WO2019197979A1 - Molécules de liaison impliquant des cellules - Google Patents

Molécules de liaison impliquant des cellules Download PDF

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Publication number
WO2019197979A1
WO2019197979A1 PCT/IB2019/052896 IB2019052896W WO2019197979A1 WO 2019197979 A1 WO2019197979 A1 WO 2019197979A1 IB 2019052896 W IB2019052896 W IB 2019052896W WO 2019197979 A1 WO2019197979 A1 WO 2019197979A1
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Prior art keywords
region
seq
polypeptide
antigen
amino acid
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PCT/IB2019/052896
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English (en)
Inventor
Seil Jang
Bum-Chan PARK
Young Woo Park
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Y-Biologics Inc.
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Priority claimed from US16/372,196 external-priority patent/US10654944B2/en
Application filed by Y-Biologics Inc. filed Critical Y-Biologics Inc.
Priority to EP19785439.1A priority Critical patent/EP3774922A4/fr
Priority to JP2020555789A priority patent/JP7076571B2/ja
Priority to RU2020136600A priority patent/RU2770620C1/ru
Priority to CN201980030028.0A priority patent/CN112074540B/zh
Priority to KR1020207032356A priority patent/KR102567489B1/ko
Priority to AU2019251422A priority patent/AU2019251422A1/en
Priority to CA3096791A priority patent/CA3096791C/fr
Publication of WO2019197979A1 publication Critical patent/WO2019197979A1/fr
Priority to AU2020257053A priority patent/AU2020257053B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure generally relates to cell engaging binding molecules, methods of making the binding molecules, compositions comprising the binding molecules, and uses thereof.
  • Antibodies and/or antibody-based agents are now therapeutic options for a wide variety of diseases and disorders.
  • a naturally occurring antibody is monospecific and binds to one epitope or antigen.
  • Multispecific antibodies combine specificities of multiple antibodies and have the capability to bind different antigens or epitopes.
  • Many technical hurdles, however, have hampered development of multispecific antibodies; as such, few multispecific antibodies have been approved as therapeutics.
  • the present disclosure provides, in part, cell engaging binding molecules having multiple binding domains, methods of making the binding molecules, and pharmaceutical compositions comprising the binding molecules. Also provided herein are methods of treatment comprising administering the binding molecules.
  • cell engaging binding molecules In some aspects, provided herein are cell engaging binding molecules.
  • binding molecule comprising:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region bind to a first antigen
  • the Fv region binds to a second antigen
  • the first antigen is different from the second antigen
  • the third polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide and/or the four polypeptide further comprise both a CH1 region and an ABS C- terminal to the second VH region and/or to the VL region, respectively.
  • the first and/or second polypeptides further comprise an ABS. In some embodiments, with regard to any of the previous binding molecules, the first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain. In specific embodiments, with regard to any of the previous binding molecules, the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • binding molecules wherein the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region. In some embodiments, the first Fab region and the second Fab region are linked to the Fv region via fusion.
  • the flexible peptide region comprises an antibody hinge region.
  • the antibody hinge region is an Immunoglobulin G (IgG) hinge region.
  • the antibody hinge region is selected from the group consisting of IgGl, IgG2, IgG3, and IgG4 hinge regions.
  • the antibody hinge region comprises an interchain disulfide bond between the third polypeptide and the fourth polypeptide.
  • the flexible peptide region further comprises a linker.
  • the linker comprises an amino acid sequence of GGGGS (G4S).
  • the linker comprises two tandem copies of the amino acid sequence of GGGGS (G4S).
  • the first Fab region and the second Fab region bind to the same epitope of the first antigen.
  • the second antigen is expressed on an immune cell.
  • the immune cell is selected from the group consisting of lymphocytes and monocytes.
  • the immune cell is an effector cell.
  • the immune cell is selected from the group consisting of a T cell, a B cell, a dendritic cell, a granulocyte, an innate lymphoid cell, a megakaryocyte, a monocyte, a myeloid- derived suppressor cell, and a natural killer (NK) cell.
  • the first antigen is a cancer antigen.
  • the cancer antigen is a tumor associated antigen (TAA) or a tumor specific antigen (TSA).
  • TAA tumor associated antigen
  • TSA tumor specific antigen
  • the first antigen is selected from a group consisting of CD 19, CD20, EGFR, Her2, and PD-L1.
  • the second antigen is CD3 or TNF alpha. In some embodiments, the second antigen is CD3 or TNF alpha.
  • the first antigen is a cancer antigen and the second antigen is CD3.
  • the cancer antigen is selected from a group consisting of CD 19, CD20, EGFR, Her2, and PD-L1.
  • a binding molecule comprising:
  • a third nucleic acid encoding a third polypeptide comprising, in the order from N- terminus to C-terminus, a first VH region and a first CH1 region and a second VH region;
  • a fourth nucleic acid encoding a fourth polypeptide comprising, in the order from N- terminus to C-terminus, a third VH region and a second CH1 region and a VL region, wherein the first polypeptide and the first VH region and the first CH1 region of the third polypeptide can form a first antigen binding Fab region;
  • first Fab region and the second Fab region bind to a first antigen
  • the Fv region binds to a second antigen
  • the first antigen is different from the second antigen
  • polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • CH3 constant heavy 3
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • ABS albumin binding domain or site
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • polypeptide and/or the four polypeptide further comprise both a CH1 region and an ABS C- terminal to the second VH region and/or to the VL region, respectively.
  • first and/or second polypeptides further comprise an ABS.
  • first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain.
  • the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region comprising an antibody hinge region.
  • the antibody hinge region comprises an interchain disulfide bond formed between the third polypeptide and the fourth polypeptide.
  • the flexible peptide region further comprises a linker.
  • the linker comprises an amino acid sequence of GGGGS (G4S).
  • the first antigen is a cancer antigen and the second antigen is CD3.
  • the first antigen is selected from a group consisting of CD19, CD20, EGFR, Her2, and PD-L1.
  • compositions comprising a binding molecule.
  • a pharmaceutical composition comprising a binding molecule and a pharmaceutically acceptable carrier, wherein the binding molecule comprises:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first VH region and a first CH1 region, and a second VH region;
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a VL region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • the second VH region of the third polypeptide and the VL region of the fourth polypeptide form an antigen binding Fv region; and wherein the first Fab region and the second Fab region bind to a first antigen, the Fv region binds to a second antigen, and the first antigen is different from the second antigen.
  • the third polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • CH3 constant heavy 3
  • ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide and/or the four polypeptide further comprise both a CH 1 region and an ABS C-terminal to the second VH region and/or to the VL region, respectively.
  • the first and/or second polypeptides further comprise an ABS. In some embodiments, with regard to any of the previous pharmaceutical compositions, the first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain. In specific embodiments, with regard to any of the previous pharmaceutical compositions, the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • provided herein are methods of treating a disease or condition comprising administering a binding molecule.
  • a method of treating a disease or condition in a subject comprising administering a therapeutically effective amount of a binding molecule to the subject, wherein the binding molecule comprises:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first VH region and a first CH1 region, and a second VH region;
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a VL region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region bind to a first antigen
  • the Fv region binds to a second antigen
  • the first antigen is different from the second antigen
  • polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • CH3 constant heavy 3
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide and/or the four polypeptide further comprise both a CH1 region and an ABS C- terminal to the second VH region and/or to the VL region, respectively.
  • the first and/or second polypeptides further comprise an ABS. In some embodiments, with regard to any of the previous methods, the first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain. In specific embodiments, with regard to any of the previous methods, the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • a binding molecule comprising:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • the second VH region of the third polypeptide and the VL region of the fourth polypeptide form an antigen binding Fv region; and wherein the first Fab region and the second Fab region binds to Programmed Death-Ligand 1 (PD-L1), and the Fv region binds to Cluster of Differentiation 3 (CD3).
  • PD-L1 Programmed Death-Ligand 1
  • CD3 Cluster of Differentiation 3
  • the third polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide and/or the four polypeptide further comprise both a CH1 region and an ABS C- terminal to the second VH region and/or to the VL region, respectively.
  • the third polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • the first and/or second polypeptides further comprise an ABS. In some embodiments, with regard to any of the previous binding molecules, the first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain. In specific embodiments, with regard to any of the previous binding molecules, the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • binding molecule wherein:
  • the antibody light chains of the first and the second polypeptide each comprise three Complementarity Determining Regions (CDRs) having amino acid sequences of SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.: 11;
  • CDRs Complementarity Determining Regions
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.: 7, and the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15; and
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.: 7, and the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region. In some embodiments, the first Fab region and the second Fab region are linked to the Fv region via fusion.
  • the flexible peptide region comprises an antibody hinge region.
  • the antibody hinge region is an Immunoglobulin G (IgG) hinge region.
  • the antibody hinge region is selected from the group consisting of IgG 1, IgG2, IgG3, and IgG4 hinge regions.
  • the antibody hinge region comprises an interchain disulfide bond between the third polypeptide and the fourth polypeptide.
  • the flexible peptide region further comprises a linker.
  • the linker comprises an amino acid sequence of GGGGS (G4S).
  • the linker comprises an amino acid sequence of GGGGSGGGGS.
  • the linker comprises an amino acid sequence of GGSGGGGSG.
  • binding molecule wherein:
  • the antibody light chains of the first and the second polypeptide each comprise a VL region that comprises the amino acid sequence of SEQ ID NO.: 8;
  • the first VH region comprises the amino acid sequence of SEQ ID NO.: 4;
  • the second VH region comprises the amino acid sequence of SEQ ID NO.: 12;
  • the third VH region comprises the amino acid sequence of SEQ ID NO.: 4
  • the VL region comprises the amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each comprise the amino acid sequence of SEQ ID NO.: 3; the third polypeptide comprises the amino acid sequence of SEQ ID NO.: 1; and the fourth polypeptide comprises the amino acid sequence of SEQ ID NO.: 2.
  • the first polypeptide and the second polypeptide each comprise the amino acid sequence of SEQ ID NO.: 95; the third polypeptide comprises the amino acid sequence of SEQ ID NO.: 96; and the fourth polypeptide comprises the amino acid sequence of SEQ ID NO.: 97.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 95; the third polypeptide has the amino acid sequence of SEQ ID NO.: 98; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 99.
  • a binding molecule comprising:
  • a third nucleic acid encoding a third polypeptide comprising, in the order from N- terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth nucleic acid encoding a fourth polypeptide comprising, in the order from N- terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • second polypeptide and the third VH region and the second CH1 region of the fourth polypeptide form a second antigen binding Fab region
  • first Fab region and the second Fab region bind to PD-L1
  • Fv region binds to CD3
  • polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • CH3 constant heavy 3
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • ABS albumin binding domain or site
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • polypeptide and/or the four polypeptide further comprise both a CH1 region and an ABS C- terminal to the second VH region and/or to the VL region, respectively.
  • the first and/or second polypeptides further comprise an ABS. In some embodiments, with regard to any of the previous methods, the first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain. In specific embodiments, with regard to any of the previous methods, the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • the antibody light chains of the first and the second polypeptide each comprise three Complementarity Determining Regions (CDRs) having amino acid sequences of SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.: 11;
  • CDRs Complementarity Determining Regions
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.: 7, and the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15; and
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.: 7, and the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region.
