WO2021195067A1 - Bi-specific antibodies for use in producing armed immune cells - Google Patents

Bi-specific antibodies for use in producing armed immune cells Download PDF

Info

Publication number
WO2021195067A1
WO2021195067A1 PCT/US2021/023655 US2021023655W WO2021195067A1 WO 2021195067 A1 WO2021195067 A1 WO 2021195067A1 US 2021023655 W US2021023655 W US 2021023655W WO 2021195067 A1 WO2021195067 A1 WO 2021195067A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
seq
ctat
cell
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2021/023655
Other languages
English (en)
French (fr)
Inventor
Michael Chen
Kuo-Hsiang Chuang
Yi-Jou CHEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytoarm Co Ltd
Original Assignee
Cytoarm Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cytoarm Co Ltd filed Critical Cytoarm Co Ltd
Priority to AU2021244375A priority Critical patent/AU2021244375A1/en
Priority to CN202180029464.3A priority patent/CN115551890A/zh
Priority to EP21774346.7A priority patent/EP4126954A4/en
Priority to JP2022558065A priority patent/JP7729833B2/ja
Priority to US17/913,775 priority patent/US20240209084A1/en
Priority to IL296566A priority patent/IL296566A/en
Publication of WO2021195067A1 publication Critical patent/WO2021195067A1/en
Anticipated expiration legal-status Critical
Priority to JP2025135194A priority patent/JP2025161873A/ja
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/33—Antibodies; T-cell engagers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4204—Epidermal growth factor receptors [EGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4205—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4209—Hepatocyte growth factor receptor [HGFR] or c-met]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/422—Ephrin Receptors [Eph]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4221—CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4222—CD38 not IgG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4223—CD44 not IgG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4274—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2809—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 immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2827—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 immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • 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
    • C07K16/2887—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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
    • C07K16/28—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
    • C07K16/3007—Carcino-embryonic Antigens
    • 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
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • 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
    • C07K16/3076—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 against structure-related tumour-associated moieties
    • C07K16/3084—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 against structure-related tumour-associated moieties against tumour-associated gangliosides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • 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
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components

