluble form.
• the present invention is further based on the discovery that bispecific antibodies targeting p95HER2 and CD 137 possess dual functions, antibody antibodydependent cell-mediated cytotoxicity (ADCC) and p95HER2-dependent T-cell activation, against p95HER-postive cancer cells, and that bispecific antibodies targeting CD73 and CD 137 possess extraordinary activity to boost T-cell effector function and inhibit tumor growth in vivo better than mono-treatment or combination treatment with each antibody.
• ADCC antibody antibodydependent cell-mediated cytotoxicity
• CD73 and CD 137 possess extraordinary activity to boost T-cell effector function and inhibit tumor growth in vivo better than mono-treatment or combination treatment with each antibody.
• the receptor tyrosine kinase HER2 (ErbB2) is one of four members (HER1- 4) of epidermal growth factor receptor family and overexpressed in different tumors, including 20-30% of breast and gastric cancers.
• HER1 -4 share a similar structure: an extracellular ligandbinding domain, a short hydrophobic transmembrane region and a cytoplasmic tyrosine kinase domain.
• Activation HER family through hetero/ homodimerization of receptors, induced by ligand binding or receptor overexpression and result in cell proliferation, differentiation, adhesion, migration and survival.
• a recombinant humanized monoclonal antibody, Trastuzumab binds to the extracellular domain of HER2 .
• Trastuzumab improves the relapse-free survival and overall survival in HER2 -positive early breast cancer patients.
• combination therapy of Trastuzumab and chemotherapy improves the survival and provides substantial clinical benefits in the HER2 -positive advanced breast cancer patients.
• only 20-30% of the HER2 -positive breast cancer patients respond to Trastuzumab when given as a monotherapy.
• the patients that do not response to Trastuzumab is caused by several potential mechanisms which lead to primary or acquired resistance, including activation the signaling of insulin-like growth factor receptor (IGF-1R), inactivation or loss the gene of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and accumulation of p95HER2.
• IGF-1R insulin-like growth factor receptor
• PTEN tensin homolog deleted on chromosome 10
• p95HER2 is the truncated forms of HER2 receptor which lack the extracellular trastuzumab-binding domain and about 30-40% of HER2-positive tumors express p95HER2. In some tumors, p95HER2 are the predominant HER2 forms. p95HER2 was generated by alternative initiation of translation from two methionine residues (611 and 687) or by proteolytic shedding of full-length HER2. The biologic role of p95HER2 has not been fully evaluated and the possible relationship between p95HER2 and full-length HER2 are unknown. p95HER2 possesses tyrosine kinase activity, which is essential for xenograft tumor growth in nude mice.
• p95HER2 Due to its exclusive expression in cancerous tissues, p95HER2 would be a good target for cancer therapy.
• a bispecific antibody that engages T cells and p95HER2-expressing cancer cells was developed and showed an effective antitumor activity in orthotopic MCF-7 breast cancer and PDX models.
• Bispecific antibodies BsAbs
• Blincyto® Blinatumomab
• Catumaxomab Removab®
• TAA of tumor cells and CD3 of T cells have been demonstrated anticancer efficacy and approved for the treatment of Philadelphia chromosome-negative B- acute lymphoblastic leukemia (B-ALL) and malignant ascites with ovarian cancer.
• B-ALL Philadelphia chromosome-negative B- acute lymphoblastic leukemia
• BiTE the poor efficacy in solid tumors and systemic cytokine release of BiTE limit their usages.
• anti-CD137 agonistic antibody may provide the better therapeutic window and safety profile as the proven efficacy against solid tumors in clinical trials and the crosslinking-dependent agonistic activity.
• novel anti-p95HER2 monoclonal antibodies that were generated and screened for the most potent binding affinity. Also provided herein are novel bispecific antibodies that simultaneously targeting p95HER2 and CD 137, to induce a superior T-cell activation in the presence of p95HER2.
• the invention provides antibodies and antigen binding fragments thereof that bind p95HER2.
• the invention provides four antibodies or antigen binding fragments thereof: anti-p95HER2 antibody clone R4-3 (p95HER2 #R4-3), anti-p95HER2 antibody clone R4-5 (p95HER2 #R4-5), anti- p95HER2 antibody clone R4-11 (p95HER2 #R4-11), anti- p95HER2 antibody clone R4-15 (p95HER2 #R4-15), anti- p95HER2 antibody clone R4-38 (p95HER2 #R4-38).
• the anti-p95HER2 antibodies provided herein include a heavy chain variable (VH) region including an amino acid sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO: 1, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25 and SEQ ID NO:33; and a light chain variable (VL) region including an amino acid sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO:2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO:26 and SEQ ID NO:34.
• VH heavy chain variable
• VL light chain variable
• antibody refers to an immunoglobulin molecule that has the ability to specifically bind to an antigen.
• the term “antibody” includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, and anti-idiotypic antibodies, unless context clearly indicates otherwise.
• antibodies provided herein include monoclonal antibodies.
• Antibodies provided herein include any isotype and class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).
• antigen binding fragment means a fragment or portion of an immunoglobulin molecule or antibody that has the ability to specifically bind to the same antigen as the immunoglobulin molecule or antibody.
• exemplary antigen binding fragments include scFv, Fab, or F(ab)2 fragments.
• antigen binding region means the part of an antibody or immunoglobulin molecule that binds to antigens or proteins by contacting the antigen or protein, for example.
• An antigen binding region generally includes heavy chain variable (VH) regions and light chain variable (VL) regions.
• An antigen binding region generally includes one or more antigen binding sites or paratopes.
• Antibodies provided herein have a VH region including an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to a sequence of SEQ ID NO:1; SEQ ID NOV; SEQ ID NO:17; SEQ ID NO:25 or SEQ ID NO: 33.
• Antibodies provided herein also include an VL region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to a sequence of SEQ ID NO:2; SEQ ID NO: 10; SEQ ID NO: 18; SEQ ID NO:26 SEQ ID NO:34.
• sequence identity or “sequence homology,” which can be used interchangeably, refer to an exact nucleotide -to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
• techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby or the amino acid sequence of a polypeptide and comparing these sequences to a second nucleotide or amino acid sequence.
• percent (%) sequence identity or “percent (%) identity,” also including “homology,” refers to the percentage of amino acid residues or nucleotides in a sequence that are identical with the amino acid residues or nucleotides in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
• two or more sequences can be compared by determining their “percent identity,” also referred to as “percent homology.”
• the percent identity to a reference sequence e.g., nucleic acid or amino acid sequences
• the percent identity to a reference sequence may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100.
• Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc.
• BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the sequences being compared.
• Default parameters are provided to optimize searches with short query sequences, for example, with the blastp program.
• the program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values in between. Percent identities between a reference sequence and a claimed sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. In general, an exact match indicates 100% identity over the length of the reference sequence.
• Additional programs and methods for comparing sequences and/or assessing sequence identity include the Needleman- Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/, optionally with default settings), the Smith- Waterman algorithm (see, e.g., the EMBOSS Water aligner available at www.ebi.ac.uk/Tools/psa/emboss_water/, optionally with default settings), the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci.
• Needleman- Wunsch algorithm see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/, optionally with default settings
• the Smith- Waterman algorithm see, e.g., the EMBOSS Water aligner available at www.ebi.ac.uk/Tool
• reference to percent sequence identity refers to sequence identity as measured using BLAST (Basic Local Alignment Search Tool).
• ClustalW is used for multiple sequence alignment. Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters.
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:1 and a VL region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9%
• SEQ ID NO:1 provides an amino acid sequence that includes the variable region of anti-p95HER2 antibody clone #4-3 heavy chain.
• SEQ ID NO: 2 provides an amino acid sequence that includes the variable region of anti- p95HER2 clone #4-3 light chain.
• the antigen binding region of an antibody or antigen binding fragment thereof generally includes complementarity determining regions (CDRs).
• CDR complementarity determining region
• “Complementarity determining region (CDR)” refers to hypervariable regions of VH and VL. CDRs include the target protein or antigen binding site of an antibody that confers specificity for protein or antigen binding.