  • the flexible peptide region comprises an antibody hinge region.
  • the antibody hinge region is an Immunoglobulin G (IgG) hinge region.
  • the antibody hinge region comprises an interchain disulfide bond between the third polypeptide and the fourth polypeptide.
  • the flexible peptide region further comprises a linker.
  • the linker comprises an amino acid sequence of GGGGS (G4S).
  • the linker comprises an amino acid sequence of GGGGSGGGGS.
  • the linker comprises an amino acid sequence of GGSGGGGSG.
  • VH region of each of the first and second Fab regions comprises an amino acid sequence of SEQ ID NO.: 4;
  • VL region of each of the first and second Fab regions comprises an amino acid sequence of SEQ ID NO.: 8; wherein the VH region of the Fv region comprises an amino acid sequence of SEQ ID NO.: 12; and
  • VL region of the Fv region comprises an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 3; the third polypeptide has the amino acid sequence of SEQ ID NO.: 1; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 2.
  • compositions comprising a binding molecule.
  • a pharmaceutical composition comprising a binding molecule and a pharmaceutically acceptable carrier, wherein the binding molecule comprises:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region binds to PD-L1, and the Fv region binds to CD3.
  • the third polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region. In some embodiments, with regard to any of the previous pharmaceutical compositions, both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • CH3 constant heavy 3
  • ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide and/or the four polypeptide further comprise both a CH1 region and an ABS C-terminal to the second VH region and/or to the VL region, respectively.
  • the first and/or second polypeptides further comprise an ABS. In some embodiments, with regard to any of the previous pharmaceutical compositions, the first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain. In specific embodiments, with regard to any of the previous pharmaceutical compositions, the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • provided herein are methods of treating a disease or condition comprising administering a binding molecule.
  • a method of treating a disease or condition in a subject comprising administering a therapeutically effective amount of a binding molecule to the subject, wherein the binding molecule comprises:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and (c) a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region binds to PD-L1, and the Fv region binds to CD3.
  • polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • CH3 constant heavy 3
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • ABS albumin binding domain or site
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide and/or the four polypeptide further comprise both a CH1 region and an ABS C- terminal to the second VH region and/or to the VL region, respectively.
  • the first and/or second polypeptides further comprise an ABS. In some embodiments, with regard to any of the previous methods, the first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain. In specific embodiments, with regard to any of the previous methods, the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • a binding molecule comprising:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region each binds to CD20 or epidermal growth factor receptor (EGFR), and the Fv region binds to CD3.
  • EGFR epidermal growth factor receptor
  • the third polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide and/or the four polypeptide further comprise both a CH1 region and an ABS C- terminal to the second VH region and/or to the VL region, respectively.
  • the first and/or second polypeptides further comprise an ABS. In some embodiments, with regard to any of the previous binding molecules, the first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain. In specific embodiments, with regard to any of the previous binding molecules, the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • the first Fab region and the second Fab region bind to CD20
  • the antibody light chains of the first and the second polypeptide each comprise three Complementarity Determining Regions (CDRs) having amino acid sequences of SEQ ID NO.:
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, and SEQ ID NO.: 29, and the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.:
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, and SEQ ID NO.: 29, and the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region.
  • the flexible peptide region comprises an antibody hinge region.
  • the antibody hinge region comprises an interchain disulfide bond between the third polypeptide and the fourth polypeptide.
  • the flexible peptide region further comprises a linker.
  • binding molecule wherein:
  • the antibody light chains of the first and the second polypeptide each comprise a VL region that comprises the amino acid sequence of SEQ ID NO.: 30;
  • the first VH region comprises the amino acid sequence of SEQ ID NO.: 26 and the second VH region comprises the amino acid sequence of SEQ ID NO.: 12;
  • the third VH region comprises the amino acid sequence of SEQ ID NO.: 26 and the VL region comprises the amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 25; the third polypeptide has the amino acid sequence of SEQ ID NO.: 23; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 24.
  • the first Fab region and the second Fab region bind to EGFR
  • the antibody light chains of the first and the second polypeptide each comprise three CDRs having amino acid sequences of SEQ ID NO.: 45, SEQ ID NO.: 46, and SEQ ID NO.: 47;
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43
  • the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.:
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43
  • the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region.
  • the flexible peptide region comprises an antibody hinge region.
  • the antibody hinge region comprises an interchain disulfide bond between the third polypeptide and the fourth polypeptide.
  • the flexible peptide region further comprises a linker.
  • binding molecule wherein:
  • the antibody light chains of the first and the second polypeptide each comprise a VL region that comprises the amino acid sequence of SEQ ID NO.: 44;
  • the first VH region comprises the amino acid sequence of SEQ ID NO.: 40
  • the second VH region comprises the amino acid sequence of SEQ ID NO.: 12;
  • the third VH region comprises the amino acid sequence of SEQ ID NO.: 40
  • the VL region comprises the amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 39; the third polypeptide has the amino acid sequence of SEQ ID NO.: 37; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 38.
  • a binding molecule comprising:
  • first Fab region and the second Fab region each binds to CD20 or EGFR, and the Fv region binds to CD3, and
  • polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • CH3 constant heavy 3
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • ABS albumin binding domain or site
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide and/or the four polypeptide further comprise both a CH1 region and an ABS C- terminal to the second VH region and/or to the VL region, respectively.
  • the first and/or second polypeptides further comprise an ABS. In some embodiments, with regard to any of the previous methods, the first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain. In specific embodiments, with regard to any of the previous methods, the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • the first Fab region and the second Fab region bind to CD20
  • the antibody light chains of the first and the second polypeptide each comprise three Complementarity Determining Regions (CDRs) having amino acid sequences of SEQ ID NO.: 31, SEQ ID NO.: 32, and SEQ ID NO.: 33;
  • CDRs Complementarity Determining Regions
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, and SEQ ID NO.: 29, and the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15; and
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, and SEQ ID NO.: 29, and the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region.
  • the flexible peptide region comprises an antibody hinge region.
  • the flexible peptide region further comprises a linker.
  • the antibody light chains of the first and the second polypeptide each comprise a VL region that comprises the amino acid sequence of SEQ ID NO.: 30;
  • the first VH region comprises the amino acid sequence of SEQ ID NO.: 26 and the second VH region comprises the amino acid sequence of SEQ ID NO.: 12;
  • the third VH region comprises the amino acid sequence of SEQ ID NO.: 26 and the VL region comprises the amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 25; the third polypeptide has the amino acid sequence of SEQ ID NO.: 23; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 24.
  • the first Fab region and the second Fab region bind to CD20
  • the antibody light chains of the first and the second polypeptide each comprise three CDRs having amino acid sequences of SEQ ID NO.: 45, SEQ ID NO.: 46, and SEQ ID NO.: 47;
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43
  • the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.:
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43
  • the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region.
  • the flexible peptide region comprises an antibody hinge region.
  • the flexible peptide region further comprises a linker.
  • the antibody light chains of the first and the second polypeptide each comprise a VL region that comprises the amino acid sequence of SEQ ID NO.: 44;
  • the first VH region comprises the amino acid sequence of SEQ ID NO.: 40
  • the second VH region comprises the amino acid sequence of SEQ ID NO.: 12
  • the third VH region comprises the amino acid sequence of SEQ ID NO.: 40
  • the VL region comprises the amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 39; the third polypeptide has the amino acid sequence of SEQ ID NO.: 37; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 38.
  • compositions comprising a binding molecule.
  • a pharmaceutical composition comprising a therapeutically effective amount of a binding molecule and a pharmaceutically acceptable carrier, wherein the binding molecule comprises:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region each binds to CD20 or EGFR, and the Fv region binds to CD3.
  • the third polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region. In some embodiments, with regard to any of the previous pharmaceutical compositions, both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • CH3 constant heavy 3
  • ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide and/or the four polypeptide further comprise both a CH1 region and an ABS C-terminal to the second VH region and/or to the VL region, respectively.
  • the first and/or second polypeptides further comprise an ABS. In some embodiments, with regard to any of the previous pharmaceutical compositions, the first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain. In specific embodiments, with regard to any of the previous pharmaceutical compositions, the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • provided herein are methods of treating a disease or condition comprising administering a binding molecule.
  • a method of treating a disease or condition in a subject comprising administering a therapeutically effective amount of a binding molecule to the subject, wherein the binding molecule comprises:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and (c) a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region each binds to CD20 or EGFR, and the Fv region binds to CD3.
  • polypeptide further comprises a constant heavy 3 (CH3) region C-terminal to the second VH region.
  • CH3 constant heavy 3
  • both the third and the fourth polypeptides further comprise CH3 regions C-terminal to the second VH region and the VL region, respectively.
  • polypeptide further comprises an albumin binding domain or site (ABS) C-terminal to the second VH region.
  • ABS albumin binding domain or site
  • the fourth polypeptide further comprises an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise ABS C-terminal to the second VH region and the VL region, respectively.
  • polypeptide further comprises a constant heavy 3 (CH3) region and an ABS C-terminal to the second VH region.
  • the fourth polypeptide further comprises a CH3 region and an ABS C-terminal to the VL region.
  • both the third and the fourth polypeptides further comprise CH3 regions and ABSs C-terminal to the second VH region and the VL region, respectively.
  • the third polypeptide and/or the four polypeptide further comprise both a CH1 region and an ABS C- terminal to the second VH region and/or to the VL region, respectively.
  • the first and/or second polypeptides further comprise an ABS. In some embodiments, with regard to any of the previous methods, the first and/or second polypeptides further comprise an ABS C-terminal to the antibody light chain. In specific embodiments, with regard to any of the previous methods, the first and/or second polypeptides further comprise an ABS at the C-terminus of the antibody light chain.
  • binding molecule comprising:
  • a first antigen binding domain comprising two antibody Fab regions, each comprising:
  • antibody CH1 region wherein the first portion does not contain an antibody CH2 region and an antibody CH3 region;
  • VL antibody variable light
  • CL antibody light chain constant region
  • a second antigen binding domain comprising an antibody Fv region comprising a VH region and an antibody variable light (VL) region
  • the second antigen binding domain binds to an antigen present on an immune cell
  • the first portion and the second portion of each Fab region of the first antigen binding domain are on the same polypeptide.
  • at least one Fab region is oriented from N-terminus to C-terminus in the following order: VH-CH1-VL-CL.
  • at least one Fab region is oriented from N-terminus to C-terminus in the following order: VL-CL-VH-CH1.
  • the first portion and the second portion of each Fab region are present on separate polypeptides.
  • the VH region and the VL region of the Fv region are on the same polypeptide.
  • the Fv region is oriented from N-terminus to C- terminus in the following order: VH-VL.
  • the Fv region is oriented from N-terminus to C-terminus in the following order: VL-VH.
  • the VH region and the VL region of the Fv region are on separate polypeptides.
  • the first antigen binding domain and the second antigen binding domain are linked by a flexible peptide region.
  • the flexible peptide region comprises an antibody hinge region.
  • the antibody hinge region is a IgG hinge region.
  • the IgG hinge region is of IgGl subtype.
  • the IgG hinge region is of IgG2 subtype.