Definitions

  • Cancer is a disease characterized by abnormal cells that divide uncontrollably and have the ability to infiltrate and destroy normal tissue and/or organ of a subject. Cancer is the second leading cause of death globally, and is responsible for an estimated 9.6 million deaths in 2018, in which the most common cancers include, lung cancer (about 2.09 million cases), breast cancer (about 2.09 million cases), colorectal cancer (about 1.80 million cases), prostate cancer (about 1.28 million cases), skin cancer (about 1.04 million cases), and gastric cancer (about 1.03 million cases).
  • Treatments for cancers may vary with the type of cancer and how advanced it is.
  • Conventional treatments for cancers include surgery, radiation therapy, and chemotherapy.
  • Such treatments usually cause a variety of complications or side effects, such as infection, blood clot, bleeding, nausea and vomiting, diarrhea, nerve or muscle damage, incontinence, and sex and fertility issues.
  • Immunotherapy provides an alternative strategy for cancer treatment that aims at specifically stimulating immune responses of a subject against cancer cells via, for example, blocking immune checkpoints, or enhancing the ability of immune cells (e.g., T cells or B cells) to target and destroy cancer cells.
  • Serious adverse effects associated with immunotherapy-medicated overstimulation or non-specific toxicity have been reported in cancer patients, including neurotoxicity, cytokine release syndrome (CRS), allergy, organ inflammation, and autoimmune disorders.
  • CRS cytokine release syndrome
  • BsAbs bispecific antibodies capable of binding to CD3 (e.g., human CD3) and a tumor associated antigen (TAA).
  • CD3 e.g., human CD3
  • TAA tumor associated antigen
  • a bi-specific antibody comprising: (a) a first antigen binding fragment that binds human CD3, and (b) a second antigen binding fragment that binds a tumor associated antigen (TAA).
  • the first antigen binding fragment comprises (i) a first heavy chain comprising a first heavy chain variable region (VH) and (ii) a first light chain comprising a first light chain variable region (VL).
  • VH first heavy chain variable region
  • VL first light chain comprising a first light chain variable region
  • the first VH comprises the same heavy chain complementary determining regions (CDRs) as a first reference antibody.
  • the first VH comprises or no more than 5 amino acid variations in CDRs relative to the first reference antibody.
  • the first VL may comprise the same light chain CDRs as the first reference antibody. In other embodiments, the first VL may comprise no more than 5 amino acid variations in the CDRs relative to the first reference antibody.
  • the first reference antibody is CTA.02. In some examples, the first reference antibody is CTA.03. In other examples, the first reference antibody is CTA.04. In yet other examples, the first reference antibody is CTA.05. Structural information of these exemplary reference antibodies are provided in Table 1 below.
  • the first heavy chain and the first light chain comprise the same VH and VL as the first reference antibody.
  • the second antigen binding fragment comprises a second heavy chain comprising (i) a second heavy chain variable region (VH), and (ii) a second light chain comprising a second light chain variable region (VL).
  • the second antigen binding fragment binds a TAA. Examples include CD20, CD19, EGFR, HER2, PSMA, CEA, EpCAM, FAP, PD-L1, CD38, CD33, cMET, CD47, TRAIL-R2, mesothelin, or GD2.
  • the second VH comprises the same heavy chain complementary determining regions (CDRs) as a second reference antibody.
  • the second VH may comprise no more than five amino acid variations in the CDRs relative to the second reference antibody.
  • the second VL may comprise the same light chain CDRs.
  • the second VL may comprise no more than 5 amino acid variations in the CDRs relative to the second reference antibody.
  • the second reference antibody is CTAT.01, CTAT.02, CTAT.03, CTAT.04, CTAT.05, CTAT.06, CTAT.07, CTAT.08, CTAT.09, CTAT.10, CTAT.ll, CTAT.12, CTAT.13, CTAT.14, CTAT.15, or CTAT.16. See Table 2 below.
  • the second antigen binding fragment comprises the same VH and same V L as the second reference antibody.
  • the first antigen binding fragment is a Fab fragment and the second antigen binding fragment is a single chain variable fragment (scFv).
  • the Fab fragment comprises the first heavy chain, which comprises the first VH and a CHI fragment, and the first light chain, which comprises the first VL and a light chain constant region.
  • the Fab fragment may comprise the first heavy chain and the first light chain, which respectively comprise the amino acid sequences of (a) SEQ ID NO: 10 and SEQ ID NO: 11, (b) SEQ ID NO: 23 and SEQ ID NO: 24, 25, or 228, (c) SEQ ID NO: 35 and SEQ ID NO: 36, or (d) SEQ ID NO: 46 and SEQ ID NO: 47.
  • the scFv of the second antigen binding fragment comprises the amino acid sequence of any one of SEQ ID NOs: 254-271.
  • the scFv is linked to the CHI fragment, w optionally is via a peptide linker.
  • the scFv is linked to the light chain constant region, optionally via a peptide linker.
  • the bi-specific antibody may comprise a first polypeptide comprising the first light chain and a second polypeptide comprising, from N-terminus to
  • the first antigen binding fragment is a single chain variable fragment
  • the second antigen binding fragment is a Fab fragment.
  • the scFv may comprise the amino acid sequence of any one of SEQ ID NOs: 250-253.
  • the Fab fragment comprises the second heavy chain, which comprises the second VH and a CHI fragment, and the second light chain, which comprises the second VL and a light chain constant region.
  • the Fab fragment comprises the first heavy chain and the first light chain, which respectively comprise the amino acid sequences of (1) SEQ ID NO:57 and SEQ ID NO: 58, (2) SEQ ID NO: 72 and SEQ ID NO: 73, (3) SEQ ID NO: 83 and SEQ ID NO: 84, (4) SEQ ID NO: 94 and SEQ ID NO: 95, (5) SEQ ID NO: 105 and SEQ ID NO: 106,
  • SEQ ID NO: 116 and SEQ ID NO: 117 (7) SEQ ID NO: 127 and SEQ ID NO: 128, (8) SEQ ID NO:138 and SEQ ID NO:139, (9) SEQ ID NO:149 and SEQ ID NO:150, (10) SEQ ID NO:160 and SEQ ID NO:161, (11) SEQ ID NO: 171 and SEQ ID NO:172, (12) SEQ ID NO: 182 and SEQ ID NO: 183, (13) SEQ ID NO: 193 and SEQ ID NO: 194, (14) SEQ ID NO:204 and SEQ ID NO:205, (15) SEQ ID NO:215 and SEQ ID NO:216, or (16) SEQ ID NO:226 and SEQ ID NO:227.
  • the scFv is linked to the CHI fragment, optionally via a peptide linker.
  • the scFv is linked to the light chain constant region, optionally via a peptide linker. Any of the peptide linker may be at least 5 amino acids in length.
  • both the first antigen binding fragment and the second antigen binding fragment are scFv antibodies.
  • the bi-specific antibody comprises a polypeptide comprising the two scFv antibodies.
  • the present disclosure provides an armed immune cell, comprising an immune cell that expresses surface CD3, and any of the bi-specific antibodies disclosed herein (e.g., those exemplified in Tables 1-3).
  • the armed immune cell displays the bi-specific antibody on the surface via interaction between the first antigen binding fragment in the bi-specific antibody and the CD3 expressed by the immune cell.
  • the immune cell is a T cell, a B cell, a monocyte, a macrophage, or a combination thereof.
  • the T cell can be a CD4+ T cell, a CD8+ T cell, a regulatory T cell, or a natural killer T cell.
  • the immune cell is a human immune cell, for example, immune cells derived from a human donor.
  • a method of producing the armed immune cell as disclosed herein may comprise cultivating a cell population comprising the immune cells in the presence of the bi-specific antibody as disclosed herein to allow for binding of the bi-specific antibody to the immune cells, thereby producing the armed immune cell.
  • the armed immune cells produced by any of the methods disclosed herein are also within the scope of the present disclosure.
  • the cell population comprises T cells, B cells, monocytes, macrophages, or a combination thereof.
  • the cell population comprises peripheral blood mononuclear cells (PBMCs) or immune cells derived from stem cells in vitro.
  • PBMCs peripheral blood mononuclear cells
  • the stem cells may be hematopoietic stem cells, umbilical cord blood stem cells, or induced pluripotent stem (iPS) cells.
  • the cultivating step is performed in a culture medium comprising a cytokine, which optionally comprises interleukin 2 (IL-2), interleukin 7 (IL-7), transforming growth factor-beta (TGF-b), or a combination thereof.
  • a cytokine which optionally comprises interleukin 2 (IL-2), interleukin 7 (IL-7), transforming growth factor-beta (TGF-b), or a combination thereof.
  • a method for treating cancer comprising administering to a subject in need thereof an effective amount of a population of any of the armed immune cells disclosed herein.
  • the subject has or suspected of having a cancer that is positive with the TAA, to which the second antigen binding fragment of the bi-specific antibody binds.
  • the subject is a human cancer patient.
  • the armed immune cells are autologous to the subject. Alternatively, the armed immune cells are allogenic to the subject.
  • Exemplary cancers include, but are not limited to, melanoma, esophageal carcinoma, gastric carcinoma, brain tumor, small cell lung cancer, non-small cell lung cancer, bladder cancer, breast cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, renal cancer, hepatocellular carcinoma, ovary cancer, prostate cancer, thyroid cancer, testis cancer, head and neck squamous cell carcinoma, leukemia, lymphoma, and myeloma.
  • the present disclosure features a nucleic acid or a set of nucleic acids (two nucleic acid molecules), which encodes or collectively encodes any of the bi-specific antibodies disclosed herein.
  • the nucleic acid or set of nucleic acids is a vector or a set of vectors, for examples, expression vector(s).
  • Host cells e.g., a bacterial cell, a yeast cell, or a mammalian cell
  • Host cells comprising any of the nucleic acid or set of nucleic acids disclosed herein are also within the scope of the present disclosure.
  • the present disclosure features a method for producing a bi-specific antibody, comprising: (i) culturing a host cell as disclosed herein under conditions allowing for expressing of the bi-specific antibody; and (ii) harvesting the bi-specific antibody.
  • armed immune cells as disclosed herein for use in cancer treatment or use of any of the armed immune cells for manufacturing a medicament for use in treating a target cancer.
  • FIGs 1A-1N are schematic diagrams of exemplary bi-specific antibody formats.
  • FIGs. 1A-1D structures of anti-CD3 Fab/anti-TAA scFv bi-specific formats.
  • FIGs. 1E-1H structures of anti-CD3 scFv/anti-TAA Fab bi-specific formats.
  • FIGs. II- IF structures of anti-CD3 scFv/anti-TAA scFv bi-specific formats.
  • FIGs. 1M structure of anti-CD3 knob/anti-TAA hole bi-specific antibody formats, which comprises a monovalent anti-CD3 antibody and a monovalent anti-TAA antibody.
  • FIG. IN anti-CD3 knob/anti-TAA scFv hole antibody, which comprises a monovalent anti-CD3 antibody and a monovalent anti-TAA scFv-Fc fusion protein.
  • FIGs. 2A-2E include schematic diagrams of DNA constructs for expressing the illustrated recombinant bi-specific antibodies.
  • FIG. 2A exemplary constructs for expressing anti-CD3Fab/anti-TAA scFv bi-specific antibodies.
  • FIG. 2B exemplary constructions for expressing anti-CD3 scFv/anti-TAA Fab bi-specific antibodies.
  • FIG. 2C exemplary constructs for expressing anti-CD3 scFv/anti-TAA scFv bi-specific antibodies.
  • FIG. 2D exemplary constructs for expressing anti-CD3 knob/anti-TAA hole bi-specific antibodies.
  • FIG. 2E exemplary constructs for expressing anti-CD3 knob/anti-TAA scFv hole antibody.
  • FIG. 3 is a chart showing binding affinity of exemplary bi-specific antibodies to T cells as measured by flow cytometry.
  • FIG. 4 is a chart depicting the cytotoxic effect of T cells armed with exemplary bi-specific antibodies or activated by OKT3 antibody against HT-29 cancer cells.
  • FIG. 5 is a chart depicting a time course of the levels of exemplary bi- specific antibodies on the surface of T cells.
  • FlGs 6A-6B include photos showing expression and assembly of the exemplary bi-specific antibodies as indicated by SDS-PAGE under non-reducing conditions (FIG. 6A) and reducing conditions (FIG. 6B).
  • Lane 3 CTA04Fab/CTAT02scFv
  • Lane 4 CTA03Fab/CTAT02scFv
  • Lane 5 CTA05Fab/CTAT02scFv.
  • FlGs. 7A-7B CTA03Fab/anti-TAA scFv bi-specific antibodies under non-reducing conditions.
  • FlGs. 7C-7D CTA03Fab/anti-TAA scFv bi-specific antibodies under reducing conditions.
  • FlGs. 7E-7F anti-CD3 scFv/CTAT03Fab bispecific antibodies under non-reducing and reducing conditions, respectively.
  • FIG. 8 is a diagram showing the binding activity of various bi-specific antibodies as indicated to T cells and to tumor cells.
  • CD3 + T cells Jurkat
  • CD19 + B cell lymphoma Raji
  • exemplary bi-specific antibodies CTA02Fab/CTAT02scFv, CTA03Fab/CTAT02scFv, CTA04Fab/CTAT02scFv, and CTA05Fab/CTAT02scFv BsAbs, and then analyzed with FITC conjugated Goat anti-Human IgG Fab antibody and flow cytometer.
  • FlGs. 9A-9L include diagrams showing binding activity of exemplary anti-CD3 Fab/anti-tumor scFv bi-specific antibodies as indicated to T cells and to tumor cells.
  • FIG. 9A CTA03Fab/CTAT02scFv binding to CD3 + T cells (Jurkat) and CD19 + B cell lymphoma (Raji).
  • FIG. 9B CTA03Fab/CTAT03scFv binding to CD3 + T cells (Jurkat) and EGFR + triple negative breast cancer (MDA-MB-231).
  • FIG. 9C CTA03Fab/CTAT04scFv binding to CD3 + T cells (Jurkat) and HER2 + breast cancer (MCF7/HER2).
  • FIG. 9A CTA03Fab/CTAT02scFv binding to CD3 + T cells (Jurkat) and CD19 + B cell lymphoma (Raji).
  • FIG. 9B CTA03Fab/CTAT03scFv binding to CD3 + T cells (J
  • FIG. 9D CTA03Fab/CTAT05scFv binding to CD3 + T cells (Jurkat) and PSMA + Prostate cancer (LNCaP).
  • FIG. 9E CTA03Fab/CTAT07scFv binding to CD3 + T cells (Jurkat) and EpCAM + Prostate cancer (LNCaP).
  • FIG. 9F CTA03Fab/CTAT08scFv binding to CD3 + T cells (Jurkat) and FAP + mouse fibroblasts cell(3T3/FAP).
  • FIG. 9G CTA03Fab/CTAT09scFv binding to CD3 + T cells (Jurkat) and PDL1 + triple negative breast cancer (MDA-MB-231).
  • FIG. 9H CTA03Fab/CTAT09scFv binding to CD3 + T cells (Jurkat) and PDL1 + triple negative breast cancer (MDA-MB-231).
  • FIG. 91 CTA03Fab/CTATllscFv binding to CD3 + T cells (Jurkat) and CD33 + human acute myeloid leukemia (HL-60).
  • FIG. 9J CTA03Fab/CTAT12scFv binding to CD3 + T cells (Jurkat) and HGFR + human lung carcinoma (A549).
  • FIG. 9K CTA03Fab/CTAT13scFv binding to CD3 + T cells (Jurkat) and CD47 + breast cancer (MCF7/HER2).
  • FIG. 10 is a chart showing the retention ability of exemplary BsAbs on T cell surface.
  • Human T cells were incubated with variant Anti-CD3Fab/anti-CD19scFv BsAbs with a Fab of 4 different anti-CD3 antibody (CTA01Fab/CTAT02scFv, CTA02Fab/CTAT02scFv, CTA03Fab/CTAT02scFv, and CTA05Fab/CTAT02scFv) for lhr, and then were cultured in medium for 5 min, 24, 48, and 72 hr. After the culture, the cells were stained with FITC conjugated Goat anti-Human IgG Fab antibody and the retention of BsAb on T cell surface was analyzed using flow cytometry.
  • CTA01Fab/CTAT02scFv CTA02Fab/CTAT02scFv
  • CTA03Fab/CTAT02scFv CTA05Fab/CTAT02scFv
  • FIGs. 11A and 1 IB include diagrams showing formation of T cells armed with exemplary BsAbs disclosed herein.
  • FIG. 11 A OKT3, CTA01Fab/CTAT02scFv, and CTA02Fab/CTAT02scFv, from left to right.
  • FIG. 11B CTA03Fab/CTAT02scFv (left) and CTA05Fab/CTAT02scFv (right).
  • PBMCs were cultured in the presence of OKT3 or the various BsAbs. The cell cultures were then stained with FITC conjugated CD8 antibody and PE conjugated goat anti-Human IgGFab, and then analyzed using flow cytometry.
  • FIGs. 12A-12D include diagrams showing formation of T cells armed with exemplary BsAbs as indicated.
  • FIG. 12A OKT3, CTA03Fab/CTAT03scFv, and CTA03Fab/CTAT04scFv (top panel, left to right), and CTA03Fab/CTAT09scFv,
  • FIG. 12B CT A03 Fab/CT AT05 scFv, CTA03Fab/CTAT07scFv, and CT AFab/CT AT08 scFv (top panel, left to right), and CTA03Fab/CTAT12scFv and CTA03Fab/CTAT13scFv (bottom, left to right).
  • FIG. 12C OKT3, CTA01scFv/CTAT03Fab, and CTA02scFv/CTA03Fab (left to right).
  • FlGs. 13A-13B include charts showing cytotoxicity activity of T cells induced by OKT3 antibody or armed with exemplary bi-specific antibodies as indicated against tumor cells.
  • FIG. 13A anti-CD3Fab/anti-CD19scFv BsAbs against B cell lymphoma.
  • FIG. 13B anti-CD3scFv/CTAT03Fab BsAbs against HT29 cells.
  • Cells were cultured with OKT3 or the exemplary BsAbs as indicated. The cell cultures were then incubated with CD19 + B cell lymphoma (Raji) at several effector cell: target cell ratios (3:1, 5:1 and 10:1) for 18 hr. Tumor cell death was determined with CytoTox 96 ® Non-Radioactive Cytotoxicity Assay (Promega, G1780).
  • FIG. 14A-14G include charges showing in vitro cytotoxic activity of anti-CD3Fab/anti-EGFRscFv BsAb armed T cells against cancer cells.
  • FIG. 14A CTA01Fab/CTAT03scFv or CTA03Fab/CTAT03scFv armed T cells against HT29 cells (EGFR+ colon cancer cells).
  • FIG. 14B CTA01Fab/CTAT03scFv or CTA03Fab/CTAT03scFv armed T cells against HCT-116 cells (EGFR+ colon cancer cells).
  • FIG. 14C antiCD3Fab/anti-HER2scFv (CTA03Fab/CTAT04scFv) armed T cells against HER2+ breast cancer cells (MCF-7/HER2).
  • FIG. 14A CTA01Fab/CTAT03scFv or CTA03Fab/CTAT03scFv armed T cells against HT29 cells (EGFR+ colon cancer cells).
  • FIG. 14B CTA01Fab/CTAT03s
  • FIG. 14D antiCD3Fab/anti-PSMAscFv (CTA03Fab/CTAT05scFv) armed T cells against PSMA + prostate cancer cells (LNCaP).
  • FIG. 14E antiCD3Fab/anti-EpCAMscFv (CTA03Fab/CTAT07scFv) armed T cells against
  • FlGs. 14F-14G antiCD3Fab/anti-FAPscFv (CTA03Fab/CTAT08scFv) armed T cells against FAP mouse fibroblasts cells (3T3) (FIG.
  • FAP + mouse fibroblasts cells 3T3/FAP
  • OKT3-cultued T cells or armed T cells were cocultured with the cancer cells at several effector cell: target cell ratios (3:1, 5:1, and 10:1) for 18 hr. Tumor cell death was determined with CytoTox 96 ® Non-Radioactive Cytotoxicity Assay (Promega, G1780).
  • FlGs. 15A-15E include charts showing in vitro cytotoxic activities of exemplary antiCD3Fab/anti-TAA scFv BsAbs against corresponding cancer cells.
  • FIG. 15A antiCD3Fab/anti-PDLlscFv (CTA03Fab/CTAT09scFv) armed T cells against triple negative breast cancer cells (MDA-MB-231).
  • FIG. 15B antiCD3Fab/anti-CD38scFv
  • FIG. 15C antiCD3Fab/anti-CD33scFv (CTA03Fab/CTATllscFv) armed T cells against CD33 + human acute myeloid leukemia cells (HL-60).
  • FIG. 15D antiCD3Fab/anti-HGFRscFv (CTA03Fab/CTAT12scFv) armed T cells against HGFR + human lung carcinoma cells (A549).
  • FIG. 15E antiCD3Fab/anti-CD47scFv (CTA03Fab/CTAT13scFv) armed T cells against CD47 + breast cancer cells (MCF7/HER2).
  • 0KT3-cultued T cells or armed T cells were cocultured with the cancer cells at several effector cell: target cell ratios (3:1, 5:1, and 10:1) for 18 hr. Tumor cell death was determined with CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780).