• VH and VL generally include three sequentially numbered CDRs.
• CDR-H1, CDR-H2, and CDR-H3 refer to three consecutively arranged CDRs of the heavy chain variable region (VH), as numbered from the N-terminus of the heavy chain polypeptide.
• CDR-L1, CDR-L2, and CDR-L3 refer to three consecutively arranged CDRs of the light chain variable region (VL), as numbered from the N-terminus of the light chain polypeptide.
• the antigen binding region of the antibody or antigen binding fragment thereof having a VH region that includes an amino acid sequence having at least about 80% identity to SEQ ID NO: 1 and a VL region that includes an amino acid sequence having at least about 80% identity to SEQ ID NO:2 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to
• the antigen binding region of the antibody or antigen binding fragment thereof having a VH region that includes an amino acid sequence having at least about 80% identity to SEQ ID NO: 1 and a VL region that includes an amino acid sequence having at least about 80% identity to SEQ ID NO:2 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:6, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at
• the antibody or antigen binding fragment thereof includes a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:9 and a VL region that includes an amino acid sequence having at least about 80%, identity at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity,
• SEQ ID NO:9 provides an amino acid sequence that includes the variable region of anti-p95HER2 antibody clone #4-5 heavy chain.
• SEQ ID NO: 10 provides an amino acid sequence that includes the variable region of anti-p95HER2 clone #4-5 light chain.
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NOV and a VL region including an amino acid sequence having at least about 80% identity to SEQ ID NO: 10 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO: 11, a CDR-H2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO: 9 and a VL region that includes an amino acid sequence having at least about 80%, identity to SEQ ID NO: 10 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO: 14, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity,
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO: 17 and a VL region that includes an amino acid sequence having at least about 80%, identity at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9%
• SEQ ID NO: 17 provides an amino acid sequence that includes the variable region of anti-p95HER2 antibody clone #4-11 heavy chain.
• SEQ ID NO: 18 provides an amino acid sequence that includes the variable region of anti-p95HER2 antibody clone #4-11 light chain.
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO: 17 and a VL region including an amino acid sequence having at least about 80% identity to SEQ ID NO: 18 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO: 19, a CDR-H2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93%
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO: 17 and a VL region including an amino acid sequence having at least about 80%, identity to SEQ ID NO: 18 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:22, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity,
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:25 and a VL region that includes an amino acid sequence having at least about 80%, identity at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9%
• SEQ ID NO:25 provides an amino acid sequence that includes the variable region of anti-p95HER2 antibody clone #4-15 heavy chain.
• SEQ ID NO: 26 provides an amino acid sequence that includes the variable region of anti-p95HER2 antibody clone #4-15 light chain.
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:25 and a VL region including an amino acid sequence having at least about 80% identity to SEQ ID NO:26 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:27, a CDR-H2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 9
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:25 and a VL region including an amino acid sequence having at least about 80%, identity to SEQ ID NO:26 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:30, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity,
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:33 and a VL region that includes an amino acid sequence having at least about 80%, identity at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9%
• SEQ ID NO:33 provides an amino acid sequence that includes the variable region of anti-p95HER2 antibody clone #4-38 heavy chain.
• SEQ ID NO: 34 provides an amino acid sequence that includes the variable region of anti-p95HER2 antibody clone #4-38 light chain.
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:33 and a VL region including an amino acid sequence having at least about 80% identity to SEQ ID NO:34 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:35, a CDR-H2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 9
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:33 and a VL region including an amino acid sequence having at least about 80%, identity to SEQ ID NO:34 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:38, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity,
• Antibodies or antigen binding fragments thereof provided herein further include an Fc domain.
• Fc domain refers to an antibody region that includes at least a hinge region, a CH2 domain, and a CH3 domain, unless context clearly indicates otherwise.
• the terms Fc domain and Fc region may be used interchangeably, unless context clearly indicates otherwise.
• the Fc domain is an IgG domain, an IgE domain, an IgM domain, and IgD domain, an IgA domain, or an IgY domain.
• Fc domains of any sequence and from any species can be used, including human, ape, monkey, mouse, rabbit, goat, sheep, guinea pig, horse, and others.
• Fc domains are engineered, i.e., non-naturally occurring or recombinant Fc domains generated using techniques of molecular biology, for example.
• the IgG domain is an IgG 1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain.
• Bispecific molecules such as bispecific antibodies (bsAbs)
• bsAbs bispecific antibodies
• mAbs monoclonal antibodies
• bispecific antibodies that include a first antigen binding region and a second antigen binding region.
• the first antigen binding region and the second antigen binding region specifically bind to different antigens or targets.
• the first antigen binding region and the second antigen binding region bind to different epitopes in the same antigen or target.
• the invention provides bispecific antibodies including a first antigen binding region and a second antigen binding region, wherein the first antigen binding region binds to p95HER2.
• the first antigen binding region includes a VH region that includes an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range in between, to a sequence selected from SEQ ID NO: 17 and a VL region that includes an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity
• the bispecific antibody includes a VH region including complementarity-determining region (CDR)-Hl, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO: 19, CDR-H2 is set forth in SEQ ID NO:20, and CDR-H3 is set forth in SEQ ID NO:21 or sequences having 90% identity to SEQ ID NO: 19, 20 or 21 and the antigen binding specificity thereof; and a VL region including CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO:22, CDR-L2 is set forth in SEQ ID NO:23, and CDR-L3 is set forth in SEQ ID NO:24 or sequences having 90% identity to SEQ ID NO: 19, 20 or 21 and the antigen binding specificity thereof.
• CDR complementarity-determining region
• the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO: 17 and an antigen binding specificity of SEQ ID NOs: 19-21 and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO: 18 and an antigen binding specificity of SEQ ID NOs:22-24.
• first and second antigen binding regions can be included in bispecific antibodies provided herein, including first and second antigen binding regions that bind to any immune checkpoint molecule, any immune stimulatory molecule, or any tumor antigen, for example.
• the second antigen binding region of bispecific antibodies provided herein binds to any immune checkpoint molecule, any immune stimulatory molecule, or any tumor antigen.
• the first and second antigen binding regions bind to the same molecule.
• the first and second antigen binding regions may bind to the same or to a different epitope of the same molecule.
• the first and second antigen binding regions bind to different molecules.
• bispecific antibodies having a first antigen binding region that binds to p95HER2 and a second antigen binding region that binds to CD 137 bind to p95HER2 and CD 137 simultaneously (FIGURE 8).
• restricting anti- p95HER2 binding activity to tumor sites that express p95HER2 by designing bispecific antibodies that are able to bind to both p95HER2 and CD 137 may reduce the risk of hepatotoxicity and its associated fatality seen in clinical trials with anti-CD137 antibodies such as Urelumab.
• anti-CD137 antibodies such as Urelumab.
• simultaneous binding to p95HER2 and CD 137 may enhance T-cell activation as a result of cross-linking (see also Example 8 below).
• first antigen binding regions and second antigen binding regions include an scFv, an F(ab)2, an Fab, or any combination thereof.
• the first antigen binding region includes an scFv and the second antigen binding region includes an Fab.
• an scFv included in bispecific antibodies provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen.
• an scFv included in bispecific antibodies provided herein binds to a CD137.
• an Fab included in bispecific antibodies provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen.
• an Fab included in bispecific antibodies provided herein binds to p95HER2.
• the scFv of bispecific antibodies provided herein includes an amino acid sequence of SEQ ID NO: 48.
• bispecific antibodies provided herein further include a linker between the VH region and the VL region of the scFv.
• Any linker can be used.
• linkers can include any amino acid sequence.
• Linkers can be of any length, such as one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, or more amino acids.
• Linkers can also include multiples of an amino acid sequence. Any number of multiples of an amino acid sequence can be included in a linker. Exemplary linker sequences are provided in SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO: 160.
• bispecific antibodies provided herein include a heavy chain sequence of SEQ ID NO:46. In another aspect, bispecific antibodies provided herein further include a light chain sequence of SEQ ID NO: 18. Heavy chain sequences such as SEQ ID NO:46, and others, can include one or more G linkers (SEQ ID NO:44), one or more G4S linkers (SEQ ID NO:45 or SEQ ID NO: 160), or any multiple of a G linker or G4S linker, although any other suitable linker can be used.