  • the IgG hinge region is of IgG3 subtype.
  • the IgG hinge region is of IgG4 subtype.
  • the flexible peptide region comprise additional amino acids.
  • the flexible peptide region further comprises a linker between the antibody hinge region and the second antigen binding domain.
  • the linker comprises an amino acid sequence of GGGGS (G4S).
  • the second antigen binding domain further comprises a first CH3 region linked to the VH region of the Fv region and a second CH3 region linked to the VL region of the Fv region.
  • the binding molecule further comprises one or more albumin binding domain or site (ABS).
  • ABS is linked to the C-terminus of the VH region of the Fv region.
  • the ABS is linked to the C-terminus of the VL region of the Fv region.
  • the C-terminus of each of the VL and VH regions of the Fv region is linked to ABS.
  • the ABS is linked to the CL region of at least one of the Fab regions.
  • the binding molecule further comprises one or more albumin domain.
  • the two Fab regions bind to different antigens. In other embodiments, the two Fab regions bind to the same antigen. In some embodiments, the two Fab regions bind to the same epitope of the same antigen. In other embodiments, the two Fab regions bind to different epitopes of the same antigen.
  • the first antigen binding domain and the second antigen binding domain bind to the same antigen.
  • the second antigen binding domain binds to the same epitope as at least one of the epitopes bound by the first antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain bind to different antigens, and wherein the first antigen binding domain binds to a first antigen and the second antigen binding domain binds to a second antigen.
  • the first antigen is a cancer antigen. In other embodiments, the first antigen is not a cancer antigen.
  • the second antigen is expressed on an immune cells including lymphocytes and monocytes. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a B cell. In other embodiments, the second antigen is expressed on a dendritic cell. In other embodiments, the second antigen is expressed on a granulocyte. In yet other embodiments, the second antigen is expressed on an innate lymphoid cell. In yet other embodiments, the second antigen is expressed on a megakaryocyte. In yet other embodiments, the second antigen is expressed on a monocyte. In yet other embodiments, the second antigen is expressed on a myeloid-derived suppressor cell. In yet other embodiments, the second antigen is expressed on a NK cell.
  • the second antigen is expressed on an effector cell. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a T helper cell, a regulatory T cell, or a cytotoxic T cell. In some embodiments, the second antigen is expressed on a T helper cell. In some embodiments, the second antigen is expressed on a regulatory T cell. In some embodiments, the second antigen is expressed on a cytotoxic T cell. In some embodiments, the second antigen is expressed on a CD8+ T cell. In some embodiments, the second antigen is expressed on a CD4+ T cell. In some embodiments, the second antigen comprises an extracellular domain.
  • the VH region of the first portion of each Fab region comprise the same CDR amino acid sequences. In other embodiments, the VH region of the first portion of each Fab region comprise different CDR amino acid sequences. [00131] In some embodiments, the VL region of the second portion of each Fab region comprise the same CDR amino acid sequences. In other embodiments, the VL region of the second portion of each Fab region comprise different CDR amino acid sequenceln some embodiments, the VH region of the first portion of each Fab region comprise the same CDR amino acid sequences, and the VL region of the second portion of each Fab region comprise the same CDR amino acid sequences. In some embodiments, the VH region of the first portion of each Fab region comprise the same CDR amino acid sequences, and the VL region of the second portion of each Fab region comprise different CDR amino acid sequences. In some embodiments, the VH region of the first portion of each Fab region comprise the same CDR amino acid sequences, and the VL region of the second portion of each Fab region comprise
  • the VH region of the first portion of each Fab region comprise different CDR amino acid sequences, and the VL region of the second portion of each Fab region comprise the same CDR amino acid sequences. In some embodiments, the VH region of the first portion of each Fab region comprise different CDR amino acid sequences, and the VL region of the second portion of each Fab region comprise different CDR amino acid sequences.
  • the second antigen is CD3.
  • the first antigen is a cancer antigen and the second antigen is CD3.
  • the first antigen is PD-L1 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.: 11;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 4; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 8; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first antigen is CD20 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 31, SEQ ID NO.: 32, and SEQ ID NO.: 33;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 26; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 30; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first antigen is EGFR and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43;
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 45, SEQ ID NO.: 46, and SEQ ID NO.: 47;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15 and the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 40; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 44; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first antigen is Her2 and the second antigen is TNF alpha.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 51; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 52; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 53; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 54.
  • binding molecule comprising:
  • a fourth polypeptide comprising a third VH region and a second CH1, and a VL region, wherein the first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region;
  • the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region.
  • the flexible peptide region comprises an antibody hinge region.
  • the antibody hinge region is a IgG hinge region.
  • the IgG hinge region is of IgGl subtype.
  • the IgG hinge region is of IgG2 subtype.
  • the IgG hinge region is of IgG3 subtype.
  • the IgG hinge region is of IgG4 subtype.
  • the flexible peptide region further comprises a linker between the antibody hinge region and the second antigen binding domain.
  • the linker comprises an amino acid sequence of GGGGS (G4S).
  • the first Fab region and the second Fab region bind to different antigens. In other embodiments, the first Fab region and the second Fab region bind to the same antigen. In some embodiments, the first Fab region and the second Fab region bind to the same epitope of the same antigen. In other embodiments, the first Fab region and the second Fab region bind to different epitopes of the same antigen.
  • the first Fab region and the second Fab region form a first antigen binding domain
  • the Fv region forms a second antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain bind to the same antigen.
  • the second antigen binding domain binds to the same epitope as at least one of the epitopes bound by the first antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain bind to different antigens, and wherein the first antigen binding domain binds to a first antigen and the second antigen binding domain binds to a second antigen.
  • the first antigen is a cancer antigen. In other embodiments, the first antigen is not a cancer antigen.
  • the second antigen is expressed on an immune cells including lymphocytes and monocytes. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a B cell. In other embodiments, the second antigen is expressed on a dendritic cell. In other embodiments, the second antigen is expressed on a granulocyte. In yet other embodiments, the second antigen is expressed on an innate lymphoid cell. In yet other embodiments, the second antigen is expressed on a megakaryocyte. In yet other embodiments, the second antigen is expressed on a monocyte. In yet other embodiments, the second antigen is expressed on a myeloid-derived suppressor cell. In yet other embodiments, the second antigen is expressed on a NK cell.
  • the second antigen is expressed on an effector cell. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a T helper cell, a regulatory T cell, or a cytotoxic T cell. In some embodiments, the second antigen is expressed on a T helper cell. In some embodiments, the second antigen is expressed on a regulatory T cell. In some embodiments, the second antigen is expressed on a cytotoxic T cell. In some embodiments, the second antigen is expressed on a CD8+ T cell. In some embodiments, the second antigen is expressed on a CD4+ T cell. In some embodiments, the second antigen comprises an extracellular domain.
  • the second antigen is CD3.
  • the first antigen is a cancer antigen and the second antigen is CD3.
  • the first antigen is PD-L1 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.: 11;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 16, SEQ ID NO.: 17, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 4; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 8; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 3; the third polypeptide has the amino acid sequence of SEQ ID NO.: l ; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.:2.
  • the first antigen is CD20 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, and SEQ ID NO.: 29;
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 31, SEQ ID NO.: 32, and SEQ ID NO.: 33;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 26; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 30; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 25; the third polypeptide has the amino acid sequence of SEQ ID NO.: 23; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 24.
  • the first antigen is EGFR and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 45, SEQ ID NO.: 46, and SEQ ID NO.: 47;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 40; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 44; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 39; the third polypeptide has the amino acid sequence of SEQ ID NO.: 37; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 38.
  • the first antigen is Her2 and the second antigen is TNF alpha.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 51; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 52; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 53; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 54.
  • provided herein are methods for making the binding molecules provided herein.
  • a method of making a binding molecule comprising transfecting one or more vectors into a host cell, wherein the one or more vectors comprise: (a) a first nucleic acid encoding a first polypeptide and a second polypeptide, each being an antibody light chain,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide can form a first antigen binding Fab region
  • the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region.
  • the flexible peptide region comprises an antibody hinge region.
  • the antibody hinge region is a IgG hinge region.
  • the IgG hinge region is of IgGl subtype.
  • the IgG hinge region is of IgG2 subtype.
  • the IgG hinge region is of IgG3 subtype.
  • the IgG hinge region is of IgG4 subtype.
  • the flexible peptide region further comprises a linker between the antibody hinge region and the second antigen binding domain.
  • the linker comprises an amino acid sequence of GGGGS (G4S).
  • the first Fab region and the second Fab region bind to different antigens. In other embodiments, the first Fab region and the second Fab region bind to the same antigen. In some embodiments, the first Fab region and the second Fab region bind to the same epitope of the same antigen. In other embodiments, the first Fab region and the second Fab region bind to different epitopes of the same antigen.
  • the first Fab region and the second Fab region form a first antigen binding domain, and the Fv region forms a second antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain bind to the same antigen.
  • the second antigen binding domain binds to the same epitope as at least one of the epitopes bound by the first antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain bind to different antigens, and wherein the first antigen binding domain binds to a first antigen and the second antigen binding domain binds to a second antigen.
  • the first antigen is a cancer antigen. In other embodiments, the first antigen is not a cancer antigen.
  • the second antigen is expressed on an immune cells including lymphocytes and monocytes. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a B cell. In other embodiments, the second antigen is expressed on a dendritic cell. In other embodiments, the second antigen is expressed on a granulocyte. In yet other embodiments, the second antigen is expressed on an innate lymphoid cell. In yet other embodiments, the second antigen is expressed on a megakaryocyte. In yet other embodiments, the second antigen is expressed on a monocyte. In yet other embodiments, the second antigen is expressed on a myeloid-derived suppressor cell. In yet other embodiments, the second antigen is expressed on a NK cell.
  • the second antigen is expressed on an effector cell. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a T helper cell, a regulatory T cell, or a cytotoxic T cell. In some embodiments, the second antigen is expressed on a T helper cell. In some embodiments, the second antigen is expressed on a regulatory T cell. In some embodiments, the second antigen is expressed on a cytotoxic T cell. In some embodiments, the second antigen is expressed on a CD8+ T cell. In some embodiments, the second antigen is expressed on a CD4+ T cell. In some embodiments, the second antigen comprises an extracellular domain.
  • the second antigen is CD3.
  • the first antigen is a cancer antigen and the second antigen is CD3.
  • the first antigen is PD-L1 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.: 11;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 4; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 8; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 3; the third polypeptide has the amino acid sequence of SEQ ID NO.: 1; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 2.
  • the first nucleic acid has a nucleotide sequence of SEQ ID NO.: 22; the second nucleic acid has a nucleotide sequence of SEQ ID NO.: 20; and the third nucleic acid has a nucleotide sequence of SEQ ID NO.:2l.
  • the first antigen is CD20 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 31, SEQ ID NO.: 32, and SEQ ID NO.: 33;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 26; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 30; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 25; the third polypeptide has the amino acid sequence of SEQ ID NO.: 23; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 24.
  • the first nucleic acid has a nucleotide sequence of SEQ ID NO.: 36; the second nucleic acid has a nucleotide sequence of SEQ ID NO.: 34; and the third nucleic acid has a nucleotide sequence of SEQ ID NO.: 35.