  • FlGs 16A-16C include diagrams showing in vivo therapeutic efficacy of exemplary anti-CD3Fab/anti-CD19scFv armed-T cell against lymphoma.
  • SCID mice were i.v. inoculated with CD19 + B cell lymphoma cells (2.5 xlO 6 cells/mice). After 3 days, T cell, CTA01Fab/CTAT02scFv armed-T cells sand CTA03Fab/CTAT02scFv armed-T cells were i.v. injected into the mice (5xl0 6 cells/mice, once a week for 4 times).
  • FIG. 16A Body weight.
  • FIG. 16B survival rate.
  • FIG. 16C incidence of hindlimb paralysis.
  • FlGs. 17A-17B include diagrams showing in vivo therapeutic efficacy of exemplary CTA03Fab/CTAT03scFv armed-T cells and CTA03Fab/CTAT04scFv armed-T cells against human triple-negative breast cancer.
  • ASID mice were s.c. inoculated with clinical human breast tumor tissues. After 19 days, T cell, CTA03Fab/CTAT03scFv armed-T cells and CTA03Fab/CTAT04scFv armed-T cells were i.v. injected into the mice (5xl0 6 cells/mice, once a week for 4 times).
  • FIG. 17A Body weight.
  • FIG. 17B tumor size.
  • FIG. 18A-18D include diagrams showing formation of BsAb Armed-NKT cells with various anti-CD3/anti-TAA BsAbs.
  • FIG. 18A OKT3 (left) and CTA03Fab/CTAT03scFv (right).
  • FIG. 18B CTA03Fab/CTAT04scFv (left) and CTA03Fab/CTAT05scFv (right).
  • FIG. 18C OKT3, CTA01scFv/CTAT03Fab, and CTA02scFv/CTAT03Fab (left to right).
  • FIG. 18D CTA03scFv/CTAT03Fab, CTA04scFv/CTAT03Fab, and CTA05scFv/CTAT03Fab (left to right).
  • NKT cells (CD8 + CD25 + ) were cultured and differentiated in the presence of the OKT3 antibody or the BsAbs as indicated. The cells were then were stained with anti-CD8 antibody, anti-CD56 antibody and FITC-conjugated anti-Human IgGFab antibody. BsAbs on cell surface were analyzed using flow cytometry.
  • FlGs 19A-19C include charts showing binding activity and toxicity of point-mutant BsAbs CTA03Fab/CTAT02scFv.
  • FIG. 19A binding activity to CD3+ T cells (Jurkat).
  • FIG. 19B binding activity to CD19 + B cells lymphoma (Raji).
  • FIG. 19C cytotoxicity of armed T cells relative to T cells cultured with OKT3. The cells were included with
  • CTA03Fab/CTAT02scFv, CTA03-01 Fab/CTAT02-01 scFv, CTA03-01 Fab/CTAT02-02scFv, CTA03-02Fab/CTAT02-02scFv and CTA03-02Fab/CTAT02-01scFv BsAbs and then stained with FITC conjugated Goat anti-Human IgG Fab antibody. Fluorescent signal on cell surface was detected using flow cytometry. For cytotoxicity analysis, T cells were cultured with OKT3 or with the various BsAbs to form armed T cells.
  • the cells were then cocultured with CD19 + B cell lymphoma (Raji) at several effector cell: target cell ratios (3:1, 5:1 and 10:1) for 18 hr. Tumor cell death was determined with CytoTox 96 ® Non-Radioactive Cytotoxicity Assay (Promega, G1780).
  • bispecific antibodies capable of binding to CD3 (e.g., human CD3) and a tumor associated antigen (TAA).
  • BsAbs are capable of attaching to the surface of CD3-positive immune cells via binding of the anti-CD3 moiety in the BsAb to the cell surface CD3 to produce armed immune cells.
  • an armed immune cell refers to an immune cell that displays a bispecific antibody as disclosed herein via binding of the anti-CD3 moiety in the bispecific antibody to a cell surface CD3 molecule.
  • an armed immune cell is capable of targeting disease cells (e.g., cancer cells) that express the TAA, thereby eliciting immune responses against the disease cells.
  • the BsAbs disclosed herein show high binding activities to both CD3 + immune cells and TAA + cancer cells and high retention levels on CD3 + immune cells for at least 72 hours. Immune cells armed with the BsAbs disclosed herein exhibited high cytotoxicity against cancer cells expressing the corresponding TAA both in vitro and in vivo. Thus, the BsAbs and the armed immune cells disclosed herein would be expected to have high anti-cancer effects.
  • bispecific antibodies capable of binding to CD3 and an TAA, armed immune cells displaying such, methods of using the bispecific antibodies for producing armed immune cells, and methods of treating cancer using the armed immune cells.
  • the present disclosure provides bispecific antibodies capable of binding to CD3 (e.g., CD3+ cells) and a tumor associated antigen (TAA) (e.g., cancer cells expressing the TAA on cell surface).
  • An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • a bispecific antibody as disclosed herein comprises two antigen-binding moieties, one of which binds CD3 such as human CD3 and the other one of which binds a tumor associated antigen such as those disclosed herein.
  • a typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding.
  • VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Rabat definition, the Chothia definition, the AbM definition, and/or the contact definition, all of which are well known in the art. See, e.g., Rabat, E.A., et al. (1991)
  • an antibody moiety disclosed herein may share the same heavy chain and/or light chain complementary determining regions (CDRs) or the same VH and/or VL chains as a reference antibody.
  • CDRs heavy chain and/or light chain complementary determining regions
  • Two antibodies having the same VH and/or VL CDRS means that their CDRs are identical when determined by the same approach (e.g., the Rabat approach, the Chothia approach, the AbM approach, the Contact approach, or the IMGT approach as known in the art. See, e.g., bioinf.org.uk/abs/).
  • Such anti-CD19 antibodies may have the same VH, the same VL, or both as compared to an exemplary antibody described herein.
  • an antibody moiety disclosed herein may share a certain level of sequence identity as compared with a reference sequence.
  • the “percent identity” of two amino acid sequences is determined using the algorithm of Rarlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Rarlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990.
  • an antibody moiety disclosed herein may have one or more amino acid variations relative to a reference antibody.
  • the amino acid residue variations as disclosed in the present disclosure can be conservative amino acid residue substitutions.
  • a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. , Molecular Cloning: A Laboratory Manual, J.
  • the bispecific antibodies disclosed herein comprise a CD3 binding moiety (anti-CD3 moiety) and a TAA binding moiety (anti-TAA moiety).
  • the anti-CD3 moiety in any of the bispecific antibodies disclosed herein comprises an antigen-binding fragment specific to a CD3 molecule, for example, human CD3.
  • the anti-CD3 moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL).
  • the anti-CD3 moiety may be derived from a reference anti-CD3 antibody.
  • Exemplary reference anti-CD3 antibodies include CTA.02, CTA.03, CTA.04, or CTA.05.
  • the structural information of these reference anti-CD3 antibodies are provided in Table 1 below (heavy chain and light chain complementary determining regions (CDRs) based on the Rabat scheme are in boldface and underlined).
  • an anti-CD3 binding moiety (and an anti-TAA binding moiety disclosed below) derived from a reference antibody refers to binding moieties having substantially similar structural and functional features as the reference antibody.
  • the binding moiety may have the same heavy and/or light chain complementary determining regions or the same VH and/or VL chains as the reference antibody.
  • the binding moiety may only have a limited number of amino acid variations in one or more of the framework regions and/or in one or more of the CDRs without significantly affecting its binding affinity and binding specificity relative to the reference antibody.
  • the anti-CD3 binding moiety may comprise the same heavy chain CDRs as those in antibody CTA.02, which are provided in Table 1 above.
  • the anti-CD3 binding moiety may have the same light chain CDRs as those in antibody CTA.02, which are also provided in Table 1 above.
  • Such an anti-CD3 binding moiety may comprise the same VH and/or VL chains as CTA.02.
  • the anti-CD3 binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTA.02.
  • the anti-CD3 binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more framework regions relative to the corresponding framework regions in CTA.02.
  • the anti-CD3 moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTA.02.
  • the anti-CD3 moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTA.02.
  • the anti-CD3 antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTA.02.
  • “individually” means that one CDR of an antibody shares the indicated sequence identity relative to the corresponding CDR of a reference antibody (e.g., the anti-CD3 reference antibodies provided in Table 1 above or any of the anti-TAA reference antibodies disclosed below). “Collectively” means that three VH or VL CDRS of an antibody in combination share the indicated sequence identity relative the corresponding three VH or VL CDRS of the reference antibody in combination.
  • the anti-CD3 moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTA.02.
  • the anti-CD3 moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTA.02 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-CD3 binding moiety may comprise the same heavy chain CDRs as those in antibody CTA.03, which are provided in Table 1 above.
  • the anti-CD3 binding moiety may have the same light chain CDRs as those in antibody CTA.03, which are also provided in Table 1 above.
  • Such an anti-CD3 binding moiety may comprise the same VH and/or VL chains as CTA.03.
  • the anti-CD3 binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTA.03.
  • the anti-CD3 binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more framework regions relative to the corresponding framework regions in CTA.03.
  • the anti-CD3 moiety disclosed herein comprises a mutation at position G58 of the VL chain relative to CTA.03, for example, an amino acid residue substitution (e.g., G58A). See, e.g., CTA.03 VL-01 in Table 1 above.
  • the anti-CD3 moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTA.03.
  • the anti-CD3 moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTA.03.
  • the anti-CD3 antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTA.03.
  • the anti-CD3 moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTA.03.
  • the anti-CD3 moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTA.03 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-CD3 moiety disclosed herein may comprise a mutation at position D57 of the VL chain relative to that of CTA.03, for example, an amino acid residue substitution such as D57E. See, e.g., CTA.03 VL-02 in Table 1.
  • the anti-CD3 binding moiety may comprise the same heavy chain CDRs as those in antibody CTA.04, which are provided in Table 1 above.
  • the anti-CD3 binding moiety may have the same light chain CDRs as those in antibody CTA.04, which are also provided in Table 1 above.
  • Such an anti-CD3 binding moiety may comprise the same VH and/or VL chains as CTA.04.
  • the anti-CD3 binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTA.04.
  • the anti-CD3 binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more framework regions relative to the corresponding framework regions in CTA.04.
  • amino acid variations e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations
  • the anti-CD3 moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTA.04.
  • the anti-CD3 moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTA.04.
  • the anti-CD3 antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTA.04.
  • the anti-CD3 moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTA.04.
  • the anti-CD3 moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTA.04 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-CD3 binding moiety may comprise the same heavy chain CDRs as those in antibody CTA.05, which are provided in Table 1 above. Alternatively or in addition, the anti-CD3 binding moiety may have the same light chain CDRs as those in antibody CTA.05, which are also provided in Table 1 above.
  • Such an anti-CD3 binding moiety may comprise the same VH and/or VL chains as CTA.05.
  • the anti-CD3 binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTA.05.
  • the anti-CD3 binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-CD3 moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTA.05.
  • the anti-CD3 moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTA.05.
  • the anti-CD3 antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTA.05.
  • the anti-CD3 moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTA.05.
  • the anti-CD3 moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTA.05 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • any of the bispecific antibodies disclosed herein further comprises a second binding moiety specific to a tumor associated antigen.
  • TAA tumor-associated antigen
  • TAA tumor-associated antigen
  • Non-limiting examples of TAA include CD5, CD19, CD20, CD22, CD23,
  • the anti-TAA binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL).
  • the anti-TAA binding moiety is specific to CD20 (e.g., human CD20).
  • the anti-TAA binding moiety is specific to CD19 (e.g., human CD19).
  • the anti-TAA binding moiety is specific to EGFR (e.g., human EGER).
  • the anti-TAA binding moiety is specific to HER2 (e.g., human HER2).
  • the anti-TAA binding moiety is specific to PSMA (e.g., human PSMA).
  • the anti-TAA binding moiety is specific to CEA (e.g., human CEA). In some examples, the anti-TAA binding moiety is specific to EpCAM (e.g., human EpCAM). In some examples, the anti-TAA binding moiety is specific to FAP (e.g., human FAP). In some examples, the anti-TAA binding moiety is specific to PDL1 (e.g., human PDL1). In some examples, the anti-TAA binding moiety is specific to CD38 (e.g., human CD38). In some examples, the anti-TAA binding moiety is specific to CD33 (e.g., human CD33).
  • the anti-TAA binding moiety is specific to HGFR (cMET) (e.g., human cMET). In some examples, the anti-TAA binding moiety is specific to CD47 (e.g., human CD47). In some examples, the anti-TAA binding moiety is specific to TRAIL- R2 (e.g., human TRAIL- R2). In some examples, the anti-TAA binding moiety is specific to mesothelin (e.g., human mesothelin). In some examples, the anti-TAA binding moiety is specific to GD2 (e.g., human GD2). In some instances, the anti-TAA moiety may be derived from a reference anti-TAA antibody.
  • Exemplary reference anti-TAA antibodies include CTAT.01-CTAT.16.
  • the structural information of these reference anti-CD3 antibodies are provided in Table 2 below (heavy chain and light chain complementary determining regions (CDRs) based on the Rabat scheme are in boldface and underlined).
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.01, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.01, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.01. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.01.
  • the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more framework regions relative to the corresponding framework regions in CTAT.01.
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.01.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.01.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.01.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.01.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.01 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.02, which are provided in Table 2 above.
  • the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.02, which are also provided in Table 2 above.
  • Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.02.
  • the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.02.
  • the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety disclosed herein comprises a mutation at position G42 of the VL chain relative to CTAT.02, for example, an amino acid residue substitution (e.g., G42A). See, e.g., CTAT.02 VL-01 in Table 2 above.
  • the anti-TAA moiety disclosed herein comprises a mutation at position D41 of the VL chain relative to CTAT.02, for example, an amino acid residue substitution (e.g., D41E). See, e.g., CTAT.02 VL-02 in Table 2 above.
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.02.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.02.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.02.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.02.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.02 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.03, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.03, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.03. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.03. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.03.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.03.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.03.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.03.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.03 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.04, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.04, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.04. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.04. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.04.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.04.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.04.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.04.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.04 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.05, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.05, which are also provided in Table 2 above.
  • Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.05.
  • the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.05.