• Tumor hypoxia, chronic inflammation, and metabolic changes give rise to extracellular ATP accumulation that promote anti-tumor activity through inductions of inflammasome assembly and proinflammatory cytokines secretion.
• cytokines ectonucleotidases
• TGF-[3 higher levels of ectonucleotidases CD39 and CD73 usually observed in solid tumors compared to the corresponding non- malignant tissues.
• Extracellular accumulated ATP was sequentially hydrolyzed by tumor associated CD39 and CD73 to adenosine, then mediates it regulatory functions upon binding to adenosine receptors expressed by tumor cells, stromal cells, and infiltrating lymphocytes in tumors.
• adenosine signaling via A2A and A2B receptors promotes proliferation, metastasis, and secretion of vascular endothelial growth factor (VEGF).
• VEGF vascular endothelial growth factor
• CAFs cancer-associated fibroblasts
• FGF2 pro-tumor fibroblast growth factor 2
• CXCL12 pro-tumor fibroblast growth factor 2
• A2A signaling inhibits effector T cell activation through dampening proximal TCR signaling and concurrently inducing co- inhibitory molecules PD-1, LAG3, and TIM3 expression.
• A2A signaling promotes differentiation, proliferation, and CTLA-4 expression of murine Treg cells.
• NK cells A2A signaling blocks maturation and cytotoxicity.
• A2A and A2B signaling downregulate the expressions of antigen presentation and costimulatory molecules, while promotes M2 macrophage differentiation.
• High levels of CD73 were found in the malignant tissues of various cancer types, including pancreatic, gastrointestinal, renal and lung cancers, and corresponded to a poor prognosis. High levels of soluble CD73 were also observed in the circulation of patients with metastatic melanoma.
• CD73 has become an attractive therapeutic target for cancers.
• antitumor activities of small molecule inhibitors and antibodies target CD73 were attributed not only to the inhibition of tumor cell growth and migration, but the involvement of adaptive immunity as well.
• combine anti-CD73 antibody enhanced antitumor activities of antibodies against other immune regulatory molecules, including CTLA-4, PD-1, and CD 137.
• the synergized antitumor activity in the combination with anti- PD-1 antibody was attribute to the activation of A2A signaling that enhance PD-1 expression on tumor-specific CD8+ T.
• tumor resistance to anti-CD137 antibody therapy was referred to the upregulation of CD73 on tumor-infiltrating CD8+ T cells, therefore a more effective antitumor activity was elicited by the combination therapy of anti-CD137 and CD73 antibody.
• anti-CD73 antibodies including, MEDI9447 (oleclumab), CPI-006, BMS-986179, and NZV930, are undergoing Phase I/II in the monotherapy or combinational therapies.
• the partial response was observed in 4.8% (1/21) patients with colon rectal cancer, and in 10% (2/20) patients with pancreatic cancer by treating with oleclumab and durvalumab (anti-PD-Ll antibody).
• the partial response was observed in 13.5% (7/52) patients with head and neck, pancreatic, prostate, anal, and renal cancer by the combination of BMS-986179 and nivolumab (anti-PD-1 antibody).
• novel anti-CD73 monoclonal antibodies that were generated and screened for the most potent binding affinity and enzymatic blockade activity. Also provided herein are a novel bispecific antibody that simultaneously targeting CD73 and CD 137 or PD-L1, to induce a superior T-cell activation upon coculturing with CD73+ tumor cells and antitumor activity in vivo.
• the invention provides antibodies and antigen binding fragments thereof that bind CD73.
• the invention provides four antibodies or antigen binding fragments thereof: anti-CD73 antibody clone 3-4 (CD73 #3-4), anti-CD73 antibody clone 3-16 (CD73 #3-16), anti-CD73 antibody clone 3-1-28 (CD73 #3-1-28), and anti-CD73 antibody clone 3-1-37 (CD73 #3-1-37).
• the invention provides three affinity-maturated antibodies derived from anti-CD73 clone 3-1-28: anti-CD73 #3-1-28 (S51A Q54E), anti-CD73 #3-1-28 (S107D S109H), anti-CD73 #3-1-28 (S107D S109T A114S).
• the anti-CD73 antibodies provided herein include a heavy chain variable (VH) region including an amino acid sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:65 and SEQ ID NO:73; and a light chain variable (VL) region including an amino acid sequence with at least 80% sequence identity to a sequence selected from SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:81, SEQ ID NO:83 and SEQ ID NO:85.
• VH heavy chain variable
• VL light chain variable
• Antibodies provided herein have a VH region including an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to a sequence of SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:65, and SEQ ID NO:73.
• Antibodies provided herein also include an VL region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to a sequence of SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:66 and SEQ ID NO:74, SEQ ID NO:81, SEQ ID NO:83 and SEQ ID NO:85.
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:49 and a VL region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9%
• the antigen binding region of the antibody or antigen binding fragment thereof having a VH region that includes an amino acid sequence having at least about 80% identity to SEQ ID NO:49 and a VL region that includes an amino acid sequence having at least about 80% identity to SEQ ID NO:50 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:51, a CDR-H2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about
• the antigen binding region of the antibody or antigen binding fragment thereof having a VH region that includes an amino acid sequence having at least about 80% identity to SEQ ID NO:49 and a VL region that includes an amino acid sequence having at least about 80% identity to SEQ ID NO:50 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:54, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity,
• the antibody or antigen binding fragment thereof includes a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:57 and a VL region that includes an amino acid sequence having at least about 80%, identity at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity,
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:57 and a VL region including an amino acid sequence having at least about 80% identity to SEQ ID NO:58 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:59, a CDR-H2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 9
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:57 and a VL region that includes an amino acid sequence having at least about 80%, identity to SEQ ID NO:58 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:62, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:65 and a VL region that includes an amino acid sequence having at least about 80%, identity at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9%
• SEQ ID NO:65 provides an amino acid sequence that includes the variable region of anti-CD73 antibody clone #3-1-28 heavy chain.
• SEQ ID NO:66 provides an amino acid sequence that includes the variable region of anti-CD73 clone #3-1-28 light chain.
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region including an amino acid sequence having at least about 80% identity to SEQ ID NO:66 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:67, a CDR-H2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 9
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region including an amino acid sequence having at least about 80%, identity to SEQ ID NO:66 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:70, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity,
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:73 and a VL region that includes an amino acid sequence having at least about 80%, identity at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9%
• SEQ ID NO:73 provides an amino acid sequence that includes the variable region of anti-CD73 antibody clone #3-1-37 heavy chain.
• SEQ ID NO:74 provides an amino acid sequence that includes the variable region of anti-CD73 clone #3-1-37 light chain.
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:73 and a VL region including an amino acid sequence having at least about 80% identity to SEQ ID NO:74 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:75, a CDR-H2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 9
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:73 and a VL region including an amino acid sequence having at least about 80%, identity to SEQ ID NO:74 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:78, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity,
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:65 and a VL region that includes an amino acid sequence having at least about 80%, identity at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9%
• SEQ ID NO:65 provides an amino acid sequence that includes the variable region of anti-CD73 antibody clone #3-1-28 heavy chain.
• SEQ ID NO:81 provides an amino acid sequence that includes the variable region of anti-CD73 clone #3-1-28 (S51A Q54E) light chain.
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region including an amino acid sequence having at least about 80% identity to SEQ ID NO:81 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:67, a CDR-H2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 9
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region including an amino acid sequence having at least about 80%, identity to SEQ ID NO:81 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:70, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity,
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:65 and a VL region that includes an amino acid sequence having at least about 80%, identity at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9%
• SEQ ID NO:65 provides an amino acid sequence that includes the variable region of anti-CD73 antibody clone #3-1-28 heavy chain.
• SEQ ID NO:83 provides an amino acid sequence that includes the variable region of anti-CD73 clone #3-1-28 (S107D S109H) light chain.