  • the first antigen is EGFR and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43;
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 45, SEQ ID NO.: 46, and SEQ ID NO.: 47;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 40; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 44; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 39; the third polypeptide has the amino acid sequence of SEQ ID NO.: 37; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 38.
  • the first nucleic acid has a nucleotide sequence of SEQ ID NO.: 50; the second nucleic acid has a nucleotide sequence of SEQ ID NO.: 48; and the third nucleic acid has a nucleotide sequence of SEQ ID NO.: 49.
  • the first antigen is Her2 and the second antigen is TNF alpha.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 51; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 52; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 53; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 54.
  • a pharmaceutical composition comprising a therapeutically effective amount of the binding molecule provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is for use in treating a disease or condition in a subject.
  • the disease or condition is a cancer.
  • cancer is lung cancer.
  • the cancer is Non Small Cell Lung Carcinoma (NSCLC).
  • NSCLC Non Small Cell Lung Carcinoma
  • the cancer is a Diffuse Large B cell
  • the disease or condition is a PD-L1 positive cancer.
  • the disease or condition is a cancer.
  • cancer is lung cancer.
  • NSCLC Non Small Cell Lung Carcinoma
  • the cancer is a Diffuse Large B cell
  • the disease or condition is a PD-L1 positive cancer.
  • a binding molecule comprising:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and (c) a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region bind to a first antigen
  • the Fv region binds to a second antigen
  • the first antigen is different from the second antigen
  • Immunoglobulin G (IgG) hinge region IgG hinge region.
  • the binding molecule of embodiment 5, wherein the antibody hinge region is selected from the group consisting of IgGl, IgG2, IgG3, and IgG4 hinge regions.
  • the binding molecule of embodiment 12, wherein the immune cell is selected from the group consisting of lymphocytes and monocytes.
  • the binding molecule of embodiment 12, wherein the immune cell is is selected from the group consisting of a T cell, a B cell, a dendritic cell, a granulocyte, an innate lymphoid cell, a megakaryocyte, a monocyte, a myeloid-derived suppressor cell, and a natural killer (NK) cell.
  • the immune cell is selected from the group consisting of a T cell, a B cell, a dendritic cell, a granulocyte, an innate lymphoid cell, a megakaryocyte, a monocyte, a myeloid-derived suppressor cell, and a natural killer (NK) cell.
  • TAA tumor associated antigen
  • TSA tumor specific antigen
  • the binding molecule of embodiment 20, wherein the cancer antigen is selected from a group consisting of CD 19, CD20, EGFR, Her2, and PD-F1.
  • a method of making a binding molecule comprising:
  • a third nucleic acid encoding a third polypeptide comprising, in the order from N- terminus to C-terminus, a first VH region and a first CH1 region and a second VH region;
  • a fourth nucleic acid encoding a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region and a VF region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide can form a first antigen binding Fab region; wherein the second polypeptide and the third VH region and the second CH1 region of the fourth polypeptide can form a second antigen binding Fab region;
  • first Fab region and the second Fab region bind to a first antigen
  • the Fv region binds to a second antigen
  • the first antigen is different from the second antigen
  • a pharmaceutical composition comprising a binding molecule and a pharmaceutically acceptable carrier, wherein the binding molecule comprises:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first VH region and a first CH1 region, and a second VH region;
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a VL region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • second polypeptide and the third VH region and the second CH1 region of the fourth polypeptide form a second antigen Fab region; wherein the second VH region of the third polypeptide and the VL region of the fourth polypeptide form an antigen binding Fv region;
  • first Fab region and the second Fab region bind to a first antigen
  • the Fv region binds to a second antigen
  • the first antigen is different from the second antigen
  • a method of treating a disease or condition in a subject comprising administering a therapeutically effective amount of a binding molecule to the subject, wherein the binding molecule comprises:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first VH region and a first CH1 region, and a second VH region;
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a VL region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region bind to a first antigen
  • the Fv region binds to a second antigen
  • the first antigen is different from the second antigen
  • a binding molecule comprising:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • second polypeptide and the third VH region and the second CH1 region of the fourth polypeptide form a second antigen binding Fab region
  • first Fab region and the second Fab region binds to Programmed Death- Ligand 1 (PD-L1), and the Fv region binds to Cluster of Differentiation 3 (CD3).
  • PD-L1 Programmed Death- Ligand 1
  • CD3 Cluster of Differentiation 3
  • the antibody light chains of the first and the second polypeptide each comprise three Complementarity Determining Regions (CDRs) having amino acid sequences of SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.: 11;
  • CDRs Complementarity Determining Regions
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.: 7, and the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.:
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.: 7, and the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.:
  • the binding molecule of embodiment 35 wherein the antibody hinge region comprises an interchain disulfide bond between the third polypeptide and the fourth polypeptide. 39. The binding molecule of embodiment 35, wherein the flexible peptide region further comprises a linker.
  • the antibody light chains of the first and the second polypeptide each comprise a VL region that comprises the amino acid sequence of SEQ ID NO.: 8;
  • the first VH region comprises the amino acid sequence of SEQ ID NO.: 4
  • the second VH region comprises the amino acid sequence of SEQ ID NO.: 12;
  • the third VH region comprises the amino acid sequence of SEQ ID NO.: 4
  • the VL region comprises the amino acid sequence of SEQ ID NO.: 16.
  • first polypeptide and the second polypeptide each comprise the amino acid sequence of SEQ ID NO.: 3; the third polypeptide comprises the amino acid sequence of SEQ ID NO.: 1 ; and the fourth polypeptide comprises the amino acid sequence of SEQ ID NO.: 2.
  • first polypeptide and the second polypeptide each comprise the amino acid sequence of SEQ ID NO.: 95; the third polypeptide comprises the amino acid sequence of SEQ ID NO.: 96; and the fourth polypeptide comprises the amino acid sequence of SEQ ID NO.: 97.
  • first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 95; the third polypeptide has the amino acid sequence of SEQ ID NO.: 98; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 99.
  • a method of making a binding molecule comprising:
  • the binding molecule comprises (i) expressing the binding molecule from one or more vectors in a host cell, wherein the one or more vectors comprise (a) a first nucleic acid encoding a first polypeptide and a second nucleic acid encoding a second polypeptide, wherein each polypeptide comprises an antibody light chain,
  • a third nucleic acid encoding a third polypeptide comprising, in the order from N- terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth nucleic acid encoding a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region bind to PD-L1
  • Fv region binds to CD3
  • the antibody light chains of the first and the second polypeptide each comprise three Complementarity Determining Regions (CDRs) having amino acid sequences of SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.: 11;
  • CDRs Complementarity Determining Regions
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.: 7, and the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.:
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.: 7, and the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.:
  • linker comprises an amino acid sequence of GGGGSGGGGS.
  • linker comprises an amino acid sequence of GGSGGGGSG.
  • VH region of each of the first and second Fab regions comprises an amino acid sequence of SEQ ID NO.: 4;
  • VL region of each of the first and second Fab regions comprises an amino acid sequence of SEQ ID NO.: 8;
  • VH region of the Fv region comprises an amino acid sequence of SEQ ID NO.: 12;
  • VL region of the Fv region comprises an amino acid sequence of SEQ ID NO.: 16.
  • a pharmaceutical composition comprising a binding molecule and a pharmaceutically acceptable carrier, wherein the binding molecule comprises:
  • a first polypeptide and a second polypeptide each comprising an antibody light chain
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region binds to PD-L1, and the Fv region binds to CD3.
  • a method of treating a disease or condition in a subject comprising administering a therapeutically effective amount of a binding molecule to the subject, wherein the binding molecule comprises:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • a binding molecule comprising:
  • a third polypeptide comprising, in the order from N-terminiis to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region each binds to CD20 or epidermal growth factor receptor (EGFR), and the Fv region binds to CD3.
  • EGFR epidermal growth factor receptor
  • the antibody light chains of the first and the second polypeptide each comprise three Complementarity Determining Regions (CDRs) having amino acid sequences of SEQ ID NO.: 31, SEQ ID NO.: 32, and SEQ ID NO.: 33;
  • CDRs Complementarity Determining Regions
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, and SEQ ID NO.: 29, and the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.:
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, and SEQ ID NO.: 29, and the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.:
  • the antibody light chains of the first and the second polypeptide each comprise a VL region that comprises the amino acid sequence of SEQ ID NO.: 30;
  • the first VH region comprises the amino acid sequence of SEQ ID NO.: 26 and the second VH region comprises the amino acid sequence of SEQ ID NO.: 12;
  • the third VH region comprises the amino acid sequence of SEQ ID NO.: 26 and the VL region comprises the amino acid sequence of SEQ ID NO.: 16.
  • the antibody light chains of the first and the second polypeptide each comprise three CDRs having amino acid sequences of SEQ ID NO.: 45, SEQ ID NO.: 46, and SEQ ID NO.: 47;
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43
  • the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.:
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43
  • the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.:
  • the antibody light chains of the first and the second polypeptide each comprise a VL region that comprises the amino acid sequence of SEQ ID NO.: 44;
  • the first VH region comprises the amino acid sequence of SEQ ID NO.: 40
  • the second VH region comprises the amino acid sequence of SEQ ID NO.: 12;
  • the third VH region comprises the amino acid sequence of SEQ ID NO.: 40
  • the VL region comprises the amino acid sequence of SEQ ID NO.: 16.
  • first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 39; the third polypeptide has the amino acid sequence of SEQ ID NO.: 37; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 38.
  • a method of making a binding molecule comprising:
  • a third nucleic acid encoding a third polypeptide comprising, in the order from N- terminus to C-terminus, a first VH region and a first CH1 region, and a second VH region;
  • a fourth nucleic acid encoding a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a VL region, wherein the first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region;
  • first Fab region and the second Fab region each binds to CD20 or EGFR, and the Fv region binds to CD3, and
  • the antibody light chains of the first and the second polypeptide each comprise three Complementarity Determining Regions (CDRs) having amino acid sequences of SEQ ID NO.: 31, SEQ ID NO.: 32, and SEQ ID NO.: 33;
  • CDRs Complementarity Determining Regions
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, and SEQ ID NO.: 29, and the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.:
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, and SEQ ID NO.: 29, and the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.:
  • the antibody light chains of the first and the second polypeptide each comprise a VL region that comprises the amino acid sequence of SEQ ID NO.: 30;
  • the first VH region comprises the amino acid sequence of SEQ ID NO.: 26 and the second VH region comprises the amino acid sequence of SEQ ID NO.: 12;
  • the third VH region comprises the amino acid sequence of SEQ ID NO.: 26 and the VL region comprises the amino acid sequence of SEQ ID NO.: 16.
  • first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 25; the third polypeptide has the amino acid sequence of SEQ ID NO.: 23; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 24.
  • the antibody light chains of the first and the second polypeptide each comprise three CDRs having amino acid sequences of SEQ ID NO.: 45, SEQ ID NO.: 46, and SEQ ID NO.: 47;
  • the first VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43
  • the second VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.:
  • the third VH region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43
  • the VL region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.:
  • the antibody light chains of the first and the second polypeptide each comprise a VL region that comprises the amino acid sequence of SEQ ID NO.: 44;
  • the first VH region comprises the amino acid sequence of SEQ ID NO.: 40, and the second VH region comprises the amino acid sequence of SEQ ID NO.: 12;
  • the third VH region comprises the amino acid sequence of SEQ ID NO.: 40
  • the VL region comprises the amino acid sequence of SEQ ID NO.: 16.