  • the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.05.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.05.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.05.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.05.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.05 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.06, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.06, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.06. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.06. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.06.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.06.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.06.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.06.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.06 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.07, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.07, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.07. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.07. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.07.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the V H CDRS of CTAT.07.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.07.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.07.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.07 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.08, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.08, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.08. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.08. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.08.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.08.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.08.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.08.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.08 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.09, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.09, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.09. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.09. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.09.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.09.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.09. In some instances, the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.09 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.10, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.10, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.10. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.10. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.10.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the V H CDRS of CTAT.10.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.10.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.10. In some instances, the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.10 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.l 1, which are provided in Table 2 above.
  • the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.ll, which are also provided in Table 2 above.
  • Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.ll.
  • the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.l 1.
  • the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.l 1.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.ll.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.10.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.ll.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.l 1 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.12, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.12, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.12. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.12. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.12.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.12.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.12.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.12.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.12 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.13, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.13, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.13. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.13. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.13.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the V H CDRS of CTAT.13.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.13.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.13.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.13 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.14, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.14, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.14. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.14. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.14.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.14.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.14.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.14.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.14 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.15, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.15, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.15. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.15. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.15.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.15.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.15.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.15.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.15 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the anti-TAA binding moiety may comprise the same heavy chain CDRs as those in antibody CTAT.16, which are provided in Table 2 above. Alternatively or in addition, the anti-TAA binding moiety may have the same light chain CDRs as those in antibody CTAT.16, which are also provided in Table 2 above. Such an anti-TAA binding moiety may comprise the same VH and/or VL chains as CTAT.16. Alternatively, the anti-TAA binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in CTAT.16. For example, the anti-TAA binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10,
  • the anti-TAA moiety may comprise a certain level of variations in one or more of the CDRs relative to those of CTAT.16.
  • the anti-TAA moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of CTAT.16.
  • the anti-TAA antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as CTAT.16.
  • the anti-TAA moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of CTAT.16.
  • the anti-TAA moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of CTAT.16 and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.
  • the bispecific antibody disclosed herein may be in any suitable format as those known in the art, for example, those disclosed in Mol. Immunol. 67(2):95-106 (2015), the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. Some examples are provided below. See also FIGs. 1A-1N.
  • the bispecific antibody disclosed herein may comprise one antigen binding moiety in Fab format and the other antigen binding moiety in single chain variable fragment (scFv) format.
  • a bispecific antibody may comprise two polypeptides, one comprising the heavy or light chain of the Fab fragment linked to the scFv fragment and the other comprising the light or heavy chain of the Fab that is not linked to the scFv fragment.
  • a Fab fragment comprises two polypeptide chains, one comprising a VH domain linked to a fragment of a heavy chain constant region (e.g., CHI) and the other one comprising a VL domain linked to a light chain constant region.
  • the heavy chain constant region fragment may be from any Ig subclass, for example, IgG, IgA, IgE, IgD, or IgM.
  • the heavy chain constant region fragment is from an IgG molecule (e.g., a human IgG molecule).
  • the light chain constant region may be a kappa chain or a lambda chain (e.g., a human kappa or lambda chain).
  • an scFv fragment comprises a VH domain and a VL domain linked by a peptide linker. See e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883.
  • the scFv fragment has, form N-terminus to C-terminus, the VH-linker-VL orientation.
  • the scFv fragment has, form N-terminus to C-terminus, the VL-linker-VH orientation.
  • the scFv fragment may be linked to the heavy chain of the Fab fragment.
  • the scFv may be linked to the light chain of the Fab fragment. See FIGs. 1A-1H.
  • the bispecific antibody disclosed herein may comprise the anti-CD3 binding moiety in Fab format and the anti-TAA binding moiety in scFv format. Exemplary illustrations are provided in FIGs. 1A-1D.
  • the anti-CD3 Fab comprises a heavy chain VH-CH1 domain and a light chain VL-CK or VL-Ck domain.
  • the anti-TAA scFv comprises a VH domain and a VL domain. FIGs. 1A-1D.
  • the anti-CD3 Fab may be linked to the anti-TAA scFv via a peptide linker disposed between the CHI domain of the anti-CD3 Fab heavy chain and the VH domain of the anti-tumor scFv.
  • a peptide linker disposed between the CHI domain of the anti-CD3 Fab heavy chain and the VH domain of the anti-tumor scFv.
  • FIG. 1A An exemplary illustration is provided in FIG. 1A.
  • the CHI domain of the anti-CD3 Fab heavy chain can be linked to the VL domain of the anti-tumor scFv as illustrated in FIG. IB.
  • the anti-TAA scFv can be linked to the CK or Ck domain of the anti-CD3 Fab light chain via the VL domain of the scFv (FIG. 1C), or via the VH domain of the anti-tumor scFv (FIG. ID).
  • VH-CH1 and VL-Ck anti-CD3 Fab heavy chain
  • VL-Ck light chains
  • anti-TAA scFv fragments are provided in Tables 1 and 2, respectively. Any combination of such is within the scope of the present disclosure.
  • the bispecific antibody disclosed herein may comprise the anti-TAA binding moiety in Fab format and the anti-CD3 binding moiety in scFv format.
  • Exemplary illustrations are provided in FIGs. 1E-1H.
  • the anti-TAA Fab comprises a heavy chain VH-CH1 domain and a light chain VL-CK or VL-Ck domain.
  • the anti-CD3 scFv comprises a VH domain and a VL domain.
  • the anti-TAA Fab may be linked to the anti-CD3 scFv via a peptide linker disposed between the CHI domain of the anti-TAA Fab heavy chain and the VH domain of the anti-CD3 scFv.
  • the CHI domain of the anti-TAA Fab heavy chain can be linked to the VL domain of the anti-CD3 scFv as illustrated in FIG. IF.
  • the anti-CD3 scFv can be linked to the CK or Ck domain of the anti-TAA Fab light chain via the VL domain of the scFv (FIG. 1G), or via the VH domain of the anti-CD3 scFv (FIG. 1H).
  • Examples of anti-TAA Fab heavy chain (VH-CH1) and light chains (VL-Ck) and examples of anti-CD3 scFv fragments are provided in Tables 2 and 1, respectively. Any combination of such is within the scope of the present disclosure.
  • the bispecific antibody disclosed herein may comprise both antigen binding moieties in scFv format. Exemplary illustrations are provided in FIGs. II to 1L.
  • the VH domain of anti-CD3 scFv may be linked to the VH domain of the anti-TAA scFv via a peptide linker (FIG. II).
  • the VH domain of anti-CD3 scFv may be linked to the VL domain of the anti-TAA scFv via a peptide linker (FIG. 1J).
  • the VL domain of anti-CD3 scFv may be linked to the VH domain of the anti-TAA scFv via a peptide linker (FIG. IK).
  • the VL domain of anti-CD3 scFv may be linked to the VH domain of the anti-TAA scFv via a peptide linker (FIG. 1L).
  • a peptide linker FIG. 1L
  • Exemplary anti-CD3 scFv fragments and exemplary anti-TAA scFv fragments are provided in Tables 1 and 2, respectively. Any combination thereof for constructing a bispecific antibody is within the scope of the present disclosure.
  • the bispecific antibodies disclosed herein may comprise one or more Fc regions, which may optionally a “knob into hole” structure, in which a knob in the CH2 domain, the CH3 domain, or both of the first heavy chain is created by replacing several amino acid side chains with alternative ones, and a hole in the juxtaposed position at the CH3 domain of the second heavy chain is created by replacing appropriate amino acid side chains with alternative ones. Exemplary illustrations are provided in FIGs. 1M and IN.
  • knob and a hole or “knobs-into-holes” are used interchangeably herein.
  • Knobs-into-holes amino acid changes is a rational design strategy known in the art for heterodimerization of the heavy (H) chains in the production of bispecific IgG antibodies. Carter, J. Immunol. Methods, 248(l-2):7-15 (2001), the relevant disclosures of which are incorporated by reference herein for the purpose and subject matter referenced herein.
  • the "knobs-into-holes" provides an approach as described in, e.g., Ridgway JBB et al, (1996) Protein Engineering, 9(7): 617-21 and US 5,731,168, the relevant disclosures of each of which are incorporated by reference herein for the purpose and subject matter referenced herein.
  • This approach has been shown to promote the formation of heterodimers of the first polypeptide and the second polypeptide chain, and hinder the assembly of corresponding homodimers.
  • a knob is created by replacing small amino side chains at the interface between CH3 domains with larger ones, whereas a hole is constructed by replacing large side chains with smaller ones.
  • the "knob” mutation comprises T366W and the "hole” mutations comprise T366S, L368A and Y407V (Atwell S et al, (1997) J. Mol. Biol. 270: 26-35).
  • the bispecific antibody may comprise an anti-CD3 binding moiety comprising a first VH-CH1-CH2-CH3 domain and a first VL-CK or VL-Ck domain, and an anti-TAA binding moiety comprising a second VH-CH1-CH2-CH3 domain and second a VL-CK or VL-Ck domain.
  • FIG. 1M The CH2 and/or CH3 in the heavy chain of the anti-CD3 binding moiety that those in the heavy chain of the anti-TAA binding moiety may comprise the knob/hole modifications, allowing for the binding between the two heavy chains.
  • the bispecific antibody may comprise an anti-Cd3 binding moiety comprising a first VH-CH1-CH2-CH3 domain and a first VL-CK or VL-C/. domain, and an anti-TAA scFv linked to a second CH2-CH3 domain.
  • the CH2 and/or CH3 in the heavy chain of the anti-CD3 binding moiety that those in the anti-TAA binding moiety may comprise the knob/hole modifications, allowing for the binding between the two heavy chains.
  • FlGs. IN. In this setting, the format of the anti-CD3 binding moiety and the format of the anti-TAA binding moiety may be switched.
  • peptide linker refers to a peptide having natural or synthetic amino acid residues for connecting two polypeptides.
  • the peptide linker may be used to connect one VH domain and one VL domain to form a single chain variable fragment (e.g.
  • scFv to connect one scFv and one Fab to form a scFv/Fab recombinant antibody; to connect two scFvs to form a scFv/scFv recombinant antibody; or to connect two monovalent antibodies (e.g., two monovalent IgGs), two monovalent antibody fragments (e.g., two monovalent scFv-Fc fusion proteins), or one monovalent antibody and one monovalent antibody fragment (e.g., one monovalent IgG and on monovalent scFv-Fc fusion protein) thereby forming a divalent antibody.
  • monovalent antibodies e.g., two monovalent IgGs
  • two monovalent antibody fragments e.g., two monovalent scFv-Fc fusion proteins
  • monovalent antibody and one monovalent antibody fragment e.g., one monovalent IgG and on monovalent scFv-Fc fusion protein
  • the peptide linker is a peptide having at least 5 amino acid residues in length, such as 5 to 100 amino acid residues in length; more preferably, 10 to 30 amino acid residues in length.
  • the peptide linker within scFv is a peptide of at least 5 amino acid residues in length, preferably 15 to 20 amino acid residues in length.
  • the linker comprises a sequence of (G2S)4.
  • the linker comprises a sequence or (G4S)3.
  • the peptide linker for linking the first antibody fragment (/. ⁇ ? ., anti-CD3 antibody fragment) and the second antibody fragment (/. ⁇ ? ., anti-TAA antibody fragment) may be any peptide suitable for connecting two polypeptides.
  • the peptide linker is a peptide having at least 5 amino acid residues in length, for example, having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
  • the peptide linker of the present recombinant antibody consists of 10 to 30 glycine (G) and/or serine (S) residues.
  • the bispecific antibodies described herein specifically bind to one or both of the corresponding target antigen (CD3 and a TAA) or an epitope thereof.
  • An antibody that “specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets.
  • An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • an antibody that specifically (or preferentially) binds to an antigen (CD3 and/or a TAA) or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding.
  • an antibody that “specifically binds” to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen (i.e.., only baseline binding activity can be detected in a conventional method).
  • a bispecific antibody as described herein has a suitable binding affinity for one or both of the target antigens (e.g., CD3 and a TAA) or antigenic epitopes thereof.
  • binding affinity refers to the apparent association constant or KA.
  • the KA is the reciprocal of the dissociation constant (KD).
  • the bispecific antibody described herein may have a binding affinity (KD) of at least 100 nM, lOnM, InM, 0.1 nM, or lower for CD3 (e.g., lower than InM or O.lnM).
  • the bispecific antibody described herein may have a binding affinity (KD) of at least 100 nM, lOnM, InM, 0.1 nM, or lower for the TAA.
  • An increased binding affinity corresponds to a decreased KD.
  • Higher affinity binding of an antibody for a first antigen relative to a second antigen can be indicated by a higher KA (or a smaller numerical value KD) for binding the first antigen than the KA (or numerical value KD) for binding the second antigen.
  • the antibody has specificity for the first antigen (e.g., a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein).
  • Differences in binding affinity can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 90, 100, 500, 1000, 10,000 or 10 5 fold.
  • any of the anti-CD3 and/or anti-TAA antibodies for making the bispecific antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.
  • Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay).
  • Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration.
  • the concentration of bound binding protein [Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation:
  • [Bound] [Free]/(Kd+[Free]) It is not always necessary to make an exact determination of K A , though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K A , and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g. , an in vitro or in vivo assay.
  • a functional assay e.g. , an in vitro or in vivo assay.
  • Exemplary bispecific antibodies as disclosed herein are provided in Table 3 below (using anti-CD3 binding moieties from CTA.03 as examples).
  • Anti-CD3 binding moieties from other anti-CD3 reference antibodies e.g., CTA.02, CTA.04, and CTA.05 are also within the scope of the present disclosure.
  • Table 3 Exemplary Bispecific Antibodies
  • compositions comprising any of the bispecific antibodies disclosed herein (or the armed immune cells also disclosed herein), which further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient may be any inert substance that is combined with an active molecule (such as the bispecific antibody or the armed immune cells) for preparing an agreeable or convenient dosage form.
  • the pharmaceutically acceptable excipient is non-toxic to recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation comprising the recombinant antibody.
  • the pharmaceutically acceptable excipient suitable to be employed in the present pharmaceutical composition include, but are not limited to, water, phosphate buffer, acetate buffer, succinate buffer, citrate buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, phosphate-buffered saline (PBS), Ringer’s solution, lactated Ringer’s solution, and a combination thereof.
  • the pharmaceutical composition may further comprise an agent for storing and/or stabilizing the recombinant antibody, e.g., amino acid reside (such as, histidine (H) or serine (S) residue), glucose, galactose, xylitol, sorbitol, mannitol, sucrose, trehalose, or antioxidant.
  • Other agents may also be added, such as antimicrobial agents, to prevent spoilage upon storage, /. ⁇ ? ., to inhibit growth of microbes such as yeasts and molds.
  • the bispecific antibodies described herein can be made by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