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region including an amino acid sequence having at least about 80% identity to SEQ ID NO:83 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:67, a CDR-H2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 9
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region including an amino acid sequence having at least about 80%, identity to SEQ ID NO:83 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:70, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity,
• the antibody or antigen binding fragment thereof has a VH region that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91 % identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:65 and a VL region that includes an amino acid sequence having at least about 80%, identity at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9%
• SEQ ID NO:65 provides an amino acid sequence that includes the variable region of anti-CD73 antibody clone #3-1-28 heavy chain.
• SEQ ID NO:85 provides an amino acid sequence that includes the variable region of anti-CD73 clone #3-1-28 (S107D S109T Al 14S) light chain.
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region including an amino acid sequence having at least about 80% identity to SEQ ID NO:85 has a CDR-H1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:67, a CDR-H2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 9
• the antigen binding region of the antibody or antigen binding fragment thereof that includes a VH region including an amino acid sequence having at least about 80% identity to SEQ ID NO:65 and a VL region including an amino acid sequence having at least about 80%, identity to SEQ ID NO:85 has a CDR-L1 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, at least about 99.9% identity, and any number or range in between, to SEQ ID NO:70, a CDR-L2 that includes an amino acid sequence having at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity,
• Antibodies or antigen binding fragments thereof provided herein further include an Fc domain.
• the Fc domain is an IgG domain, an IgE domain, an IgM domain, and IgD domain, an IgA domain, or an IgY domain.
• the IgG domain is an IgG 1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain.
• bi-functional proteins including bispecific antibodies.
• bi-functional protein refers to a protein that has at least two functions.
• a non-limiting example of a bi-functional protein includes a bispecific antibody that is capable of binding to two antigens.
• Bispecific antibodies provided herein can include isolated, functional scFv fragments that bind to CD 137 and that are fused to the C-terminus of the Fc domain of an anti-CD73 antibody, for example.
• a C-terminally positioned scFv that binds CD 137 in fusion constructs provided herein is fused to an Fc domain of an antibody that binds to other immunoregulatory molecules, such as CD40 or CTLA-4, for example.
• a C-terminally positioned scFv can bind to an immunoregulatory molecule, such as CD40 or CTLA-4, for example.
• the invention provides a bispecific antibody including a first antigen binding region and a second antigen binding region, wherein the first antigen binding region binds to CD 73.
• the first antigen binding region includes a VH region that includes an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range in between, to a sequence selected from SEQ ID NO:65 and a VL region that includes an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99%
• the bispecific antibody includes a VH region including complementarity-determining region (CDR)-Hl, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO:67, CDR-H2 is set forth in SEQ ID NO:68, and CDR-H3 is set forth in SEQ ID NO:69 or sequences having 90% identity to SEQ ID NO:67, 68 or 69 and the antigen binding specificity thereof; and a VL region including CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO:71, and CDR-L3 is set forth in SEQ ID NO:72 or sequences having 90% identity to SEQ ID NO:70, 71 or 72 and the antigen binding specificity thereof.
• CDR complementarity-determining region
• the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO:65 and an antigen binding specificity of SEQ ID NOs:67-69 and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO:66 and an antigen binding specificity of SEQ ID NOs:70-72.
• the first antigen binding region includes a VH region that includes an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range in between, to a sequence selected from SEQ ID NO: 65 and a VL region that includes an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range in between, to about 100 to 120 amino acids of an N-terminal sequence of a sequence selected from SEQ ID NO
• the bispecific antibody includes a VH region including complementarity-determining region (CDR)-Hl, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO:67, CDR-H2 is set forth in SEQ ID NO:68, and CDR-H3 is set forth in SEQ ID NO:69 or sequences having 90% identity to SEQ ID NO:67, 68 or 69 and the antigen binding specificity thereof; and a VL region including CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO: 70, CDR-L2 is set forth in SEQ ID NO: 84, and CDR-L3 is set forth in SEQ ID NO:72 or sequences having 90% identity to SEQ ID NO:70, 84 or 72 and the antigen binding specificity thereof.
• CDR complementarity-determining region
• the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO:65 and an antigen binding specificity of SEQ ID NOs:67-69 and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO:83 and an antigen binding specificity of SEQ ID NOs:70, 84 and 72.
• the first antigen binding region includes a VH region that includes an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range in between, to a sequence selected from SEQ ID NO: 65 and a VL region that includes an amino acid sequence having at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.9% identity, and any number or range in between, to about 100 to 120 amino acids of an N-terminal sequence of a sequence selected from SEQ ID NO
• the bispecific antibody includes a VH region including complementarity-determining region (CDR)-Hl, CDR-H2, and CDR-H3, wherein CDR-H1 is set forth in SEQ ID NO:67, CDR-H2 is set forth in SEQ ID NO:68, and CDR-H3 is set forth in SEQ ID NO:69 or sequences having 90% identity to SEQ ID NO:67, 68 or 69 and the antigen binding specificity thereof; and a VL region including CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 is set forth in SEQ ID NO:70, CDR-L2 is set forth in SEQ ID NO:85, and CDR-L3 is set forth in SEQ ID NO:72 or sequences having 90% identity to SEQ ID NO:70, 86 or 72 and the antigen binding specificity thereof.
• CDR complementarity-determining region
• the VH region has an amino acid sequence having at least 80% identity to SEQ ID NO:65 and an antigen binding specificity of SEQ ID NOs:67-69 and the VL region has an amino acid sequence having at least 80% identity to SEQ ID NO:85 and an antigen binding specificity of SEQ ID NOs:70, 86 and 72.
• the second antigen binding region of bispecific antibodies provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen.
• first and second antigen binding regions can be included in bispecific antibodies provided herein, including first and second antigen binding regions that bind to any immune checkpoint molecule, any immune stimulatory molecule, or any tumor antigen, for example.
• the second antigen binding region of bispecific antibodies provided herein binds to any immune checkpoint molecule, any immune stimulatory molecule, or any tumor antigen.
• the first and second antigen binding regions bind to the same molecule.
• the first and second antigen binding regions may bind to the same or to a different epitope of the same molecule.
• the first and second antigen binding regions bind to different molecules.
• bispecific antibodies having a first antigen binding region that binds to CD73 and a second antigen binding region that binds to CD 137 bind to CD73 and CD 137 simultaneously (FIGURE 22).
• restricting anti-CD73 binding activity to tumor sites that express CD73 by designing bispecific antibodies that are able to bind to both CD73 and CD 137 may reduce the risk of hepatotoxicity and its associated fatality seen in clinical trials with anti-CD137 antibodies such as Urelumab.
• anti-CD137 antibodies such as Urelumab.
• simultaneous binding to CD73 and CD 137 may enhance T-cell activation as a result of cross-linking (see also Example 21 below).
• first antigen binding regions and second antigen binding regions include an scFv, an F(ab)2, an Fab, or any combination thereof.
• the first antigen binding region includes an scFv and the second antigen binding region includes an Fab.
• an scFv included in bispecific antibodies provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen.
• an scFv included in bispecific antibodies provided herein binds to a CD137.
• an Fab included in bispecific antibodies provided herein binds to an immune checkpoint molecule, an immune stimulatory molecule, or a tumor antigen.
• an Fab included in bispecific antibodies provided herein binds to CD73.
• the antibody includes a heavy chain sequence having the amino acid sequence of SEQ ID NO:88, 89, 90 or 91 or a sequence having 90% identity thereto and the binding specificity thereof.
• the bispecific antibody further includes a light chain having the amino acid sequence of SEQ ID NO:66.
• the scFv of bispecific antibodies provided herein includes an amino acid sequence of SEQ ID NO:91.
• bispecific antibodies provided herein further include a linker between the VH region and the VL region of the scFv.
• Any linker can be used.
• linkers can include any amino acid sequence.
• Linkers can be of any length, such as one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, or more amino acids.
• Linkers can also include multiples of an amino acid sequence. Any number of multiples of an amino acid sequence can be included in a linker.
• bispecific antibodies provided herein include a heavy chain sequence of SEQ ID NO:88, SEQ ID NO:89, or SEQ ID NO:91.