  • a pharmaceutical composition comprising a therapeutically effective amount of a binding molecule and a pharmaceutically acceptable carrier, wherein the binding molecule comprises:
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region each binds to CD20 or EGFR, and the Fv region binds to CD3.
  • a method of treating a disease or condition in a subject comprising administering a therapeutically effective amount of a binding molecule to the subject, wherein the binding molecule comprises:
  • a first polypeptide and a second polypeptide each comprising an antibody light chain
  • a third polypeptide comprising, in the order from N-terminus to C-terminus, a first variable heavy (VH) region and a first constant heavy 1 (CH1) region, and a second VH region; and
  • a fourth polypeptide comprising, in the order from N-terminus to C-terminus, a third VH region and a second CH1 region, and a variable light (VL) region,
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region
  • first Fab region and the second Fab region each binds to CD20 or EGFR, and the Fv region binds to CD3.
  • FIG. 1A illustrates a binding molecule (ALiCE) provided herein.
  • FIG. 1B illustrates an exemplary binding molecule provided herein that contains CH3 regions.
  • FIG. 1C illustrates an exemplary binding molecule provided herein that contains albumin binding sites (ABS).
  • FIG. 1A illustrates a binding molecule (ALiCE) provided herein.
  • FIG. 1B illustrates an exemplary binding molecule provided herein that contains CH3 regions.
  • FIG. 1C illustrates an exemplary binding molecule provided herein that contains albumin binding sites (ABS).
  • FIG. 1D illustrates an exemplary binding molecule provided herein that has a binding domain targeting a cancer antigen and also illustrates exemplary options for the flexible peptide region provided herein.
  • FIG. 1E illustrates an exemplary binding molecule provided herein that has a binding domain targeting a cancer antigen and a binding domain targeting CD3.
  • FIG. 2A illustrates the assembly pattern of ACE-00.
  • “BiP” illustrates the binding immunoglobubin protein (BiP), which binds CH1 or VH domains of ACE-00 that are exposed.
  • FIG. 2B shows the results of the SDS-PAGE performed to identify the assembly pattern of ACE- 00.
  • the arrows indicate the band of ACE-00-VL in the“ACE-00+ Light chain” sample and the two bands of ACE-00-VH and ACE-00- VL in the“ACE-00- VH+ACE-00-VL+ Light chain” samples under the reducing condition.
  • FIG. 2C shows the co-immunoprecipitation (co-IP) results of wild-type heavy chain (HC), CH1 -truncated heavy chain (ACHl) and VH-CH I - truncated heavy chain (AVH-CHl) of adalimumab.
  • Fig. 2D illustrates that BiP can regulate the assembly and secretion of heavy chain by interaction with VH and/or CH1 domain of heavy chain.
  • “ERAD” represents endoplasmic-reticulum-associated protein degradation.
  • FIG. 2E illustrates contribution of the antibody VH domain to antibody assembly and contribution of the ACE-00-VH chain to the proper assembly of ACE-00 molecule.
  • “X” represents no assembly; “O” represents assembly.
  • FIG. 2F illustrates the structure of ACE-00.
  • FIG. 2G shows the results of affinity chromatography for ACE-00 and ACE-00-VL2 proteins using HitrapTM KappaSelect (GE healthcare, USA).
  • FIG. 2H shows the results of capillary electrophoresis performed to identify the molecular size differentiation between ACE-00- VL2 and ACE-00.
  • FIG. 21 shows the results of capillary electrophoresis showing the conformation of ACE-00 and ACE-00-VL2 molecules.
  • Solid arrows indicate results of ACE-00; dashed arrows indicate results of ACE-00- VL2.
  • FIG. 2J shows the results of capillary isoelectric focusing performed to corroborate the heterodimerization between ACE-00- VH chain and ACE-00- VL chain.
  • FIG. 2K shows the results of SDS-PAGE and capillary electrophoresis performed to identify the assembly pattern of ACE-00. “R” represents reducing;“NR” represents non reducing.
  • FIG. 2L shows the results of size exclusion chromatography of ACE-00.
  • FIG. 3 shows the result of SDS-PAGE (under reducing (left) and non-reducing (right) conditions) performed to identify the expression and assembly of ACE-02, ACE-02-VL2, ACE- 03, ACE-03 -VL2, ACE-00 and ACE-01.
  • ACE-02 contains the second antigen as humanized 12F6 (hl2F6, an anti-CD3 antibody)
  • ACE-03 contains the second antigen as humanized OKT3 (hOKT3, an anti-CD3 antibody).
  • the arrows indicate the bands of assembled ACE-02, ACE-03 and ACE-03-VL2 respectively under non-reducing condition.
  • FIG. 4A illustrates the structure of ACE-04. “A” refers to anti-PD-Ll. UCHT1 is an anti-CD3 antibody. FIG. 4B illustrates the structure of ACE-05. “A” refers to anti-PD-Ll.
  • OKT3 is an anti-CD3 antibody.
  • FIG. 4C show the results of SDS-PAGE performed to identify the assembly pattern of ACE-04, ACE-04- VL2, ACE-05, and ACE-05-VL2. The arrows indicate the bands of ACE-04 and ACE-05 under reducing and non-reducing conditions.
  • FIG. 4C show the results of SDS-PAGE performed to identify the assembly pattern of ACE-04, ACE-04- VL2, ACE-05, and ACE-05-VL2. The arrows indicate the bands of ACE-04 and ACE-05 under reducing and non-reducing conditions.
  • FIG. 4D shows the results of the SDS-PAGE performed to identify the assembly pattern of ACE-05 (top) and illustrates the potential regulatory mechanisms in ACE-05 assembly (bottom).
  • FIGs. 4E-4F show the results of SDS-PAGE and capillary electrophoresis performed to identify the conformations of ACE-05 as well as the heterodimerization efficiency between ACE-05 -VH and ACE-05-VL chains.
  • “M” represents marker;“R” represents reducing;“NR” represents non reducing;“IN” represents input;“FT” represents flow through;“W” represents washing;“Elu.” represents elution.
  • FIG. 4G shows the size exclusion chromatography performed to identify the purity of ACE-05.
  • FIG. 4H shows the results of size exclusion chromatography for gel filtration analysis of ACE-05.
  • FIG. 41 shows the results of cationic exchange chromatography (CEX) performed to identify the structure conformations of ACE-05.
  • CEX cati
  • FIG. 5 shows the results of SDS-PAGE performed to identify the assembly pattern of ACE-09 and ACE-05 at 37 °C and 32 °C (top) and summarizes features of ACE-09 and ACE-05 (bottom).
  • lanes that are labeled with“ACE-05” show results of ACE-05; the rest of the lanes show the results of ACE-09;“R” represents reducing;“N.R” represents“non reducing”;“IP” represents input;“F.T” represents flow through;“W” represents washing;“OP” represents output.
  • FIG. 6A illustrates the structure of ACE- 10.
  • FIGs. 6B-6C show the expression and assembly analysis of the ACE- 10 molecule (under reducing (left) and non -reducing (right) conditions).
  • the arrows indicate the bands of ACE- 10 under reducing and non reducing conditions.
  • “ACE-10 dialysis” represents ACE-10 generated via transfection with dialyzed DNA.
  • the arrow in the results from the anti-kappa condition indicates ACE-10-VL/ACE-10-LC dimer and ACE-10-VH/ACE-10-LC dimer complex under non reducing condition; the arrow in the results from the anti-CHl condition indicates the assembled ACE- 10 under non reducing condition.
  • FIG. 7A illustrates the structure of ACE-l 1.
  • FIG. 7B shows the results of SDS-PAGE performed to identify the expression and assembly pattern of ACE-l 1 and ACE-l 1-VL2 (analyzed by Coomassie Blue staining (left) and Western blot (right)).
  • M represents marker.
  • the arrows indicate the bands of assembled ACE-l 1 under non reducing condition.
  • FIG. 8 shows the results of SDS-PAGE performed to identify the assembly pattern of ACE- 12, ACE-05 and ACE-09.
  • the arrows indicate the bands of ACE-05-VH + LC dimer and ACE-05-VL + LC dimer under non-reducing condition using anti-kappa (left) and anti-CHl (right) antibodies in Western blot.
  • FIG. 9 shows the results of the Enzyme-Linked Immunosorbent Assay (ELISA) for determining the affinity of ACE-00 and ACE-00-VL2 to TNF alpha.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • FIGs. 10A-10C show the analysis of binding affinity of ACE-05 to PD-L1 (lOA)and CD3 (10B-10C) using ELISA.
  • FIG. 11 shows the analysis of binding affinity of ACE-05 and ACE-09 to CD3 using ELISA.
  • FIGs. 12A-12C show the analysis of binding kinetics of ACE-05 to PD-L1 (12A, 12C) and CD3 (12B, 12C) using Surface Plasmon Resonance (SPR).
  • FIG. 12D shows the kinetics analysis of ACE-05 binding simultaneously to PD-L1 and CD3 using Surface Plasmon
  • FIG. 13A shows PD-L1 expression levels in HEK293E-PD-L1 cells and the parental HEK293E cells (right panel) and CD3 expression levels in Jurkat luciferase reporter cells as measured by various CD3 antibodies (left panel).
  • FIGs. 13B-13E show the results of the T cell redirecting (activity) assay for ACE-05 and BiTE-05.
  • FIG. 13F shows T cell activation in the presence of ACE-05, BiTE-05, or YBL-007 in different T cell stages.
  • FIGs. 13G-13H show the results of the PD-1/PD-L1 blockage assay for ACE-05.
  • FIG. 13I-13J show T cell activation by ACE-05, BiTE-05, UCHT1 (an anti-CD3 antibody from BioLegend, USA), or OKT3 (an anti- CD3 antibody from BioLegend, USA).
  • FIG. 13K shows the results of T cell cytotoxicity assay for determining ACE-05 mediated T cell cytotoxicity.
  • FIG. 13L shows the results of T cell cytotoxicity mediated by ACE-05, BiTE-05, YBL-007, or UCHT1. “IgG” represents normal human IgG used as a negative control.
  • FIGs. 13M shows the T cell cytotoxicity on tumor cells when in direct contact with PBMC cells in the presence of ACE-05 or YBL-007.
  • FIG. 13N shows IL-2 and INF-g levels in the presence of ACE-05, BiTE-05 or YBL-007 in co-cultivated PBMC and HCC827 cells.
  • IgG represents normal human IgG used as a negative control.
  • FIG. 130 shows the thermodynamic stability results of ACE-05, BiTE-05, YBL-007, and UCHT1.
  • FIGs. 14A-14B show the results of T cell redirecting assay performed for determining ACE- 10 mediated T cell activation.
  • “hlgG” represents normal human IgG used as a control.
  • FIG. 15 shows the results of T cell cytotoxicity assay for determining ACE- 11 mediated T cell cytotoxicity.
  • FIG. 16A-16B show the results of ACE-05 pharmacokinetic study in Sprague-Dawley (SD) rat.