  • the anti-CD3 antibody and/or the anti-TAA antibody for use in making the bispecific antibodies may be produced by the conventional hybridoma technology.
  • the anti-CD3 and/or anti-TAA antibody may be identified from a suitable library (e.g. , a human antibody library).
  • high affinity fully human CD3 and/or TAA binders may be obtained from a human antibody library, for example, affinity maturation libraries (e.g., having variations in one or more of the CDR regions).
  • affinity maturation libraries e.g., having variations in one or more of the CDR regions.
  • the bispecific antibodies disclosed herein may be produced by the conventional recombinant technology.
  • DNA encoding a monoclonal antibodies specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • the DNA may be placed into one or more expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • nucleic acids encoding the one or both chains of a bispecific antibody as described herein can be cloned into one expression vector, each nucleotide sequence being in operable linkage to a suitable promoter.
  • each of the nucleotide sequences encoding the heavy chain and light chain is in operable linkage to a distinct prompter.
  • the nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter.
  • an internal ribosomal entry site IRS
  • the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells.
  • the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolated heavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of the antibody.
  • a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art.
  • the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors/promoter would depend on the type of host cells for use in producing the antibodies.
  • promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk vims promoter.
  • CMV cytomegalovirus
  • a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR
  • SV40 simian virus 40
  • E. coli lac UV5 promoter E. coli lac UV5 promoter
  • herpes simplex tk vims promoter the herpes simplex tk vims promoter.
  • Regulatable promoters can also be used.
  • Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad.
  • Regulatable promoters that include a repressor with the operon can be used.
  • the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci.
  • tetracycline repressor tetR
  • VP 16 transcription activator
  • tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells.
  • hCMV human cytomegalovirus
  • a tetracycline inducible switch is used.
  • tetracycline repressor alone, rather than the tetR-mammalian cell transcription factor fusion derivatives can function as potent trans -modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy, 10(16):1392-1399 (2003)).
  • tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.
  • the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
  • a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
  • enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
  • transcription termination and RNA processing signals from SV40 for mRNA stability
  • SV40 polyoma origins of replication and ColEl for proper episomal replication
  • polyadenylation signals useful to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.
  • FIGs. 2A-2E Exemplary constructs for producing the bispecific antibodies in various configuration as disclosed herein are provided in FIGs. 2A-2E.
  • One or more vectors comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the antibodies.
  • the host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof.
  • Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification.
  • polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.
  • methods for preparing an antibody described herein involve a recombinant expression vector that encodes both chains of a bispecific antibody as described herein.
  • the recombinant expression vector can be introduced into a suitable host cell (e.g., a dhfr- CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection.
  • a suitable host cell e.g., a dhfr- CHO cell
  • Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium.
  • the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.
  • two recombinant expression vectors are provided, each encoding one chain of a bispecific antibody disclosed herein.
  • Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr- CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection.
  • each of the expression vectors can be introduced into a suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of the antibody.
  • the antibody produced therein can be recovered from the host cells or from the culture medium.
  • the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of the antibody.
  • the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of the antibody.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium.
  • some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.
  • any of the nucleic acids encoding the bispecific antibody as described herein, vectors (e.g., expression vectors) containing such; and host cells comprising the vectors are within the scope of the present disclosure.
  • Methods for producing such bispecific antibodies e.g., using host cells via the recombinant technology are also within the scope of the present disclosure.
  • immune cells armed with any of the bispecific antibodies disclosed herein (e.g., those comprising Fab fragment and/or scFv chains provided in Tables 1 and 2, or the exemplary bispecific antibodies provided in Table 3 above).
  • the bispecific antibody can be displayed on the surface of CD3+ immune cells via binding to the cell surface CD3 molecule.
  • the immune cells may be any type of immune cells (e.g., human immune cells) expressing surface CD3 or a mixture thereof. Examples include, but are not limited to, T cell, a B cell, a monocyte, and/or macrophage. In some instances, the T cell is a traditional CD4+ and/or CD8+ T cells. In some instances, the T cell is a regulatory T cell (Treg). In other instances, the T cell is a natural killer T cell (NKT).
  • the immune cells may be obtained from a donor such as a humor donor (e.g., a healthy donor). Alternatively, the immune cells may be obtained from a cell line or differentiated from stem cells, for example, hematopoietic stem cells, bone marrow cells, umbilical cord blood cells, or induced pluripotent stem cells.
  • any of the armed immune cells may be produced by incubating suitable immune cells with any of the bispecific antibodies disclosed herein (e.g., those comprising Fab fragment and/or scFv chains provided in Tables 1 and 2, or the exemplary bispecific antibodies provided in Table 3 above) under suitable conditions for a suitable period of time.
  • anti-CD3 antibody alone e.g., OKT3
  • incubation of the bispecific antibodies disclosed herein with immune cells result in production of armed immune cells having the bispecific antibody displayed on the cell surface.
  • the bispecific antibody may induce proliferation and/or differentiation of immune cells, such as induce differentiation of naive T cells into effector cells via binding to the CD3 molecule on T cells via its anti-CD3 binding moiety.
  • the armed immune cells thus produced is capable of targeting cancer cells via recognizing the TAA molecule expressed on the cancer cells by the anti-TAA binding moiety of the bispecific antibody, which is displayed on the surface of the armed immune cells.
  • the armed immune cells disclosed herein can be produced using peripheral blood mononuclear cells (PBMCs).
  • PBMCs can be isolated from a donor (e.g., a human donor) using a conventional method.
  • the methods suitable for isolating PBMCs from a donor include, but are not limited to, density centrifugation (e.g., FICOLL ® Paque), cell preparation tube (CPT), and SEPMATETM tube.
  • the PBMCs can be isolated from a whole blood sample obtained from a donor via density centrifugation according to the manufacturer’s directions.
  • the isolated PBMCs can then be cultivated with the bispecific antibody in a suitable cell culture medium for least 7 days, such as 7, 8, 9, 10, 11, 12, 13 14, or more days; preferably, for at least 14 days.
  • a suitable cell culture medium for least 7 days, such as 7, 8, 9, 10, 11, 12, 13 14, or more days; preferably, for at least 14 days.
  • the number of CD3 + immune cells such as T cells (e.g., CD4/CD8 T cells and/or NKT cells) multiplies after cultivation for 7 days.
  • cultivation is continued for 14 days, and the number of CD3 + T cells increases for 3 folds.
  • immune cells from cell culture may be used for making the armed immune cells disclosed herein.
  • the in vitro cultured immune cells may be from an established cell line.
  • the immune cells may be differentiated from suitable stem cells, for example, hematopoietic stem cells, bone marrow cells, umbilical cord blood cells, or induced pluripotent stem cells, following conventional methods.
  • a suitable amount of immune cells may be cultured in a suitable cell culture medium in the presence of about 500 ng to about 3,000 ng (e.g., 500, 600, 700,
  • the cell culture medium may comprise one or more cytokines for sustaining the growth of immune cells such as T cells and/or stimulating the activation of the immune cells. Examples include, but are not limited to, IL-Ib, IL-2, IL-4, IL-6, IL-7, IL-12, IL-18, IL-21, IL-23, IL-25, IL-27, IL-31, interferon-gamma (IFN-g), TGF-b, or a combination thereof. Additionally or alternatively, the medium may comprise an antibody or a carbohydrate for the activation purpose, such as an anti-CD28 antibody or a mannose.
  • cytokines for sustaining the growth of immune cells such as T cells and/or stimulating the activation of the immune cells. Examples include, but are not limited to, IL-Ib, IL-2, IL-4, IL-6, IL-7, IL-12, IL-18, IL-21, IL-23, IL-25, IL-27, IL-31, interfer
  • IL-2 may be used in the culture medium to cultivate PBMCs to produce, e.g., armed CD8 + T cells.
  • IL-2 and IL-7 may be used in the culture medium to cultivating PBMCs for producing, e.g., armed CD4+ T cells.
  • IL-2, an anti-CD28 antibody, and mannose may be used in the cell culture medium.
  • armed immune cells produced by any of the methods disclosed herein are also within the scope of the present disclosure.
  • the present disclosure provides a method for treating cancer using the armed immune cells disclosed herein.
  • an effective amount of the armed immune cells or a pharmaceutical composition comprising such can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time.
  • a suitable route such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time.
  • the armed immune cells are autologous to the subject.
  • the armed immune cells are allogenic to the subject.
  • the subject to be treated by the methods described herein can be a mammal, more preferably a human or a non-human primate. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.
  • a human subject who needs the treatment may be a human patient having, at risk for, or suspected of having a target disease/disorder characterized by carrying tumor cells expressing the target TAA, to which a bispecific antibody binds.
  • Exemplary cancers include, but are not limited to, melanoma, esophageal carcinoma, gastric carcinoma, brain tumor, small cell lung cancer, non-small cell lung cancer, bladder cancer, breast cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, renal cancer, hepatocellular carcinoma, ovary cancer, prostate cancer, thyroid cancer, testis cancer, head and neck squamous cell carcinoma, leukemia, lymphoma, and myeloma.
  • B-cell malignancies often involve CD19+ (e.g., B-cell acute lymphoblastic leukemia) and/or CD20+ cancer cells (e.g., B-cell Non-Hodgkin’s lymphoma).
  • EGFR is expressed on various types of cancer, such as lung cancer and colon cancer.
  • HER2 is associated with, for example, breast cancer.
  • PSMA is associated, for example, prostate cancer.
  • CEA is associated with various types of cancer, including colon, rectum, and pancreatic cancer.
  • EpCAM, FAP, CD47, and TRAIL-R2 are associated with solid tumors.
  • PDL1 is associated with various cancers, such as bladder cancer, non-small cell lung cancer, breast cancer, small cell lung cancer, etc.
  • CD38 is associated with, for example, multiple myeloma.
  • CD33 is associated with, for example, AML.
  • cMET HGFR
  • Mesothelin is associated with mesothelioma.
  • GD2 is associated with neuroblastoma. Accordingly, choosing a bispecific antibody disclosed herein that has a suitable anti-TAA binding moiety to treat a particular type of cancer is within the knowledge of a medical practitioner.
  • a subject having a target cancer can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds.
  • the subject to be treated by the method described herein may be a human cancer patient who has undergone or is subjecting to an anti-cancer therapy, for example, chemotherapy, radiotherapy, immunotherapy, or surgery.
  • a subject suspected of having any of such target disease/disorder might show one or more symptoms of the disease/disorder.
  • a subject at risk for the disease/disorder can be a subject having one or more of the risk factors for that disease/disorder.
  • an effective amount refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Determination of whether an amount of the antibody achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
  • Empirical considerations such as the half-life, generally will contribute to the determination of the dosage.
  • antibodies that are compatible with the human immune system such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system.
  • Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a target disease/disorder.
  • sustained continuous release formulations of an antibody may be appropriate.
  • formulations and devices for achieving sustained release are known in the art.
  • dosages for an antibody as described herein may be determined empirically in individuals who have been given one or more administration(s) of the antibody. Individuals are given incremental dosages of the agonist. To assess efficacy of the agonist, an indicator of the disease/disorder can be followed.
  • the particular dosage regimen, dose, timing and repetition will depend on the particular individual and that individual's medical history, as well as the properties of the individual agents (such as the half-life of the agent, and other considerations well known in the art).
  • the appropriate dosage of armed immune cells as described herein will depend on the specific bispecific antibody on the immune cells, the type of immune cells (or compositions thereof) employed, the type and severity of the disease/disorder, the patient's clinical history and response to the agonist, and the discretion of the attending physician.
  • the clinician will administer armed immune cells, until a dosage is reached that achieves the desired result. Methods of determining whether a dosage resulted in the desired result would be evident to one of skill in the art.
  • Administration of one or more doses of armed immune cells can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.
  • the administration of the armed immune cells may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a target disease or disorder.
  • the amount of the armed immune cells such as armed T cells administered to the subject can be about lxlO 4 to lxlO 7 cells/Kg body weight of the subject. In certain embodiments, the amount of armed immune cells such as armed T cells can be administered to the subject from about lxlO 5 to lxlO 6 cells/Kg body weight of the subject.
  • the dose can be administered in a single dose, or alternatively in more than one dose.
  • treating refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder.
  • Alleviating a target disease/disorder includes delaying the development or progression of the disease, or reducing disease severity or prolonging survival. Alleviating the disease or prolonging survival does not necessarily require curative results.
  • "delaying" the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated.
  • a method that “delays” or alleviates the development of a disease, or delays the onset of the disease is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
  • “Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and/or recurrence.
  • armed immune cells can be administered via intravenous infusion.
  • the armed immune cells disclosed herein may be co-used with another anti-cancer agent, for example, a chemotherapeutic agent, an immunotherapeutic agent, or a combination thereof.
  • the armed immune cells disclosed herein may be used in combination with an immune checkpoint inhibitor, such as an anti-PD- 1 antibody or an anti-PDLl antibody.
  • an immune checkpoint inhibitor such as an anti-PD- 1 antibody or an anti-PDLl antibody.
  • the term “combination,” “combined,” and related terms refers to the simultaneous or sequential administration of multiple therapeutic agents in accordance with this disclosure.
  • the armed immune cells as disclosed herein may be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form.
  • the PBMCs can be isolated from the subject.
  • the subject may be any mammal, for example, a human, mouse, rat, chimpanzee, rabbit, monkey, sheep, goat, cat, dog, horse, or pig.
  • the subject is a human.
  • the methods suitable for isolating PBMCs from the subject include, but are not limited to, density centrifugation (e.g., FICOLL ® Paque), cell preparation tube (CPT), and SEPMATETM tube.
  • the isolated PBMCs are cultured in a medium containing the present recombinant antibody for a sufficient period of time (e.g., at least 7 days) so as to produce the TAA-specific T cells.
  • the bispecific antibody is capable of inducing the activation of T cells by its anti-CD3 antibody fragment.
  • the thus-produced armed T cells has the bispecific antibody bound on the surface thereof, and accordingly, may specifically target the cancer cells via the anti-TAA antibody fragment of the bispecific antibody.
  • the armed immune cells such as armed T cells produced in step (b) can be administered to the subject so as to treat cancer.
  • the amount of T cells administered to the subject is from about lxlO 4 to lxlO 7 cells/Kg body weight of the subject. In certain embodiments, the amount of T cells is administered to the subject from about lxlO 5 to lxlO 6 cells/Kg body weight of the subject.
  • the dose can be administered in a single dose, or alternatively in more than one dose.
  • the method may further isolating the T cell from the product of step (b) by a method suitable for isolating or purifying immune cells, for example, affinity column, or magnetic beads.
  • a method suitable for isolating or purifying immune cells for example, affinity column, or magnetic beads.
  • Treatment efficacy may be examined via routine practice.
  • kits comprising any of the armed immune cells such as armed T cells or any of the bispecific antibodies disclosed herein. Such kits can be used for treating or alleviating a target cancer as disclosed herein. Such kits can include one or more containers comprising the armed immune cells or a bispecific antibody as those described herein.
  • the kit can comprise instructions for use in accordance with any of the methods described herein.