• bispecific antibodies provided herein further include a light chain sequence of SEQ ID NO:66 or SEQ ID NO:81.
• Heavy chain sequences such as SEQ ID NO:88, SEQ IDNO:89, SEQ IDNO:91, and others, can include one or more G linkers (SEQ ID NO:44), one or more G4S linkers (SEQ ID NO:45 and SEQ ID NO: 160), or any multiple of a G linker or G4S linker, although any other suitable linker can be used.
• G linkers SEQ ID NO:44
• G4S linkers SEQ ID NO:45 and SEQ ID NO: 160
• any multiple of a G linker or G4S linker although any other suitable linker can be used.
• the invention provides an antibody-drug conjugate including: a therapeutic agent, and any one of the antibodies or antigen binding fragments thereof described herein, or any one of the bispecific antibodies described herein.
• the antibody, antigen binding fragment thereof or the bispecific antibody include anti-p95HER2 antibody, antigen binding fragment thereof, bispecific antibody targeting p95HER2 and a second target, anti-CD73 antibody, antigen binding fragment thereof, and bispecific antibody targeting CD73 and a second target, such as those described herein.
• Immunoconjugates can also be defined by the generic formula in reverse order: A-L-C.
• the therapeutic agent is covalently linked to the antibody, antigen binding fragment or bispecific antibody via a linker.
• therapeutic agent any drug, compound or small molecule, useful for the treatment of cancer, that can be linked to the binding agent described herein.
• the therapeutic agent is a chemotherapeutic agent.
• Chemotherapeutic and antineoplastic agents are well known cytotoxic agents, and include but are not limited to: (i) anti-microtubules agents comprising vinca alkaloids (vinblastine, vincristine, vinflunine, vindesine, and vinorelbine), taxanes (cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel), epothilones (ixabepilone), and podophyllotoxin (etoposide and teniposide); (ii) antimetabolite agents comprising anti-folates (aminopterin, methotrexate, pemetrexed, pralatrexate, and raltitrexed
• Derivatives of these compounds include epirubicin and idarubicin; pirarubicin, aclarubicin, and mitoxantrone, bleomycins, mitomycin C, mitoxantrone, and actinomycin; (vi) enzyme inhibitors agents comprising FI inhibitor (Tipifamib), CDK inhibitors (Abemaciclib, Alvocidib, Palbociclib, Ribociclib, and Seliciclib), PrI inhibitor (Bortezomib, Carfilzomib, and Ixazomib), Phi inhibitor (Anagrelide), IMPDI inhibitor (Tiazofurin), LI inhibitor (Masoprocol), PARP inhibitor (Niraparib, Olaparib, Rucaparib), HDAC inhibitor (Belinostat, Panobinostat, Romidepsin, Vorinostat), and PIKI inhibitor (Idelalisib); (vii) receptor antagonist agent comprising ERA receptor antagonist (Atra
• the invention provides a pharmaceutical composition including any one of the antibodies or antigen binding fragments thereof described herein, any one of the bispecific antibodies described herein or any one of the antibody-drug conjugates described herein, and at least one pharmaceutical acceptable carrier.
• the pharmaceutical composition includes an anti-p95HER2 antibody, an antigen binding fragment thereof, a bispecific antibody targeting p95HER2 and a second target, an antibody-drug conjugate thereof, an anti-CD73 antibody, an antigen binding fragment thereof, a bispecific antibody targeting CD73 and a second target, or an antibodydrug conjugate thereof, such as those described herein.
• “pharmaceutical composition” refers to a formulation comprising an active ingredient, and optionally one or more pharmaceutically acceptable carriers, diluents or excipients.
• active ingredient can interchangeably refer to an “effective ingredient” and is meant to refer to any agent that is capable of inducing a sought-after effect upon administration.
• the active ingredient includes a biologically active molecule.
• biologically active molecule refers to a molecule that has a biological effect in a cell.
• the active molecule an antibody, an antibody fragment, a bispecific antibody or an antibody-drug conjugate, such as those described herein or a morpholino.
• pharmaceutically acceptable it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof, nor to the activity of the active ingredient of the formulation.
• Pharmaceutically acceptable carriers, excipients or stabilizers are well known in the art, for example Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).
• Pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
• carrier examples include, but are not limited to, liposome, nanoparticles, ointment, micelles, microsphere, microparticle, cream, emulsion, and gel.
• excipient examples include, but are not limited to, anti-adherents such as magnesium stearate, binders such as saccharides and their derivatives (sucrose, lactose, starches, cellulose, sugar alcohols and the like) protein like gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate and parabens.
• anti-adherents such as magnesium stearate
• binders such as saccharides and their derivatives (sucrose, lactose, starches, cellulose, sugar alcohols and the like) protein like gelatin and synthetic polymers
• the pharmaceutically acceptable carrier is conjugated to the C- terminus of one or more polypeptides of the antibody or antigen binding fragment. Any suitable means of conjugating the pharmaceutically acceptable carrier can be used, including covalent conjugation and use of linkers, for example.
• the invention provides a method of treating cancer including administering to a subject in need thereof an effective amount of any one of the antibodies or antigen binding fragments thereof described herein, any one of the bispecific antibodies described herein, or any one of the antibody-drug conjugates described herein, thereby treating cancer.
• the terms “treat,” “treatment,” “therapy,” “therapeutic,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect, including, but not limited to, alleviating, delaying or slowing the progression, reducing the effects or symptoms, preventing onset, inhibiting, ameliorating the onset of a diseases or disorder, obtaining a beneficial or desired result with respect to a disease, disorder, or medical condition, such as a therapeutic benefit and/or a prophylactic benefit.
• Treatment includes any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject, including a subject which is predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and/or (c) relieving the disease, i.e., causing regression of the disease.
• a therapeutic benefit includes eradication or amelioration of the underlying disorder being treated.
• a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
• treatment or compositions for treatment are administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
• the methods of the present disclosure may be used with any mammal or other animal.
• treatment results in a decrease or cessation of symptoms.
• a prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
• the term “subject” refers to any individual or patient on which the methods disclosed herein are performed.
• the term “subject” can be used interchangeably with the term “individual” or “patient.”
• the subject can be a human, although the subject may be an animal, as will be appreciated by those in the art.
• other animals including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.
• the term "effective amount” or “therapeutically effective amount” refers to that amount of an antibody, an antigen binding fragment thereof, or other composition described herein that is sufficient to induce the intended effect, including but not limited to disease treatment, as defined herein.
• the therapeutically effective amount may vary depending upon the intended treatment application (e.g., in vivo), or the patient and disease condition being treated, e.g., the weight and age of the patient, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
• the term also applies to a dose that will induce a particular response in a target cell.
• antibodies of the invention or antigen binding fragments thereof are used as a monotherapy or combined with other therapeutic agents such as radiotherapy, cytotoxic chemotherapy, immunotherapy including immune checkpoint inhibitors, and other immunoregulatory agents, such as vaccines, interleukins, cytokines, chemokines, and biologies as a combination therapy.
• Exemplary interleukins for immune therapy include IL- 1 , IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, and IL-23.
• Exemplary cytokines for immune therapy include interferons, TNF-a, TGF-[3, G-CSF, and GM-CSF.
• Exemplary chemokines for immune therapy include CCL3, CCL26, and CXCL7.
• Exemplary biologies include CAR T-cell therapy, tumor-infiltrating lymphocyte (TIL) therapy, and monoclonal antibodies, such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, blinatumomab, bevacizumab, and cetuximab.
• TIL tumor-infiltrating lymphocyte
• Antibodies also include checkpoint inhibitors, including PD-1 inhibitors, such as pembrolizumab, nivolumab, and cemiplimab, for example, PD-L1 inhibitors, such as atezolizumab, avelumab and durvalumab, for example, CTLA-4 inhibitors, such as iplimumab, for example, and other checkpoint inhibitors, such as an anti B7-H3 antibody, an anti-KIR antibody and an anti-LAG3 antibody, for example.