  • FIG. 17A shows the results of the HCC827 (PD-L1 positive tumor) xenograft study in a humanized mouse model.
  • FIG. 17B shows the anti-tumor effects of ACE-05 and other test articles in PBMC donor A and donor B.
  • FIG. 17C shows body weight changes of donor A and donor B.
  • FIG. 17D shows individual anti-tumor efficacy responses.
  • FIG. 17E shows individual body weight loss (%) (side-effect).
  • FIG. 17F shows the comparison of anti-tumor efficacy of BiTE and ACE-05 and the comparison of the parental PD-L1 antibody and ACE-05.
  • FIGs. 18A-18D show the results of the dose limit study showing the anti-tumor effects of ACE-05 and other test articles in PBMC donor A and donor B in a humanized mouse model inoculated with HCC827 (PD-L1 positive tumor) xenograft.
  • FIG. 18A shows dose response of anti-tumor efficacy for ACE-05 and BiTE-05.
  • FIG. 18B and FIG. 18C show anti-tumor efficacy of individual mice in each dose group for ACE-05 and BiTE-05, respectively.
  • FIG. 18D shows individual body weight loss (%) (side-effect) of ACE-05 and BiTE-05 treated group.
  • ALiCE antibody like cell engagers
  • ALiCE molecules provided herein have two antigen binding domains.
  • the first antigen binding domain has two Fab regions.
  • the second antigen binding domain has a Fv region.
  • a typical ALiCE molecule is depicted in FIG. 1A.
  • the first antigen binding domain comprises Fab regions, and the second antigen binding domain is attached (directly or indirectly) to the first antigen binding domain generally at the position where CH2 and CH3 domains would generally be located in a native antibody structure.
  • the C terminus of heavy chain comprises a VH domain rather than a CH2 domain and the C-terminus of the second heavy chain comprises a VL domain rather than a domain.
  • the binding molecules disclosed herein provide many advantages over conventional antibodies and existing multispecific antibodies (e.g., bispecific antibodies). Due to its multiple antigen binding domains and overall configuration design, the binding molecules provided herein can be used as a cell engager to bring multiple cells together. For example, the first antigen binding domain can bind to an antigen expressed on a first cell and the second antigen binding domain can bind to an antigen expressed on a second cell, and thereby bring the two cells together.
  • one of the engaged cells is an immune cell, e.g., a cytotoxic T cell.
  • the binding molecules provided herein are particularly useful for directing and activating an immune cell.
  • the bivalent Fab portion of ALiCE molecule retains the functionality of conventional antibodies
  • the second Fc-less monovalent antigen-binding region i.e., the Fv region
  • the Fv region can recognize, engage, redirect, and/or activate effector cells of the immune system, such as T cells.
  • ACE-05 an AFiCE molecule composed of anti-PD- Fl and anti-CD3 domains, shows synergistic effects for both PD-F1 -dependent (mediated) T cell activation and PD-l and PD-F1 blockade efficacy.
  • the absence of a fully functional Fc region, or the absence of complete CH2 and/or CH3 region abolishes or reduces certain undesirable Fc-mediated effector cytotoxicity.
  • the native interaction between the VH and VF chains of the Fv portion facilitates heterodimerization of the AFiCE molecule without imparting undesirable immunogenicity through artificial engineering.
  • PK studies presented herein indicate higher stability of AFiCE molecules than other formats such as BiTE (bispecific T-cell engager) or DART (dual-affinity re-targeting)
  • BiTE bispecific T-cell engager
  • DART dual-affinity re-targeting
  • binding molecule refers to a protein comprising a portion (e.g ., one or more binding regions such as CDRs) that binds to a target or an antigen and, optionally, a scaffold or framework portion (e.g., one or more scaffold or framework regions) that allows the binding portion to adopt a conformation that promotes binding of the binding protein to a polypeptide, fragment, or epitope.
  • a binding molecule is said to specifically bind or selectively bind to an antigen, for example, when the dissociation constant (K D ) is ⁇ 10 7 M.
  • the binding molecule may specifically bind to an antigen with a K D of from about 10 7 M to about 10 12 M. In certain embodiments, the binding molecule may specifically bind to an antigen with high affinity when the K D is ⁇ l0 8 M or K D is ⁇ l0 9 M. In one embodiment, the binding molecule may specifically bind to a purified human antigen with a K D of from 1 x 10 9 M to 10 x 10 9 M as measured by OCTET ® . In yet another embodiment, the binding molecule specifically binds to a human antigen expressed on cells with a K D of from 0.1 x 10 9 M to 10 x 10 9 M.
  • the binding molecule specifically binds to a human antigen expressed on cells with a K D of about 0.1 x 10 9 M , about 0.5 x 10 9 M, about 1 x 10 9 M, about 5 x 10 9 M, about 10 x 10 9 M, or any range or interval thereof.
  • the term“binding molecule” includes antibodies and molecules derived from antibodies.
  • antibody “immunoglobulin,” or“Ig” is used interchangeably herein, and is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain antibodies, and fragments thereof, as described below.
  • An antibody can be human, humanized, chimeric and/or affinity matured, as well as an antibody from other species, for example, mouse and rabbit, etc.
  • antibody is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997).
  • the specific molecular antigen can be bound by an antibody provided herein, including a polypeptide or an epitope.
  • Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • Non-limiting examples of functional fragments include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab’) fragments, F(ab) 2 fragments, F(ab’) 2 fragments, disulfide-linked Fvs (dsFv),
  • scFv single-chain Fvs
  • Fab fragments F(ab’) fragments
  • F(ab) 2 fragments F(ab’) 2 fragments
  • dsFv disulfide-linked Fvs
  • antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody).
  • antigen e.g., one or more CDRs of an antibody.
  • Such antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A
  • the antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.
  • Antibodies may be agonistic antibodies or antagonistic antibodies.
  • An“antigen” is a structure to which an antibody can selectively bind.
  • a target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound.
  • the target antigen is a polypeptide.
  • an antigen is associated with a cell, for example, is present on or in a cell, for example, an immune cell.
  • the terms“antigen-binding fragment,”“antigen-binding domain,”“antigen-binding region,” and similar terms refer to that portion of a binding molecule, which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g., the CDRs).
  • binding refers to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope.
  • the ratio of dissociation rate (k 0ff ) to association rate (k on ) of a binding molecule (e.g., an antibody) to a monovalent antigen (k off /k on ) is the dissociation constant K D , which is inversely related to affinity.
  • K D the dissociation constant
  • the value of K D varies for different complexes of antibody and antigen and depends on both k on and k 0ff .
  • the dissociation constant K D for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art.
  • the affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen.
  • binding molecules described herein terms such as“bind to,” “that specifically bind to,” and analogous terms are also used interchangeably herein and refer to binding molecules of antigen binding domains that specifically bind to an antigen, such as a polypeptide.
  • a binding molecule or antigen binding domain that binds to or specifically binds to an antigen may be cross-reactive with related antigens.
  • a binding molecule or antigen binding domain that binds to or specifically binds to an antigen does not cross-react with other antigens.
  • a binding molecule or antigen binding domain that binds to or specifically binds to an antigen can be identified, for example, by immunoassays, Octet ® , Biacore ® , or other techniques known to those of skill in the art.
  • a binding molecule or antigen binding domain binds to or specifically binds to an antigen when it binds to an antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELIS As).
  • RIA radioimmunoassays
  • ELIS As enzyme linked immunosorbent assays
  • a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding binding specificity.
  • the extent of binding of a binding molecule or antigen binding domain to a“non-target” protein is less than about 10% of the binding of the binding molecule or antigen binding domain to its particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA.
  • FACS fluorescence activated cell sorting
  • RIA fluorescence activated cell sorting
  • Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
  • a binding molecule or antigen binding domain that binds to an antigen includes one that is capable of binding the antigen with sufficient affinity such that the binding molecule is useful, for example, as a diagnostic agent in targeting the antigen.
  • a binding molecule or antigen binding domain that binds to an antigen has a dissociation constant (K D ) of less than or equal to 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM.
  • K D dissociation constant
  • a binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among the antigen from different species (e.g., between human and cyno species).
  • Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • Bmax refers to the maximum binding affinity extrapolated from experimental results. Bmax can be calculated using known curve fitting methods in the art, for example, curve fitting methods provided in GraphPad Prism software 7.
  • the affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (K D ).
  • K D dissociation constant
  • Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
  • a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following.
  • the“K D ” or“K D value” may be measured by assays known in the art, for example by a binding assay.
  • the K D may be measured in a RIA, for example, performed with the Fab version of an antibody of interest and its antigen (Chen et ah, 1999, J. Mol Biol 293:865-81).
  • the K D or K D value may also be measured by using biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by Octet®, using, for example, a Octet® QK384 system, or by Biacore®, using, for example, a Biacore® TM-2000 or a Biacore® TM-3000.
  • An“on-rate” or“rate of association” or“association rate” or“k on ” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the Octet® QK384, the Biacore® TM-2000, or the Biacore® TM-3000 system.
  • BLI biolayer interferometry
  • SPR surface plasmon resonance
  • reducing refers to a condition in which interchain or intrachain disulfide (S-S) bridges within a protein are denatured or reduced, for example, by the addition of 2-Mercaptoethanol (2-ME) or dithiothreitol (DTT), resulting in multiple polypeptide chains.
  • non reducing refers to a condition in which interchain or intrachain disulfide (S-S) bridges within a protein remain intact in the absence of denaturing or reducing agents such as 2-Mercaptoethanol (2-ME) or dithiothreitol (DTT).
  • the binding molecules or antigen binding domains can comprise“chimeric” sequences in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et ai, 1984, Proc. Natl. Acad. Sci. USA 81:6851-55).
  • the binding molecules or antigen binding domains can comprise portions of“humanized” forms of nonhuman (e.g., murine) antibodies that are chimeric antibodies that include human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • a nonhuman species e.g., donor antibody
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • a humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the binding molecules or antigen binding domains can comprise portions of a“fully human antibody” or“human antibody,” wherein the terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin. “Fully human” antibodies, in certain embodiments, can also encompass antibodies which bind polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence. The term“fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. ( see Kabat et al. (1991) Sequences of Proteins of
  • A“human antibody” is one that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381 ; Marks et al, 1991, J. Mol. Biol. 222:581) and yeast display libraries (Chao et al, 2006, Nature Protocols 1 : 755-68).
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol.
  • the binding molecules or antigen binding domains can comprise portions of a“recombinant human antibody,” wherein the phrase includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, L. D. et al. (1992) Nucl. Acids Res.
  • a“recombinant human antibody” wherein the phrase includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic and
  • human antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest. Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the binding molecules or antigen binding domains can comprise a portion of a“monoclonal antibody,” wherein the term as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on the antigen.
  • a“monoclonal antibody,” as used herein is an antibody produced by a single hybridoma or other cell. The term “monoclonal” is not limited to any particular method for making the antibody.
  • the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et ah, 1975, Nature 256:495, or may be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat.
  • The“monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature 352:624-28 and Marks et al., 1991, J. Mol. Biol. 222:581-97, for example. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are well known in the art. See, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).
  • a typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • the 4-chain unit is generally about 150,000 daltons.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and g chains and four CH domains for m and e isotypes.