  • the included instructions can comprise a description of administration of the armed immune cells or use of the bispecific antibody to produce the armed immune cells, to treat, delay the onset, or alleviate a target disease as those described herein.
  • the kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease.
  • the instructions comprise a description of administering an antibody to an individual at risk of the target disease.
  • the instructions relating to the use of the armed immune cells such as armed T cells or the bispecific antibody generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • Instructions supplied in the kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • the label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating the disease, such as cancer or immune disorders (e.g., an autoimmune disease). Instructions may be provided for practicing any of the methods described herein.
  • kits of this invention are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g. , sealed Mylar or plastic bags), and the like.
  • packages for use in combination with a specific device such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump.
  • a kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • a sterile access port for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.
  • At least one active agent in the composition is armed immune cells or a bispecific antibody as those described herein.
  • Kits may optionally provide additional components such as buffers and interpretive information.
  • the kit comprises a container and a label or package insert(s) on or associated with the container.
  • the invention provides articles of manufacture comprising contents of the kits described above.
  • recombinant antibodies respectively having the structures as depicted in FIGs 1A-1L were prepared using the DNA constructs illustrated in FIGs 2A-2E.
  • the constructs comprise, from N-terminus to C-terminus, (a) Igk leader sequence (LS), anti-CD3 VL-Ck domain or anti-CD3 VL-Ck domain, internal ribosomal entry site (IRES), LS, anti-CD3 VH-CH1 domain, peptide linker, and anti-TAA scFv (e.g., anti-EGFR scFv) (FIGs. 1A-1D) and FIG.
  • Igk leader sequence LS
  • anti-CD3 VL-Ck domain or anti-CD3 VL-Ck domain internal ribosomal entry site (IRES)
  • LS anti-CD3 VH-CH1 domain
  • peptide linker e.g., anti-EGFR scFv
  • FIG. 2A top two constructs; or (b) LS, anti-CD3 VL-Ck domain or anti-CD3 VL-Ck domain, peptide linker, anti-TAA scFv (e.g., anti-EGFR scFv), IRES, LS, and anti-CD3 VH-CH1 domain (FIG. IB and FIG. 2A, bottom two constructs.
  • anti-CD3 VL-Ck domain or anti-CD3 VL-Ck domain, peptide linker, anti-TAA scFv (e.g., anti-EGFR scFv), IRES, LS, and anti-CD3 VH-CH1 domain FIG. IB and FIG. 2A, bottom two constructs.
  • the constructs comprise, from N-terminus to C-terminus, (a) LS, anti-TAA VL-Ck domain (e.g., anti-EGFR VL-Ck domain), IRES, LS, anti-TAA VH-CH1 domain (e.g., anti-EGFR VH-CH1 domain), peptide linker, and anti-CD3 VH-VL domain or anti-CD3 VL-VH domain (FIGs. 1E-1H and FIG.
  • LS top two constructs
  • anti-TAA VL-Ck domain e.g., anti-EGFR VL-Ck domain
  • peptide linker e.g., anti-CD3 VH-VL domain or anti-CD3 VL-VH domain
  • IRES e.g., IRES
  • LS e.g., anti-EGFR VH-CH1 domain
  • anti-EGFR VH-CH1 domain e.g., anti-EGFR VH-CH1 domain
  • the constructs comprise, from N-terminus to C-terminus, LS, anti-TAA scFv (e.g., anti-EGFR scFv), peptide linker, and anti-CD3 VH-VL domain or anti-CD3 VL-VH domain (FIGs. 1I-IL and Fig. 2C).
  • the anti-CD3 knob constructs comprise, iron N-terminus to C-terminus, LS, anti-CD3 VL-Ck domain or anti-CD3 VL-Ck domain, IRES, LS, and anti-CD3 VH-CHl-knob Fc, while the anti-tumor hole comprised in sequence, LS, anti-TAA VL-Ck (e.g., anti-EGFR VL-Ck domain), IRES, LS, and anti-TAA VH-CHl-hole Fc (e.g., anti-EGFR VH-CHl-hole Fc) (FIG. 2D).
  • the anti-CD3 knob construct comprised in sequence, LS, anti-CD3 VL-Ck domain or anti-CD3 VL-Ck domain, IRES, LS, and anti-CD3 VH-CHl-knob Fc, while the anti-tumor hole comprised in sequence, LS, anti-TAA scFv (e.g., anti-EGFR scFv), peptide linker, and hole Fc (FIG. 2E).
  • CTA02scFv/CTAT03Fab previously named anti-EGFR Fab/CAT.02 scFv
  • CTA01scFv/CTAT03Fab previously named anti-EGFR Fab/aCD3 scFv, structural information of which is provided in WO2018177371, the relevant disclosures of which is incorporated by reference for the subject matter and purpose referenced herein
  • WO2018177371 the relevant disclosures of which is incorporated by reference for the subject matter and purpose referenced herein
  • Both the CTA02scFv/CTAT03Fab and CTA01scFv/CTAT03Fab comprised an anti-EGFR Fab and an anti-CD3 scFv, in which the VH-CH1 and VL-Ck domains of the anti-EGFR Fab respectively had the amino acid sequences of SEQ ID NOs: 83 and 84.
  • the anti-CD3 scFv of the CTA02scFv/CTAT03Fab had the amino acid sequence of SEQ ID NO: 9
  • the anti-CD3 scFv of the CTA01scFv/CTAT03Fab is provided in WO2018177371 (CTAOl is the same antibody named as OKT3 in WO2018177371).
  • Example 2 Effect of Recombinant Bi-Specific Antibodies on T cells This example investigates bioactivities of the recombinant bi- specific antibodies disclosed herein.
  • the binding affinities of the recombinant antibodies CTA02scFv/CTAT03Fab and CTA01scFv/CTAT03Fab were examined in this example by flow cytometry. Both the CTA02scFv/CTAT03Fab and the CTA01scFv/CTAT03Fab were capable of binding to
  • CD3-positive T cells The binding affinity of the CTA02scFv/CTAT03Fab was higher than that of theCTA01scFv/CTAT03Fab.
  • FIG. 3 The binding affinity results are provided in Table 4 below.
  • the cytotoxic effect of the recombinant antibody CTA02scFv/CTAT03Fab or the CTA01scFv/CTAT03Fab on cancer cells was evaluated in this example. It was found that about 2.5%, 18.2% and 26.9% of HT-29 cells were killed by CD3 + /CD8 + T cells activated by murine OKT3 at the effect cells to target cells ratio (E/T ratio) of 3:1, 5:1 and 10:1, respectively; about 13.8%, 34.8% and 65.7% of HT-29 cells were killed by CD3 + /CD8 + T cells armed with the CTA01scFv/CTAT03Fab, and about 28.1%, 44.4% and 76.7% of HT-29 cells were killed by T cells armed with the CTA02scFv/CTAT03Fab at the same E/T ratio (FIG. 4 and Table 5). Table 5 Cytotoxic effect of specified antibodies
  • the CTA02scFv/CTAT03Fab and the CTA01scFv/CTAT03Fab were respectively incubated with T cells in the presence of 20% FBS for 24 hours.
  • the T cells were then analyzed by flow cytometry to evaluate the residual amounts of the antibodies on the surface of T cells.
  • the results from this assay indicate that the amounts of the antibodies on the surface of the T cells declined over time.
  • the CTA02scFv/CTAT03Fab was less affected by degradation.
  • About 84.5% of the CTA02scFv/CTAT03Fab still remained on the T cell surface after 24 hours, while the level of theCTA01scFv/CTAT03Fab remained on the T cell surface dropped to about 40% after 24 hours.
  • a panel of various anti-CD3/anti-tumor-associated antigen (TAA) bispecific antibodies were constructed by genetic engineering. These BsAbs were derived from 4 anti-CD3 antibodies and 16 anti-TAA antibodies (CD20(CTAT01), CD19(CTAT02), EGFR(CTAT03), HER2(CTAT04), PSMA(CTAT05), CEA(CTAT06), EpCAM(CTAT07), FAP(CTAT08), PDL1(CTAT09), CD38(CTAT10), CD33(CTAT11), HGFR(CTAT12), CD47(CTAT13), TRAIL- R2(CTAT14), mesothelin (CTAT15) and GD2(CTAT16)). See Tables 1 and 2 above.
  • the BsAbs were produced via recombinant technology in mammalian host cells, collected, and examined by SDS-PAGE under reduced conditions and non-reduced conditions. Briefly, protein electrophoresis with 8% SDS-PAGE in non-reducing conditions and reducing conditions were performed to analyze the structure and molecular weight of various BsAbs comprising an anti-CD3 fragment and an anti-TAA fragment.
  • FIGs. 6A-6B show the expression and assembly of BsAbs each comprising a Fab of 4 different anti-CD3 antibody (CTA02, CTA03, CTA04, and CTA05; see Table 1 above) and an anti-CD 19 scFv (CTAT02; see Table 2 above).
  • FIGs. 7A-7D show the expression and assembly of BsAbs each comprising an anti-CD3 Fab (CTA03; see Table 1 above) and a scFv of 16 different anti-tumor antibodies (CD20(CTAT01), CD19(CTAT02), EGFR (CTAT03), HER2(CTAT04), PSMA(CTAT05), CEA(CTAT06), EpCAM (CTAT07), FAP(CTAT08), PDL1(CTAT09), CD38(CTAT10), CD33(CTAT11), HGFR(CTAT12), CD47(CTAT13), TR AIL-R2(CT AT 14), mesothelin (CTAT15) and GD2(CTAT16); see Table 2 above).
  • FIGs. 7E and 7F show expression of BsAbs each comprising a scFv of one of the four anti-CD3 antibodies (CTA02-CTA05) and a Fab fragment of anti-EGFR CTAT03.
  • the binding activities of the various anti-CD3/anti-tumor BsAbs to T cells and tumor cells were analyzed using flow cytometry.
  • the BsAbs specific to CD3 and CD19 (CTA02Fab/CTAT02scFv, CTA03Fab/CTAT02scFv, CTA04Fab/CTAT02scFv, and CTA05Fab/CTAT02scFv) all showed binding activity to CD3 + T cells (Jurkat) and CD19 + B cell lymphoma (Raji), indicating that T cells armed with such BsAbs could be used to target CD19+ disease cells such as CD19 + B cell lymphoma.
  • CTA03Fab/CTAT03scFv showed binding activity to CD3 + T cells (Jurkat) and EGFR + triple negative breast cancer (MDA-MB-231) (FIG. 9B);
  • CTA03Fab/CTAT04scFv showed binding activity to CD3 + T cells (Jurkat) and HER2 + breast cancer (MCF7/HER2) (FIG. 9C);
  • CTA03Fab/CTAT08scFv showed binding activity to CD3 + T cells (Jurkat) and FAP + mouse fibroblasts cell (3T3/FAP) (FIG. 9F);
  • CTA03Fab/CTAT09scFv showed binding activity to CD3 + T cells (Jurkat) and PDL1 + triple negative breast cancer (MDA-MB-231) (FIG. 9G);
  • CTA03Fab/CTAT10scFv showed binding activity to CD3 + T cells (Jurkat) and CD38 + B cell lymphoma (Raji) (FIG. 9H);
  • CTA03Fab/CTATl IscFv showed binding activity to CD3 + T cells (Jurkat) and CD33 + human acute myeloid leukemia (HL-60) (FIG. 91);
  • CTA03Fab/CTAT12scFv showed binding activity to CD3 + T cells (Jurkat) and HGFR + human lung carcinoma (A549) (FIG. 9J);
  • CTA03Fab/CTAT13scFv showed binding activity to CD3 + T cells (Jurkat) and CD47 + breast cancer (MCF7/HER2) (FIG. 9K).
  • FIG. 9L shows the targeting ability against CD3+ T cells (Jurkat) and EGFR+ colon cancer (HT-29) of BsAbs consisting of a scFv of 4 different anti-CD3 antibody (CTA02, CTA03, CTA04, CTA05) and an anti-EGFR Fab (CTAT03).
  • CTA02scFv/CTAT03Fab, CTA03scFv/CTAT03Fab, CTA04scFv/CTAT03Fab, and CTA05scFv/CTAT03Fab all possessed targeting abilities against CD3+ T cells (Jurkat) and EGFR+ colon cancer (HT-29).
  • the retention time of BsAb on T cell surface was analyzed using an in vitro incubation platform. Briefly, human T cells were incubated with various anti-CD3Fab/anti-CD19scFv (CTA01Fab/CTAT02scFv, CTA02Fab/CTAT02scFv, CTA03Fab/CTAT02scFv and CTA05Fab/CTAT02scFv) for lhr, and then were cultured in medium for 5 min, 24, 48, and 72 hr. After the culture, the residual amount of BsAb on T cell surface was detected using flow cytometry.
  • PBMCs Human peripheral blood mononuclear cells from a healthy donor were cultured and differentiated into T cells in the presence of the OKT3 antibody, or in the presence of exemplary BsAbs disclosed herein (using CTA01Fab/CTAT02scFv, CTA02Fab/CTAT02scFv, CTA03Fab/CTAT02scFv and CTA05Fab/CTAT02scFv as examples). All groups were cultured under the same conditions (in an incubator with 5% CO2 supply and a stable humidity level at 37°C). After 7 days, all groups were analyzed using flow cytometry to measure production of BsAb armed-T cells.
  • the OKT3 anti-CD3 antibody induced differentiation of PBMCs into only normal T cells but not armed-T cells.
  • the OKT3 anti-CD3 antibody induced differentiation of PBMCs into only normal T cells but not armed-T cells.
  • CTA01 Fab/CTAT02scFv, CTA02Fab/CTAT02scFv, CTA03Fab/CTAT02scFv and CTA05Fab/CTAT02scFv BsAbs all successfully produced armed-T cells.
  • PBMCs were cultured and differentiated into T cells with OKT3 or exemplary anti-CD3 Fab/anti-Tumor scFv BsAbs (CTA03Fab/CTAT03scFv, CTA03Fab/CTAT04scFv, CTA03Fab/CTAT05scFv, CTA03Fab/CTAT07scFv, CTA03Fab/CTAT08scFv, CTA03Fab/CTAT9scFv, CTA03Fab/CTAT10scFv, CTA03Fab/CTATllscFv, CTA03Fab/CTAT12scFv, and CTA03Fab/CTAT13scFv).
  • CTA03Fab/CTAT03scFv CTA03Fab/CTAT04scFv
  • CTA03Fab/CTAT05scFv CTA03Fab/CTAT07scFv
  • CTA03Fab/CTAT08scFv CTA03Fab/CTAT9scFv
  • FIGs 12A and 12B show that the OKT3 antibody led to differentiation of PBMCs into normal T cells, but not armed-T cells were produced.
  • PBMCs were cultured and differentiated into T cells with OKT3 or various anti-CD3 scFv/anti-Tumor Fab BsAbs (CTA01scFv/CTAT03Fab, CTA02scFv/CTAT03Fab, CTA03scFv/CTAT03Fab, CTA04scFv/CTAT03Fab, and CTA05scFv/CTAT03Fab). All groups were cultured under the same conditions (in an incubator with 5% C02 and a stable humidity level at 37°C). After 7 days, all groups were analyzed using flow cytometry to reveal whether BsAb armed-T cells were successfully generated. FIGs.
  • 12C and 12D shows that the traditional OKT3 method caused PBMCs to differentiate only into normal T cells, but not armed-T cells.
  • the cytotoxicity activity of T cells armed with anti-CD3/anti-CD19 BsAbs against CD19 + B cell lymphoma (Raji) were investigated in this example.
  • T cells cultured with the OKT3 antibody was analyzed using an in vitro cytotoxicity assay. No significant cytotoxicity of T cells cultured with OKT3 against CD19 + B cell lymphoma (Raji) was observed.
  • FIG. 13B shows that the T cells incubated with OKT3 did not efficiently kill EGFR + colon cancer (HT-29), but the armed T cells cultured with CTA01scFv/CTAT03Fab, CTA02scFv/CTAT03Fab, CTA03scFv/CTAT03Fab, CTA04scFv/CTAT03Fab, and CTA05scFv/CTAT03Fab efficiently killed EGFR + colon cancer (HT-29).
  • FIG. 13B shows that the T cells incubated with OKT3 did not efficiently kill EGFR + colon cancer (HT-29), but the armed T cells cultured with CTA01scFv/CTAT03Fab, CTA02scFv/CTAT03Fab, CTA03scFv/CTAT03Fab, CTA04scFv/CTAT03Fab, and CTA05scFv/CTAT03Fab efficiently killed EGFR + colon cancer (HT-29).
  • FIG. 13B shows that the T cells incubated with OK
  • the tumor cell killing activity of armed T cells generated with various anti-CD3 Fab/anti-Tumor scFv BsAb including CTA03Fab/CTAT03scFv, CTA03Fab/CTAT04scFv, CTA03Fab/CTAT05scFv, CTA03Fab/CTAT07scFv , CTA03Fab/CTAT08scFv, CTA03Fab/CTAT9scFv, CTA03Fab/CTAT10scFv, CTA03Fab/CTATllscFv, CTA03Fab/CTAT12scFv and CTA03Fab/CTAT13scFv) were further analyzed. As shown in FIGs.
  • CTA03Fab/CTAT03scFv armed-T cells efficiently killed EGFR + colon cancer cells HT29 and HCT-116.
  • FIG. 14C shows that CTA03Fab/CTAT04scFv armed-T cells efficiently killed HER2 + Breast cancer (MCF7/HER2).
  • FIG. 14D shows that CTA03Fab/CTAT05scFv armed-T cells efficiently killed PSMA +
  • FIG. 14E shows that CTA03Fab/CTAT07scFv armed-T cells efficiently killed EpCAM + Prostate cancer (LNCaP). Further, FIGs. 14F-14G show that CTA03Fab/CTAT08scFv armed-T cells efficiently killed FAP + mouse fibroblasts cell (3T3/FAP).
  • FIG. 15A shows that CTA03Fab/CTAT09scFv armed-T cells efficiently killed PDL1 + triple negative breast cancer (MDA-MB-231).
  • FIG. 15B shows that CTA03Fab/CTAT10scFv armed-T cells efficiently killed CD38 + B cell lymphoma (Raji).
  • FIG. 15C shows that CTA03Fab/CTATllscFv armed-T cells efficiently killed CD33 + human acute myeloid leukemia (HL-60).
  • FIG. 15D shows that CTA03Fab/CTAT12scFv armed-T cells efficiently killed HGFR + human lung carcinoma (A549).
  • FIG. 15E shows that CTA03Fab/CTAT13scFv armed-T cells efficiently killed CD47 + Breast cancer (MCF7/HER2).
  • CTA01Fab/CTAT02scFv and CTA03Fab/CTAT02scFv were evaluated.
  • CTA01Fab/CTAT02scFv armed-T cells sand CTA03Fab/CTAT02scFv armed-T cells were i.v. injected into SCID mice bearing with B cell lymphoma (Raji). Body weight, survival rate and incidence of hindlimb paralysis were recorded.
  • FIGs. 16A-16C show that CTA03Fab/CTAT02scFv armed-T cells had the best therapeutic efficacy to effectively inhibit cancer.
  • Example 8 In Vivo Anti-Tumor Activity of CTA03Fab/CTAT03scFv Armed-T cells and CTA03Fab/CTAT04scFv Armed-T cells
  • CTA03Fab/CTAT03scFv In vivo tumor inhibition of anti-CD3Fab/anti-EGFRscFv (CTA03Fab/CTAT03scFv) armed-T cells and anti-CD3Fab/anti-HER2scFv (CTA03Fab/CTAT04scFv) armed-T cells were evaluated.
  • CTA03Fab/CTAT03scFv armed-T cells and CTA03Fab/CTAT04scFv armed-T cells were i.v. injected into patient-derived xenograft (PDX) mice models bearing with human triple-negative breast cancer. Body weight and tumor size were recorded.
  • PDX patient-derived xenograft
  • PBMCs Human peripheral blood mononuclear cells from a healthy donor were cultured and differentiated into NKT cells (CD8 + CD56 + ) with the OKT3 traditional method, or with CTA03Fab/CTAT03scFv, CTA03Fab/CTAT04scFv and CTA03Fab/CTAT05scFv BsAbs. All groups were cultured in the same environment (an incubator with 5% CO2 and a stable humidity level at 37°C). After 7 days, all groups were analyzed using flow cytometry to reveal whether BsAb armed-NKT cells were successfully generated.
  • FIGs. 18A and 18B show that OKT3 method induced PBMCs to differentiate into only normal NKT cells, but not formation of armed-T cells. Differently, CTA03Fab/CTAT03scFv, CTA03Fab/CTAT04scFv and CTA03Fab/CTAT05scFv BsAbs all successfully generated armed-NKT cells.
  • PBMCs peripheral blood mononuclear cells
  • NKT cells CD8 + CD56 +
  • OKT antibody or with CTA01scFv/CTAT03Fab, CTA02scFv/CTAT03Fab, CTA03scFv/CTAT03Fab, CTA04scFv/CTAT03Fab, and CTA05scFv/CTAT03Fab BsAbs. All groups were cultured in the same environment (an incubator with 5% CO2 and a stable humidity level at 37°C). After 7 days, all groups were analyzed using flow cytometry to reveal whether BsAb armed-NKT cells were successfully generated.
  • Point mutations were introduced into CTA03Fab/CTAT02scFv BsAb by genetic engineering, resulting in BsAbs CTA03-01Fab/CTAT02-01scFv (CTA03Fab(VLG58A) / CTAT02scFv(VLG42A)), CTA03-01Fab/CTAT02-02scFv (CTA03Fab(VLG58A) / CTAT02scFv( VLD4 IE)), CTA03-02Fab/CTAT02-02scFv (CTA03Fab(VLD57E) / CTAT02scFv( VLD4 IE)), and CTA03-02Fab/CTAT02-01scFv (CTA03Fab(VLD57E) / CTAT02scFv(VLG42A)). More specifically, point mutations G58A and D57E were introduced into the VL of CTA03 and G42A and D41E were introduced into the VL of CTAT02. See Table 2 above.
  • FIGs. 19A-19B show that CTA03-01 Fab/CTAT02-01 scFv, CTA03 -01 Fab/CTAT02-02scFv, CTA03-02Fab/CTAT02-02scFv and CTA03-02Fab/CTAT02-01scFv BsAbs all possessed targeting abilities against CD3 + T cells (Jurkat) and CD19 + B cells lymphoma (Raji). Additionally, binding of the BsAbs to the target cells is in a dose-dependent manner.
  • FIG. 19C shows that T cells incubated with OKT3 did not show cytotoxicity against CD19 + B cell lymphoma (Raji).
  • the armed-T cells cultured with CTA03-01Fab/CTAT02-01scFv, CTA03-01Fab/CTAT02-02scFv, CTA03-02Fab/CTAT02-02scFv or CTA03-02Fab/CTAT02-01scFv efficiently killed CD19 + B cell lymphoma (Raji), and the cytotoxicity were all better than the parent CTA03Fab/CTAT02scFv BsAb.
  • inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
  • inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the hinge domain is a hinge domain of a naturally occurring protein.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one,