• PD-1 inhibitors such as pembrolizumab, nivolumab, and cemiplimab
• PD-L1 inhibitors such as atezolizumab, avelumab and durvalumab
• CTLA-4 inhibitors such as iplimumab
• other checkpoint inhibitors such as an anti B7-H3 antibody, an anti-KIR antibody and an anti-LAG3 antibody, for example.
• methods of treating cancer in a subject are provided herein.
• Methods of treating cancer include administering to a subject an amount of any bispecific antibody or an antigen binding fragment thereof provided herein, effective for treating the cancer.
• Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. In 2015, about 90.5 million people had cancer, about 14.1 million new cases occur a year and it caused about 8.8 million deaths (15.7% of deaths).
• the most common types of cancer in males are lung cancer, prostate cancer, colorectal cancer and stomach cancer. In females, the most common types are breast cancer, colorectal cancer, lung cancer and cervical cancer.
• the term “cancer” refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to others sites (secondary sites, metastases) which differentiate cancer (malignant tumor) from benign tumor.
• neoplasm or “tumor” including grammatical variations thereof, means new and abnormal growth of tissue, which may be benign or cancerous. In a related aspect, the neoplasm is indicative of a neoplastic disease or disorder, including but not limited, to various cancers.
• cancers can include prostate, pancreatic, biliary, colon, rectal, liver, kidney, lung, testicular, breast, ovarian, pancreatic, brain, and head and neck cancers, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.
• the cancer is prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, head and neck cancer, renal cell carcinoma (RCC), ovarian cancer, kidney cancer, urinary bladder cancer, uterine cancer, cervical cancer, ovarian cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, oral cavity cancer, pharynx cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, or leukemia.
• NSCLC non-small cell lung cancer
• RRCC renal cell carcinoma
• provided herein are isolated amino acid sequences encoding any of the antibodies, antigen binding fragment thereof or bispecific antibodies described herein.
• polypeptide refers to any chain of at least two amino acids, linked by a covalent chemical bound.
• polypeptide can refer to the complete amino acid sequence coding for an entire protein or to a portion thereof.
• a "protein coding sequence” or a sequence that "encodes" a particular polypeptide or peptide is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
• a coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.
• a transcription termination sequence will usually be located 3' to the coding sequence.
• amino acid sequences are as set forth in SEQ ID NOs:l-40 and 46-91.
• isolated nucleic acid sequences encoding the amino acid sequences provided herein.
• nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, anti-sense DNA strands, shRNA, ribozymes, nucleic acids conjugated and oligonucleotides.
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• a nucleic acid may be present as a single- stranded or doublestranded and linear or covalently circularly closed molecule.
• a nucleic acid can be isolated.
• isolated nucleic acid means, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.
• a nucleic can be employed for introduction into, i.e., transfection of, cells, in particular, in the form of RNA which can be prepared by in vitro transcription from a DNA template.
• the RNA can moreover be modified before application by stabilizing sequences, capping, and polyadenylation.
• nucleic acid sequences encoding any one ofthe amino acid sequences of SEQ ID NOs: l-40 and 46-91.
• the invention further provides the expression, purification and characterization of anti-p95HER2 and anti-CD73 antibodies, as detailed in the examples below.
• a signal sequence can be included in expression constructs for antibodies provided herein. Any suitable signal sequence can be used, such as a sequence of SEQ ID NO:43.
• Panning was performed using p95HER2-overexpressing HEK293 cells in the first round, then using recombinant p95HER2 protein produced by Expi-293 cells in the following three rounds selection. Briefly, for first round cell panning, the omniMab phage library was mixed with 1-fold 1%BSA in PBS. The phage/PBS mixture were added to the tube that contented wild type Expi293 cells for subtraction. Then wild type Expi293 cells were collected with centrifugation and washed cells three times with 0.5%BSA in PBS to collect the phage that didn’t recognize Expi293 cells.
• the heavy chains and light chains of positive p95HER2 binders identified by FACS were amplified, digested and subcloned into an in-house generated IgG expression vector carrying the IgGl constant region (SEQ ID NO:41).
• plasmids were prepared and transfected into ExpiCHO cells (Invitrogen) for antibody production. After 6 days of culture, antibody was affinity purified from culture supernatant by Protein A chromatography. Purified antibody was concentrated and dialyzed in PBS buffer. Antibody concentration was determined using Nano Drop2000 spectrophotometer. Antibody purity and integrity were determined by NuPAGE (ThermoFisher, Cat. No. NP0321BOX) under non-reducing or reducing conditions and visualized by Coomassie Brilliant Blue G-250 staining solution.
• FIGURE 2 shows the PAGE results of purified anti-p95HER2 antibody leads. Results indicated that proteins have a molecular weight of about 145 kDa under non-reducing conditions, and heavy chain and light chain have a molecular weight of 55kDa and 25kDa, respectively, under reducing conditions. Most of antibody leads showed greater than 90% purity from one-step Protein A chromatography.
• FIGURES 3A-3B show the binding activity of purified anti-p95HER2 antibody leads by flow cytometer.
• binding epitope mappings were conducted by PEPperPRINT Inc. To determine the binding epitopes, GS linker elongated full-length p95HER2 peptide sequence was converted into linear 5, 10 and 15 amino acid peptides each overlapped with 4, 9 and 14 amino acids.
• the linear peptide microarrays were incubated with the antibody leads at a concentration of 1 pg/ml followed by staining with secondary (Goat anti-human IgG (H+L) DyLight680) and control (Mouse monoclonal anti-HA (12CA5) DyLight800) antibodies as well as read-out with Odyssey Imaging System (LI-COR, Inc). Quantification of spot intensities and peptide annotation were done with PepSlide® Analyzer (SICASYS Software GmbH). The epitope for p95HER2 antibodies were shown and summarized in the FIGURE 4A.
• the chip was prepared as instructed by the manufacturer and was maintained at 30°C throughout the analysis.
• the excitation and detection wavelengths are 630 nm and 700 nm, respectively.
• the purity and integrity of one-column Protein-A purified anti- p95HER2-CD137 antibodies reach about 95% (FIGURE 6B).
• the affinity of anti-p95HER2-CD137 bsAbs were determined by biolayer interferometry analysis (ForteBIO, USA). To evaluate the binding affinity of anti-p95HER2- CD137 bsAbs against CD137, the bsAbs were loaded at 5 pg/mL on AHC (Anti-Human IgG Fc Capture, ForteBIO, Cat. No.18-5060) for signal reach to approximate 0.5 nm. Sensors then were exposed to 7 series of concentration analytes which was CD137 (Cat#41B-H5256) extracellular domain fused with mouse Fc tag recombinant protein.
• AHC Anti-Human IgG Fc Capture, ForteBIO, Cat. No.18-5060
• the binding activities of anti- p95HER2-CD137 bispecific antibody was determined by ForteBIO® (Menlo Park, Calif.) biosensor analysis.
• the custom synthesis biotinylated Her2 p95 peptide was loaded onto SA (Streptavidin, ForteBIO, Cat#18-5019) biosensor at 50 pg/mL for 5 minutes. Sensors then were exposed to antibody samples at 100 nM for 5 minutes follow by associating with the mouse Fc tagged human CD137 protein (AcroBiosystems, Cat#41B-H5256) at 100 nM for 5 minutes.
• the binding chart were produced by Octet Data Acquisition and Analysis Software. In summary, these results show that anti-p95HER2-CD137 bispecific antibodies simultaneously recognize p95HER2 and CD 137, as determined by ForteBIO® biosensor analysis (FIGURE 8).
• Human T cells were isolated using a RosetteSepTM Human T Cell Enrichment Cocktail (STEMCELL Cat. No.15061). Purified T cells (I MO 5 cells) were cocultured with p95HER2-expressing NCI-H292 lung cancer cells (I MO 4 cells) in the presence of anti-CD3 (OKT3)-coated polybeads at T-cell:bead ratio 1 :1. Three days later, T cell activation was measured by IFN-y production as measured by ELISA.
• CD16-NK92 effector cells were cocultured with CD137- expressing HEK293 target cells (2x 10 4 cells) at E/T ratio 1, 3 and 10 in a well of U-bottom 96- well plate.
• cytotoxicity activity was determined by CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780).