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end.
  • VL variable domain
  • CL constant domain
  • the VL is aligned with the VH
  • the CL is aligned with the first constant domain of the heavy chain (CH1).
  • Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • the pairing of a VH and VL together forms a single antigen-binding site.
  • the term“Fab” or“Fab region” refers to an antibody region that binds to antigens.
  • a conventional IgG usually comprises two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure.
  • Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain. More specifically, the variable region and the constant region of the heavy chain in a Fab region are VH and CH1 regions, and the variable region and the constant region of the light chain in a Fab region are VL and CL regions.
  • the VH, CH1, VL, and CL in a Fab region can be arranged in various ways to confer an antigen binding capability according to the present disclosure.
  • VH and CH1 regions can be on one polypeptide, and VL and CL regions can be on a separate polypeptide, similarly to a Fab region of a conventional IgG.
  • VH, CH 1 , VL and CL regions can all be on the same polypeptide and oriented in different orders as described in more detail the sections below.
  • variable region refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the variable region of the heavy chain may be referred to as“VH.”
  • the variable region of the light chain may be referred to as“VH.”
  • the term“variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
  • variable regions consist of less variable (e.g ., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long.
  • FRs framework regions
  • hypervariable regions that are each about 9-12 amino acids long.
  • the variable regions of heavy and light chains each comprise four FRs, largely adopting a b sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the b sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)).
  • the constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
  • the variable regions differ extensively in sequence between different antibodies.
  • the variable region is a human variable region.
  • variable region residue numbering according to Kabat or“amino acid position numbering as in Kabat”, and variations thereof, refer to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 and three inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a“standard” Kabat numbered sequence.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra).
  • The“EU numbering system” or“EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra).
  • The“EU index as in Kabat” refers to the residue numbering of the human IgG 1 EU antibody.
  • Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.
  • An“intact” antibody is one comprising an antigen-binding site as well as a CL and at least heavy chain constant regions, CH1, CH2 and CH3.
  • the constant regions may include human constant regions or amino acid sequence variants thereof.
  • an intact antibody has one or more effector functions.
  • “Antibody fragments” comprise a portion of an intact antibody, such as the antigen binding or variable region of the intact antibody. Examples of antibody fragments include, without limitation, Fab, Fab’, F(ab’) 2 , and Fv fragments; diabodies and di-diabodies (see, e.g., Holliger et al, 1993, Proc. Natl. Acad. Sci. 90:6444-48; Lu et al., 2005, J. Biol. Chem.
  • the term“heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy-terminal portion includes a constant region.
  • the constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (a), delta (d), epsilon (e), gamma (g), and mu (m), based on the amino acid sequence of the heavy chain constant region.
  • the distinct heavy chains differ in size: a, d, and g contain approximately 450 amino acids, while m and e contain approximately 550 amino acids.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgM immunoglobulin M
  • subclasses of IgG namely IgGl, IgG2, IgG3, and IgG4.
  • the term“light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy-terminal portion includes a constant region.
  • the approximate length of a light chain is 211 to 217 amino acids.
  • K kappa
  • l lambda
  • CDR Determining Region
  • A“CDR” refers to one of three hypervariable regions (Hl, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH b-sheet framework, or one of three hypervariable regions (Ll, L2 or L3) within the non-framework region of the antibody VL b-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences.
  • CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems.
  • the Rabat Complementarity Determining Regions are based on sequence variability and are the most commonly used (see, e.g., Rabat et al., supra). Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17).
  • the end of the Chothia CDR-H1 loop when numbered using the Rabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Rabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35 A and 35B are present, the loop ends at 34).
  • the AbM hypervariable regions represent a compromise between the Rabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2
  • IMGT ImMunoGeneTics
  • Ig immunoglobulins
  • TCR T cell receptors
  • the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain.
  • “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody.
  • An additional numbering system (AHon) has been developed by Honegger and Pluckthun, 2001, J. Mol. Biol. 309: 657-70.
  • CDR complementary determining region
  • individual CDRs e.g.,“CDR-H1, CDR-H2
  • the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the Rabat, Chothia, or Contact method.
  • the particular amino acid sequence of a CDR is given.
  • Hypervariable regions may comprise“extended hypervariable regions” as follows: 24-36 or 24-34 (Ll), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26- 35A (Hl), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH.
  • constant region or“constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor.
  • the term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site.
  • the constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.
  • FR refers to those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.
  • Fc region herein is used to define a C-terminal region of an
  • immunoglobulin heavy chain including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl- terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • A“functional Fc region” possesses an“effector function” of a native sequence Fc region. Exemplary“effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis;
  • A“variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion).
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide.
  • the variant Fc region herein can possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith.
  • a polypeptide“extracellular domain” or“ECD” refers to a form or a portion of the polypeptide that is essentially free of the transmembrane and cytoplasmic domains.
  • an ECD may have less than 1 % of such transmembrane and/or cytoplasmic domains and can have less than 0.5% of such domains.
  • an“epitope” is a term in the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind.
  • An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope.
  • an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a“conformational,”“non-linear” or“discontinuous” epitope).
  • a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure.
  • a binding molecule binds to a group of amino acids regardless of whether they are folded in a natural three dimensional protein structure.
  • a binding molecule requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.
  • polypeptide and“peptide” and“protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
  • polypeptides containing one or more analogs of an amino acid including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a“polypeptide” can occur as a single chain or as two or more associated chains.
  • vector refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, in order to introduce a nucleic acid sequence into a host cell.
  • a binding molecule e.g., an antibody
  • Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art.
  • both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • the introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art.
  • nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA
  • immunoblotting for expression of gene products or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • the term“host” as used herein refers to an animal, such as a mammal (e.g., a human).
  • the term“host cell” as used herein refers to a particular subject cell that may be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • An“isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • An“isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule, such as a cDNA molecule can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid molecules encoding an antibody as described herein are isolated or purified.
  • the term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure molecule may include isolated forms of the molecule.
  • Polynucleotide or“nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA.
  • the nucleotides can be
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • Oligonucleotide refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length.
  • the terms“oligonucleotide” and“polynucleotide” are not mutually exclusive.
  • a cell that produces a binding molecule of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced.
  • the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction.
  • RNA transcripts The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as“upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as“downstream sequences.”
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • carrier can also refer to a diluent, adjuvant (e.g., Freund’s adjuvant (complete or incomplete)), excipient, or vehicle.
  • adjuvant e.g., Freund’s adjuvant (complete or incomplete)
  • excipient or vehicle.
  • Such carriers, including pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • Oral compositions including
  • compositions can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington and Gennaro, Remington’s Pharmaceutical Sciences ( 18th ed. 1990).
  • Compositions, including pharmaceutical compounds may contain a binding molecule (e.g., an antibody), for example, in isolated or purified form, together with a suitable amount of carriers.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia. European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • the term“effective amount” as used herein refers to the amount of binding molecule (e.g., an antibody) or pharmaceutical composition provided herein which is sufficient to result in the desired outcome.
  • a subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human).
  • the subject is a human.
  • the subject is a mammal, e.g., a human, diagnosed with a condition or disorder.
  • the subject is a mammal, e.g., a human, at risk of developing a condition or disorder.
  • administer refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.
  • the terms“treat,”“treatment” and“treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or condition resulting from the administration of one or more therapies.
  • the terms“about” and“approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.
  • the term“and/or” as used in a phrase such as“A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term“and/or” as used in a phrase such as“A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • binding molecules comprising multiple antigen binding domains (e.g., two antigen binding domains).
  • the multiple antigen binding domains of the binding molecules provided herein are useful for engaging cells, bringing a cell to an immune cell, or redirecting an immune cell.
  • the binding molecules provided herein comprises two antigen binding domains, wherein the first antigen binding domain comprises two antibody Fab regions and the second antigen binding domain comprises an antibody Fv region.
  • Each of the two Fab regions contains two portions: a first portion having an antibody variable heavy (VH) region and an antibody CH1 region; and a second portion having a second portion comprising an antibody light chain (EC) comprising an antibody variable light (VF) region and an antibody light chain constant region (CF).
  • VH antibody variable heavy
  • EC antibody light chain
  • VF antibody variable light
  • CF antibody light chain constant region
  • Each of the two Fab regions binds to an antigen.
  • the Fv region in the second antigen binding domain comprises a VH region and an antibody variable light (VF) region.
  • the two Fab regions are linked to the Fv region.
  • the present disclosure provides a binding molecule comprising:
  • a first antigen binding domain comprising two antibody Fab regions, each comprising:
  • antibody CH1 region wherein the first portion does not contain an antibody CH2 region and an antibody CH3 region;
  • VF variable light
  • CF antibody light chain constant region
  • a second antigen binding domain comprising an antibody Fv region comprising a VH region and an antibody variable light (VF) region
  • a Fab region (i.e., antigen-binding fragment) is an antibody region that binds to antigens.
  • a conventional IgG usually comprises two Fab regions, each residing on one of the two arms of the Y -shaped IgG structure.
  • Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain. More specifically, the variable region and the constant region of the heavy chain in a Fab region are VH and CH1 regions, and the variable region and the constant region of the light chain in a Fab region are VL and CL regions.
  • VH, CH1, VL, and CL in a Lab region can be arranged in various ways to confer an antigen binding capability according to the present disclosure.
  • VH and CH1 regions can be on one polypeptide, and VL and CL regions can be on a separate polypeptide, similarly to a Lab region of a conventional IgG.
  • VH, CH1, VL and CL regions can all be on the same polypeptide and oriented in different orders as described in more detail below.
  • a Lv region is an antigen binding region that comprises a VH region and a VL region.
  • the VH and VL regions in a Lv region can be arranged in various ways to confer an antigen binding capability according to the present disclosure. Lor example, VH and VL region can be on the same or separate polypeptides. If the VH and VL regions are on the same polypeptide, they can be oriented in different orders as described in more detail below.
  • variable region refers to a portion of the light or heavy chains of an antibody that is generally located at the amino -terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the variable region of the heavy chain may be referred to as“VH.”
  • the variable region of the light chain may be referred to as“VH.”
  • variable region refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Lc receptor.
  • the term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site.
  • the constant region can determine the mechanism used to destroy antigen.
  • Antibodies are divided into five major classes, IgM, IgG, Iga, IgD, and IgE, based on their constant region structure and immune function.
  • IgG is the class of immunoglobulin characterized by g heavy chains. It is the most abundant class of immunoglobulin found in the plasma.
  • the constant region of a light chain is referred to as“CL.”
  • the multiple heavy-chain C domains (CH domains) are numbered from the amino-terminal end to the carboxy terminus, for example CH1, CH2, CH3 and so on. Any CL and CH1 regions of these antibody classes can be used in the present disclosure.
  • CL and CH1 regions provided herein are of IgG type (e.g., IgGl).
  • a representative CL region of the Fab region provided herein has the following amino acid sequence:
  • a representative CH1 region of the Fab region provided herein has the following amino acid sequence:
  • the language that“the first portion does not contain an antibody CH2 region and an antibody CH3 region” is used herein to mean that the first portion does not contain a complete antibody CH2 region or a complete CH3 region. However, this language does not exclude the embodiments wherein a part of a CH2 region and/or a CH3 region is included in the first portion.