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pregnancy & Childbirth (AREA)
  • Reproductive Health (AREA)
  • Gynecology & Obstetrics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
PCT/US2021/023655 2020-03-23 2021-03-23 Bi-specific antibodies for use in producing armed immune cells Ceased WO2021195067A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU2021244375A AU2021244375A1 (en) 2020-03-23 2021-03-23 Bi-specific antibodies for use in producing armed immune cells
CN202180029464.3A CN115551890A (zh) 2020-03-23 2021-03-23 用于产生武装免疫细胞的双特异性抗体
EP21774346.7A EP4126954A4 (en) 2020-03-23 2021-03-23 BISPECIFIC ANTIBODIES FOR USE IN THE PRODUCTION OF ARMED IMMUNE CELLS
JP2022558065A JP7729833B2 (ja) 2020-03-23 2021-03-23 武装免疫細胞を産生する際に使用するための二重特異性抗体
US17/913,775 US20240209084A1 (en) 2020-03-23 2021-03-23 Bi-specific antibodies for use in producing armed immune cells
IL296566A IL296566A (en) 2020-03-23 2021-03-23 Bi-specific antibodies for use in producing armed immune cells
JP2025135194A JP2025161873A (ja) 2020-03-23 2025-08-14 武装免疫細胞を産生する際に使用するための二重特異性抗体