• #R4-1 le5-#54 Compared to isotype control, #R4-1 le5-#54, but not #R4-11 and #R4-l l-#54, induced the ADCC in CD137-HEK293 cells. This was corresponding to the ADCC-enhancing mutations in the IgGl Fc region of #R4-l le5-#54 (FIGURE 10A). #R4-l l-#54 induced the ADCC activity against p95HER2-NCI-H292 cells as well as its parental #R4- 11 mAb (FIGURE 10B).
• Tumor sizes (mm 3 ) were measured twice per week and calculated as (lengthxwidthxwidth)/2. Tumor inhibition ratio was calculated by the following equations based on the tumor volume (TGIv) or tumor weight (TGIw).
• TGIv (%) [l-(Vt-Vo of treatment group) / (Vt-Vo of control group)] x 100%, which Vt: mean tumor volume after treatment; Vo: mean tumor volume before treatment.
• TGIw (%) (mean tumor weight of control group - mean tumor weight of treatment group) / mean tumor weight of control group x 100%.
• the bispecific anti- p95HER2-CD137 antibody (5 mg per kg body weight) was administered via an intravenous bolus injection to SCI-beige mice. Peripheral blood was collected at 0.5, 24, 72, 168, 264, 336, 504, 672 and 840 hours post injection. Plasma concentrations of the antibody were determined by ELISA. MaxiSorp plates (Invitrogen) were coated with CD137-Fc fusion protein (AP Biosciences, 0.5 pg/mL), followed by application of serially diluted plasma samples and anti-CD73-CD137#54 bsAb as the standard curve.
• Bound antibodies were detected by CD73-Flag-His antigen (AP Biosciences) and HRP-conjugated anti-Flag antibody using TMB substrate. Plasma antibody concentrations were calculated by the interpolation method. PK parameters were calculated using PKSolver software. Anti- p95HER2-CD137 showed PK profile with ti/2 336 hours and AUC 63896 pg/ml*h (FIGURE 12).
• phagemid library was built up by AP Biosciences Inc. (APBio Inc.) from a collection of peripheral blood mononuclear cells from over a hundred healthy donors.
• First round panning was performed using Hyperphage (M13K07ApIII, Progen, Heidelberg, Germany). Solution phase panning was used for CD73 specific binders’ selection and isolation from the OmniMab library. Solution phase panning was performed using recombinant CD73 protein carries flag tag and polyhistidine tag at the C-terminus which produced by mammalian cell expression system used in first-round selection. Second round enrichment was also used solution phase panning. CD73 -expressing Expi293 cells and breast cancer cell line MDA-MB-231 were used for third round enrichment separately. The specific CD73 binders were screened and isolated by direct ELISA and FACS after three round panning (FIGURES 13A-13C).
• the phage clones recognize human CD73 specifically after three rounds human CD73 specific enrichment process from OmniMab phage library. Phagecontaining supernatant were applied to CD73 ELISA, and positive binders were numbered. MDA-MB-231 breast cancer cell line was stained with CD73 phages supernatant (50 pl/well) which were isolated from MDA-MB-231 cell-based panning to examine CD73 binding activity. FIGURE 13B showed ranking of binders binding activity. Binders were isolated and sent for sequencing to confirm the sequence and diversity of the heavy chain.
• the heavy chains and light chains of positive CD73 binders identified by ELISA and FACS were amplified, digested and subcloned into an IgG expression vector generated by APBio and carrying the engineered IgG 1 constant region (SEQ ID NO:87). After sequence validation, plasmids were prepared and transfected into ExpiCHO cells (Invitrogen) for antibody production. After 6 days of culture, antibody secreted into serum-free medium was affinity purified from culture supernatant by Protein A chromatography. Purified antibody was concentrated, followed by dialysis in PBS buffer.
• the final concentration of dialyzed protein was determined using a NanoDrop2000 spectrophotometer and the purity and integrity were determined by SDS-PAGE with or without reducing reagent. Purified antibodies were analyzed by NuPAGE (ThermoFisher, Cat. No. NP0321BOX) under non-reducing or reducing conditions. Once the dye front has reached the bottom of the gel, turn off the voltage and remove the gel from the glass plates. Place the gel in Coomassie Brilliant Blue G-250 staining solution in a plastic container and rock for several hours. The destain can be poured off into the designated waste container in the fume hood and replaced several times until the bands can be clearly visualized against the background of the gel.
• FIGURE 14 shows representative PAGE gel analysis of different batches of purified anti-CD137 antibody leads.
• the purity and integrity of one-step Protein A-purified anti-CD73 antibody leads was analyzed by SDS-PAGE under non-reducing and reducing conditions. Results indicated that proteins have a molecular weight of about 145 kDa under non-reducing conditions, and heavy chain and light chain have a molecular weight of 55kDa and 25kDa, respectively, under reducing conditions. More than 90% purity could be obtained by one step of Protein A chromatography.
• MDA-MB-231 cells (2x l0 4 cells) were incubated with anti-CD73 antibody in a well of 96-well tissue-cultured plate at 4°C for 30 minutes.
• AMP (12.5 pM, Sigma, Cat. No. A1752) was added and incubated at 4°C for additional one hour.
• Supernatants were transferred to 96-well white plate, and residual AMP was detected by AMP-Glo assay followed manufacturer’s instruction (Promega, Cat. No. V5011).
• the percentage of enzyme activity inhibition was calculated as [(Cells+AMP+antibody) - (Cells+AMP)] / [AMP - (Cells+AMP)] x lOO.
• MDA-MB-231 cells (4.8x 10 6 cells) seeded in a well of 24-well plate for 40 hours, then cultured medium were collected and incubated with antibodies at 37°C for 1 hour.
• AMP (12.5 pM, Sigma, Cat. No. A1752) was added and incubated at 37°C for additional 2 hours, followed by the same procedures described above to detect residual AMP and calculate the percentage of enzyme inhibition.
• the binding affinity of anti-CD73 antibody did not necessarily correspond to their blockade of enzymatic activity. Although possess the better binding activity than clone #3-4, the weaker blockade of CD73 enzymatic activity was observed in clone #3-6, #3-27, #3-34, #3-1-14, #3-1-21, and #3-1-31. Interestingly, clone #3-1-28 showed the most potent blockade of CD73 enzymatic activity even with a comparable binding activity to clone #3-4, and #3-1-28 was selected for affinity maturation (FIGURE 15 and FIGURE 16). As illustrated in FIGURE 15 the binding activity of anti-CD73 antibody leads was assessed by flow cytometer.
• MDA-MB-231 cells (1 x 105 cells) were surface stained with anti-CD73 antibody (4 pg/ml) for 30 minutes, then incubated with A488-conjugated anti-human IgG secondary antibody for 15 minutes on ice. Cells were analyzed by flow cytometer, and A488 fluorescence intensity was shown by histograms (left) and number (right table). As illustrated in FIGURE 16 the blockade of CD73 enzymatic activity by anti-CD73 antibody leads was assessed. The enzymatic activity of CD73 expressed by MDA-MB-231 on the cell surface was blocked by anti-CD73 antibody leads.
• the CD73 enzyme block activity of anti-CD73 clone #3-l-28-vl, #3-l-28-v2, and #3-l-28-v3 was better than their parental clone #3-1-28 (FIGURE 18).
• the IC50 of anti-CD73#3-l-28-vl (0.213 nM) and anti- CD73#3-l-28-v2 (0.245 nM) was about 2 folds lower than that of parental #3-1-28 (0.49 nM) (FIGURE 18A).
• Affinity-matured antibodies can exhibit increased biological efficacy. Regardless of whether an antibody is isolated from a hybridoma or a human Fv phage library. An increased affinity may also allow for a reduced dosage of a therapeutic antibody and toxic side effects may be reduced. Germline hotspot residues are most likely to have a major impact on affinity based on statistical analysis of independent monoclonal antibodies derived from the same germline genes recognizing the same epitopes. Germline hotspots in the antibody complementarity determining regions (CDR) are naturally prone to hypermutations (Neuberger and Milstein, 1995).