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202062993080P 2020-03-23 2020-03-23
US62/993,080 2020-03-23

Publications (1)

Publication Number Publication Date
WO2021195067A1 true WO2021195067A1 (en) 2021-09-30

Family

ID=77892613

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/023655 Ceased WO2021195067A1 (en) 2020-03-23 2021-03-23 Bi-specific antibodies for use in producing armed immune cells

Country Status (8)

Country Link
US (1) US20240209084A1 (https=)
EP (1) EP4126954A4 (https=)
JP (2) JP7729833B2 (https=)
CN (1) CN115551890A (https=)
AU (1) AU2021244375A1 (https=)
IL (1) IL296566A (https=)
TW (1) TWI904147B (https=)
WO (1) WO2021195067A1 (https=)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11434291B2 (en) 2019-05-14 2022-09-06 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
WO2023019179A1 (en) * 2021-08-11 2023-02-16 Cytoarm Co., Ltd. Process for producing armed immune cells
WO2023143478A1 (en) * 2022-01-27 2023-08-03 Crown Bioscience Inc. Novel anti-cd4 and anti-pd-l1 bispecific antibodies
US12006366B2 (en) 2020-06-11 2024-06-11 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
US12565529B2 (en) 2021-05-24 2026-03-03 Provention Bio, Inc. Methods for treating type 1 diabetes

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118005806B (zh) * 2024-02-08 2026-01-30 北京市眼科研究所 嵌合抗原受体、人多能干细胞分化的表达嵌合抗原受体的小胶质细胞及其应用
CN121248779A (zh) * 2024-06-25 2026-01-02 冕屹立生物技术(上海)有限公司 靶向人cd3分子的抗体及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060292142A1 (en) * 1993-06-01 2006-12-28 Bluestone Jeffrey A Methods and materials for modulation of the immunosuppressive activity and toxicity of monoclonal antibodies
US20100317834A1 (en) * 2004-10-21 2010-12-16 Xencor, Inc. IgG Immunoglobulin Variants with Optimized Effector Function
US20180194841A1 (en) * 2014-11-17 2018-07-12 Regeneron Pharmaceuticals, Inc. Methods for tumor treatment using cd3xcd20 bispecific antibody

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090215992A1 (en) * 2005-08-19 2009-08-27 Chengbin Wu Dual variable domain immunoglobulin and uses thereof
AU2013302696B9 (en) * 2012-08-14 2018-08-09 Ibc Pharmaceuticals, Inc. T-cell redirecting bispecific antibodies for treatment of disease
US9611325B2 (en) * 2014-07-21 2017-04-04 Wuhan Yzy Biopharma Co., Ltd. Construction and application of bispecific antibody HER2xCD3
US10519248B2 (en) * 2014-07-25 2019-12-31 Memorial Sloan Kettering Cancer Center Bispecific HER2 and CD3 binding molecules
CN104829725A (zh) * 2015-01-21 2015-08-12 武汉友芝友生物制药有限公司 一种双特异性抗体cd133×cd3的构建及应用
US11649435B2 (en) * 2015-08-28 2023-05-16 The Trustees Of The University Of Pennsylvania Methods and compositions for cells expressing a chimeric intracellular signaling molecule
US11702477B2 (en) * 2015-11-06 2023-07-18 Orionis Biosciences BV Bi-functional chimeric proteins and uses thereof
WO2018049261A1 (en) * 2016-09-09 2018-03-15 Icellhealth Consulting Llc Oncolytic virus expressing immune checkpoint modulators
CN106632681B (zh) * 2016-10-11 2017-11-14 北京东方百泰生物科技有限公司 抗egfr和抗cd3双特异抗体及其应用
WO2018177371A1 (en) * 2017-03-29 2018-10-04 Taipei Medical University Antigen-specific t cells and uses thereof
EP3802595A1 (en) * 2018-06-07 2021-04-14 OncoOne Research & Development GmbH Anti-oxmif/anti-cd3 antibody for cancer treatment

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060292142A1 (en) * 1993-06-01 2006-12-28 Bluestone Jeffrey A Methods and materials for modulation of the immunosuppressive activity and toxicity of monoclonal antibodies
US20100317834A1 (en) * 2004-10-21 2010-12-16 Xencor, Inc. IgG Immunoglobulin Variants with Optimized Effector Function
US20180194841A1 (en) * 2014-11-17 2018-07-12 Regeneron Pharmaceuticals, Inc. Methods for tumor treatment using cd3xcd20 bispecific antibody

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4126954A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11434291B2 (en) 2019-05-14 2022-09-06 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
US12006366B2 (en) 2020-06-11 2024-06-11 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
US12565529B2 (en) 2021-05-24 2026-03-03 Provention Bio, Inc. Methods for treating type 1 diabetes
WO2023019179A1 (en) * 2021-08-11 2023-02-16 Cytoarm Co., Ltd. Process for producing armed immune cells
WO2023143478A1 (en) * 2022-01-27 2023-08-03 Crown Bioscience Inc. Novel anti-cd4 and anti-pd-l1 bispecific antibodies

Also Published As

Publication number Publication date
EP4126954A4 (en) 2024-08-21
TWI904147B (zh) 2025-11-11
JP2025161873A (ja) 2025-10-24
US20240209084A1 (en) 2024-06-27
CN115551890A (zh) 2022-12-30
EP4126954A1 (en) 2023-02-08
JP2023519851A (ja) 2023-05-15
AU2021244375A1 (en) 2022-10-13
JP7729833B2 (ja) 2025-08-26
IL296566A (en) 2022-11-01
TW202202522A (zh) 2022-01-16

Similar Documents

Publication Publication Date Title
JP7729833B2 (ja) 武装免疫細胞を産生する際に使用するための二重特異性抗体
US20200093861A1 (en) Antigen binding receptor formats
BR112019020168A2 (pt) Receptores de ligação ao antígeno, células t transduzidas, polinucleotídeo isolado, vetor, kits, métodos para tratar uma doença e para induzir a lise de uma célula alvo e uso do receptor de ligação ao antígeno
CN113784980B (zh) 人源化抗Claudin18.2嵌合抗原受体及其用途
TWI797610B (zh) 人源化cd19抗體及其應用
US20240123068A1 (en) Cd19 binders, car-t constructs comprising the same, and methods of using the same
US20240052031A1 (en) Cea6 binding molecules and uses thereof
US20250127894A1 (en) Process for producing armed immune cells
US20240050569A1 (en) Mesothelin binding molecules and uses thereof
US20240226154A9 (en) Car-t constructs comprising a novel cd19 binder combined with il18 and methods of using the same
WO2025179294A2 (en) Immune engineering amplification
WO2025067496A1 (zh) 靶向baff-r的全人源抗体、car-t细胞及其应用
EP4501953A1 (en) Antibody that specifically binds to cd7 and use thereof in preparing chimeric antigen receptor
WO2022020945A1 (en) Anti-cd22 single domain antibodies and therapeutic constructs
WO2023199927A1 (ja) がん治療におけるｐｄ－１シグナル阻害剤との組み合わせによる抗ｔｓｐａｎ８－抗ｃｄ３二重特異性抗体の使用
JP2026513651A (ja) Ａｄｇｒｅ２キメラ受容体ｎｋ細胞組成物及び使用方法
WO2025235598A1 (en) Chimeric antigen receptors targeting glypican-2 and/or cd276 and their use for treating solid tumors
EP4263601A1 (en) Epcam binding molecules and uses thereof
WO2022133074A1 (en) Muc1 binding molecules and uses thereof
CN113528560A (zh) 靶向白蛋白的嵌合抗原受体及其使用方法

Legal Events

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

Ref document number: 21774346

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022558065

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2021244375

Country of ref document: AU

Date of ref document: 20210323

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021774346

Country of ref document: EP

Effective date: 20221024