• CDR complementarity determining regions
• the heavy chains and light chains of positive CD73 binders identified by FACS were amplified, digested and subcloned into an IgG expression vector carrying the engineered IgGl constant region. Vectors were transfected into the mammalian cell expression system for IgG production.
• clone #1919-003, #1911-022, and #1911-030 were selected for further development of bispecific antibodies (having a general structure as illustrated in FIGURE 19).
• clone #1919-003, #1911- 022, and #1911-030 were re-named hereafter as #3-l-28-vl, #3-l-28-v2, and #3-l-28-v3, respectively.
• CD73 binding activities of phage clones from small-scale affinity maturation library were determined by staining of CD73 -expressing MDA- MB-231 cells and analyzed by flow cytometer. Clones with the better binding activity compared to parental phage clone #3-1-28 were numbered. As shown in FIGURE 17B, the binding activities of antibodies converted from phage clones numbered in FIGURE 17A were measured by CD73 ELISA. The clones in bold #1919-002, #1919-003, #1919-004, #1911-022, and #1911 -030 showed the better binding activity than parental #3-1 -28 antibody.
• binding kinetics of antibodies selected in FIGURE 17B were measured by biolayer interferometry.
• the affinities of #3-l-28-vl, #3-l-28-v2, and #3- l-28-v3 were improved about 2 folds compared to parental #3-1-28.
• anti-CD73 mAbs and bsAbs were determined by biolayer interferometry analysis (ForteBIO, USA).
• AHC Anti-Human IgG Fc Capture, ForteBIO, Cat. No.18-5060
• Sensors then were exposed to his-tagged human CD73 (AcroBiosystems, Cat. No. CD3-H52H7)-containing solutions that 2-fold serial diluted from the top concentration 30 nM for total 7 points. Measurements were performed as follows: 1 minute baseline in kinetic buffer, 5 minutes association in hCD73-His-containing solutions, and 5 minutes dissociation into kinetic buffer. The kinetic parameters were calculated and produced by Octet Data Acquisition and Analysis Software.
• binding affinity of anti-CD73 #3-l-28-vl, #3-l-28-v2, and #3-1-28- v3 show about 2-fold improvement after affinity maturation compared to parental #3-1-28 (FIGURE 17C).
• the improved CD73 binding affinities of #3-l-28-vl, #3-l-28-v2, and #3-1- 28-v3 remains after conversion to anti-CD73-CD137 bispecific antibodies (FIGURES 21A- 21B).
• the chip was prepared as instructed by the manufacturer and was maintained at 30°C throughout the analysis.
• the excitation and detection wavelengths are 630 nm and 700 nm, respectively.
• the purity and integrity of one-column Protein-A purified anti-CD73-CD137 antibodies reach about 95% (FIGURES 20A-20B).
• the binding activities of the AP601 was determined by ForteBIO® (Menlo Park, Calif.) biosensor analysis.
• the His tagged human CD73 protein (AcroBiosystems, Cat. No. CD3-H52H7) were loaded onto HIS1K (Anti-Penta-HIS) biosensor (Cat. No.18-5120) at 5 pg/mL for 5 minutes. Sensors then were exposed to antibody samples at 100 nM for 5 minutes follow by associating with the mouse Fc tagged human CD 137 protein (AcroBiosystems, Cat. No. 41B-H5256) at 100 nM for 5 minutes.
• the binding chart were produced by Octet Data Acquisition and Analysis Software.
• Human T cells were isolated using a RosetteSepTM Human T Cell Enrichment Cocktail (STEMCELL Cat. No.15061). Purified T cells were labeled with CFSE (ThermoFisher, Cat. No. C34554) and stimulated with human T-Activator CD3/CD28 dynabeads (ThermoFisher, Cat. No. 1113 ID) at T-cell:bead ratio 1 :0.5 in the presence of AMP (400 pM, Sigma, Cat. No. A1752). Three days later, T-cell activation was determined by IFN- y production measured by ELISA and cell proliferation analyzed by flow cytometry.
• CFSE ThermoFisher, Cat. No. C34554
• human T-Activator CD3/CD28 dynabeads ThermoFisher, Cat. No. 1113 ID
• T-cell activation was determined by IFN- y production measured by ELISA and cell proliferation analyzed by flow cytometry.
• CD73-DEPENDENT T CELL ACTIVATION INDUCED BY ANTI-CD73-CD137 BISPECIFIC ANTIBODY
• Human T cells were isolated using a RosetteSepTM Human T Cell Enrichment Cocktail (STEMCELL Cat. No.15061). Purified T cells (l *10 5 cells) were cocultured with CD73-expressing MDA-MB-231 breast and NCI-H292 lung cancer cells (I MO 4 cells) in the presence of anti-CD3 (OKT3)-coated polybeads at T-cell:bead ratio 1 : 1. Three days later, T cell activation was measured by IFN-y production as measured by ELISA.
• FIG. 25A CD73 expression level on MDA-MB-231 breast cancer cells and NCI-H292 lung cancer cells were checked by flow cytometry. More robust IFN-y production was induced by anti-CD73-CD137 bsAb compared to the mono- or combination therapy with anti-CD73 and anti-CD137 #54 antibodies (FIGURES 25B-25C).
• Tumor sizes were measured twice per week and calculated as (lengthxwidthxwidth)/2. Tumor inhibition ratio was calculated by the following equations based on the tumor volume (TGI V ) or tumor weight (TGIw).
• TGI V (%) [l-(Vt-Vo of treatment group) / (Vt-Vo of control group)] x 100%, which Vt: mean tumor volume after treatment; Vo: mean tumor volume before treatment.
• TGI W (%) (mean tumor weight of control group - mean tumor weight of treatment group) / mean tumor weight of control group x 100%.
• TILs tumor-infiltrating lymphocytes
• dissected tumors were minced and digested with RPMI- 1640 medium containing collagenase IV (Worthington, Cat. No. LS004188), hyaluronidase (Sigma, Cat. No. H6254), and DNase I (Sigma, Cat. No. D5025) by gentleMACS (Miltenyi Biotec Inc.), followed by density gradient separation with Lymphoprep (STEMCELL, Cat. No. 07811).
• Isolated TILs were stained with cell-type specific markers and analyzed by flow cytometer.
• the bispecific anti-CD73#3-l-28-v2-CD137#54 antibody (5 mg per kg body weight) was administered via an intravenous bolus injection to SCI-beige mice. Peripheral blood was collected at 0.5, 24, 72, 168, 240, 336, 504, and 672 hours post injection. Plasma concentrations of the antibody were determined by ELISA. MaxiSorp plates (Invitrogen) were coated with CD137-Fc fusion protein (AP Biosciences, 0.5 pg/mL), followed by application of serially diluted plasma samples and anti-CD73-CD137#54 bsAb as the standard curve.
• Bound antibodies were detected by CD73-Flag-His antigen (AP Biosciences) and HRP-conjugated anti-Flag antibody using TMB substrate. Plasma antibody concentrations were calculated by the interpolation method. PK parameters were calculated using PKSolver software. Anti- CD73#3-l-28-v3 showed a worse PK profile with ti/2 103 hours and AUC 16438 pg/ml*h, while anti-CD73 #3-1-28, #3-l-28-vl, #3-l-28-v2-CD137#54 bsAbs showed a comparable 11/2 and AUC (FIGURE 27).
• NH2 -terminally truncated HER-2/neu protein relationship with shedding of the extracellular domain and with prognostic factors in breast cancer. Cancer Res 58, 5123-5129.
• Cleavage of the HER2 ectodomain is a pervanadate-activable process that is inhibited by the tissue inhibitor of metalloproteases- 1 in breast cancer cells. Cancer Res 59, 1196-1201.
• pl85HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor. Mol Cell Biol 9, 1165-1172.
• mice have increased antitumor immunity and are resistant to experimental metastasis. Cancer Res 71, 2892-2900.
• Serum CD73 is a prognostic factor in patients with metastatic melanoma and is associated with response to anti- PD-1 therapy.
• trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 20, 719-726.
• PKSolver An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput Methods Programs Biomed 99, 306-314.

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