  • CH2 and/or CH3 variants or truncations that do not exhibit full CH2 and/or CH3 activity may be included.
  • Assays such as Fc receptor binding assays or ADCC activity assays or other well-known assays for determining Fc region related functions may be used herein to determine if CH2 and/or CH3 activities (or Fc region activities) are fully retained.
  • the first portion and the second portion of each Fab region are present on separate polypeptides.
  • Each of the two Fab regions can also be optionally a single chain Fab region.
  • the first portion and the second portion of both Fab regions of the first antigen binding domain are on the same polypeptide.
  • the first portion and the second portion of one of the two Fab regions are on the same polypeptide.
  • the Fab region can be oriented from N-terminus to C-terminus in the following order: VH-CH1-VF-CF.
  • a single chain Fab region can be oriented from N-terminus to C-terminus in the following order: VL-CL-VH-CH1.
  • the VH region and the VL region of the Fv region are on separate polypeptides.
  • the Fv region of the second antigen binding domain is a single chain Fv (i.e., the VH region and the VF region of the Fv region are on the same polypeptide).
  • the Fv region can be oriented from N- terminus to C-terminus in the following order: VH-VF, or can be oriented from N-terminus to C- terminus in the following order: VF-VH.
  • each Fab region is on separate polypeptides and the VH and VF regions of the Fv region are also on separate polypeptides.
  • the first antigen binding domain and the second antigen binding domain are linked by a flexible peptide region.
  • the flexible peptide region comprises an antibody hinge region.
  • the antibody hinge region is an IgG hinge region.
  • the IgG hinge region provided herein may be selected from antibody hinge regions of various IgG subtypes. Table 2 below lists exemplary IgG subtypes with core hinge sequences that may be included in the flexible peptide region provided herein.
  • the IgG hinge region is of IgGl subtype. In other more specific embodiments, the IgG hinge region is of IgG2 subtype. In yet other more specific embodiments, the IgG hinge region is of IgG3 subtype. In yet other more specific embodiments, the IgG hinge region is of IgG4 subtype. In some specific embodiments, the flexible peptide region provided herein comprises an amino acid sequence of SEQ ID NO: 55.
  • the flexible peptide region provided herein comprises an amino acid sequence of SEQ ID NO: 56. In other specific embodiments, the flexible peptide region provided herein comprises an amino acid sequence of SEQ ID NO: 57. In some specific embodiments, the flexible peptide region provided herein comprises an amino acid sequence of SEQ ID NO: 58.
  • the flexible peptide region comprise additional amino acids.
  • the flexible peptide region further comprises a linker (e.g ., G4S) between the antibody hinge region and the second Fv antigen binding domain.
  • a linker e.g ., G4S
  • Flexible linker between antibody hinge region and second Fv domain may influence binding affinity of the second Fv domain. Improved binding affinity of second Fv domain can lead to increased redirectional efficiency of immune cells (e.g., effector cells including T cells) to target cells (e.g., cancer cells).
  • the second Fv domain need to bend to be able to interact with and bind to a surface antigen presented on immune cells (e.g., effector cells including T cells), because the paratope of the second Fv domain of AFiCE is structurally masked by the first Fab domain of AFiCE. Therefore, to reduce steric hindrance and optimize the binding of the second Fv domain to immune cells (e.g., effector cells including T cells), flexible linker such as G4S can be introduced between the antibody hinge region and the second Fv domain.
  • the linker comprises an amino acid sequence of GGGGS (G4S).
  • the linker comprises an amino acid sequence of (G4S)n where n is an integer.
  • the linker comprises an amino acid sequence of (G4S)i. In some more specific embodiments, the linker comprises an amino acid sequence of (G4S) 2 . In other more specific embodiments, the linker comprises an amino acid sequence of (G4S) 3 . In yet other more specific embodiments, the linker comprises an amino acid sequence of (G4S) 4 .
  • Other methods for designing and constructing linkers with different flexibilities are described in more detail in, e.g., Klein et al., Protein Engineering, Design & Selection, 2014, 27(10): 325-330, and
  • the binding molecules provided herein can optionally comprise a CH3 domain.
  • FIG. 1B illustrates such an exemplary binding molecule.
  • the second antigen binding domain further comprise a first CH3 region linked to the VH region of the Fv region and a second CH3 region linked to the VL region of the Fv region.
  • the CH regions are linked to the C-terminus of the VH and VL regions of the Fv region. The presence of the CH3 regions provides the Fc receptor binding capability of the binding molecules provided herein.
  • the CH3 regions linked to the Fv region are engineered to facilitate or enforce the association between the two CH3 regions using existing technologies such as knobs-into-holes (KiH) technology or electrostatic steering.
  • knobs-into- holes was originally proposed as a model for the packing of amino acid side chains between adjacent a-helices, and was later demonstrated to be an effective design strategy for engineering antibody heavy chain homodimers for heterodimerization.
  • a‘knob’ variant can be first obtained by replacement of a small amino acid with a larger one in one IgG CH3 domain (e.g., T to Y substitution).
  • the knob was designed to insert into a‘hole’ in another IgG CH3 domain created by replacement of a large residue with a smaller one (e.g., Y to T substitution).
  • knob-into-holes technology is described in detail with several examples in e.g., WO 96/027011, Ridgway et ah, Protein Eng 9 (1996) 617-621, and Merchant et ah, Nat Biotechnol 16 (1998) 677-681, each of which is incorporated herein by reference in its entirety.
  • Other well-known technologies for modifying CH3 regions to facilitate or enforce the association between the two CH3 regions are also contemplated in the present disclosure.
  • Albumin e.g., human serum albumin
  • albumin has been used to increase the serum half-life of biological drugs. See Dennis et ah, The Journal of Biological Cheminstry, 2002, 277 (38):
  • HSA human serum albumin
  • Alumin can be used in a few ways.
  • One exemplary approach is to directly couple an albumin domain (e.g., HSA) to the binding molecule provided herein, either genetically or chemically.
  • Another exemplary approach is to use an albumin binding domain or site (ABD or ABS).
  • the binding molecules provided herein may also optionally include one or more albumin binding domain or albumin binding site (ABD or ABS).
  • FIG. 1C illustrates such exemplary binding molecules.
  • the ABS of the binding molecules provided herein mediates binding with endogenous albumin, thereby helping prolong the half- life and/or enhancing the therapeutic effects of the binding molecules provided herein.
  • the ABS of the binding molecules provided herein may also help improve the pharmacokinetics, through the non-covalent association to albumin.
  • the ABS is linked to the C-terminus of the VH region of the Fv region.
  • the ABS is linked to the C-terminus of the VL region of the Fv region.
  • the C-terminus of each of the VL and VH regions of the Fv region is linked to ABS.
  • the ABS is linked to the CL region of at least one of the Fab regions.
  • the binding molecule can further optionally comprises one or more albumin domain (e.g., HSA).
  • an albumin domain is linked to the C- terminus of the VH region of the Fv region.
  • an albumin domain is linked to the C-terminus of the VL region of the Fv region.
  • the C-terminus of each of the VL and VH regions of the Fv region is linked to an albumin domain.
  • an albumin domain is linked to the CL region of at least one of the Fab regions.
  • the two Fab regions and Fv region of the binding molecules provided herein can each bind to an antigen. In some embodiments, the two Fab regions bind to different antigens.
  • the two Fab regions bind to the same antigen. In some embodiments, the two Fab regions bind to the same epitope of the same antigen. In other embodiments, the two Fab regions bind to different epitopes of the same antigen.
  • the first antigen can be the same or different from the antigen (a second antigen) bound by the Fv region.
  • the first antigen binding domain and the second antigen binding domain bind to the same antigen.
  • the second antigen binding domain binds to the same epitope as at least one of the epitopes bound by the first antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain bind to different antigens, and wherein the first antigen binding domain binds to a first antigen and the second antigen binding domain binds to a second antigen.
  • FIG. 1E provides an illustration of such an ALiCE molecule wherein the first antigen binding domain (the two Fab regions) binds to a cancer antigen, and the second antigen binding domain binds to an immune cell such as a T cell through an antigen like CD3.
  • Such ALiCE molecules can engage an immune cell (e.g., T cell) to a cancer cell and thus be used as a therapeutic for cancer treatment.
  • the binding molecules provided herein are bispecific binding molecules which comprises (a) two Fab regions (in the first antigen binding domain) that provides the binding affinity to the first antigen and (b) a Fv region (in the second antigen binding domain) that provides the binding affinity to the second antigen.
  • the first antigen binding domain can bind to an extracellular domain of a surface protein on one cell
  • the second antigen binding domain can bind to an extracellular domain of a surface protein on an immune cell, and thereby bring the two cells together.
  • the first antigen binding domain (with the two Fab regions) can bind to a cancer cell.
  • the first antigen is a cancer antigen (e.g., PD-L1). In other embodiments, the first antigen is not a cancer antigen.
  • the second antigen is expressed on an immune cells including lymphocytes and monocytes. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a B cell. In other embodiments, the second antigen is expressed on a dendritic cell. In other embodiments, the second antigen is expressed on a granulocyte. In yet other embodiments, the second antigen is expressed on an innate lymphoid cell. In yet other embodiments, the second antigen is expressed on a megakaryocyte. In yet other embodiments, the second antigen is expressed on a monocyte. In yet other embodiments, the second antigen is expressed on a myeloid-derived suppressor cell. In yet other embodiments, the second antigen is expressed on a NK cell.
  • the second antigen is expressed on an effector cell. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a T helper cell, a regulatory T cell, or a cytotoxic T cell. In some embodiments, the second antigen is expressed on a T helper cell. In some embodiments, the second antigen is expressed on a regulatory T cell. In some embodiments, the second antigen is expressed on a cytotoxic T cell. In some embodiments, the second antigen is expressed on a CD8+ T cell. In some embodiments, the second antigen is expressed on a CD4+ T cell. In some embodiments, the second antigen comprises an extracellular domain.
  • the second antigen is CD3.
  • the first antigen is a cancer antigen and the second antigen is CD3.
  • the first antigen is PD-L1 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.: 11;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 4; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 8; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first antigen is CD20 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 31, SEQ ID NO.: 32, and SEQ ID NO.: 33;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 26; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 30; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first antigen is EGFR and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43;
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 45, SEQ ID NO.: 46, and SEQ ID NO.: 47;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15 and the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 40; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 44; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first antigen is Her2 and the second antigen is TNF alpha.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 51; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 52; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 53; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 54.
  • the first antigen binding domain is at the N-terminus and maintains the native antibody structure at the N-terminus, while the second antigen binding domain is at the C-terminus and C-terminal CH2 and CH3 domains of both heavy chains are each substituted with a single VH and VL domain, respectively.
  • FIG. 1 A is an illustration of such an exemplary ALiCE molecule.
  • first portion and the second portion of each Fab region are on separate polypeptides
  • the first antigen binding domain binds to a first antigen
  • the VH region and the VL region of the Fv region are on separate polypeptides
  • the second antigen binding domain binds to a second antigen present on an immune cell
  • the first antigen and the second antigen are different antigens.
  • the binding molecule provided herein comprises:
  • a first antigen binding domain comprising two antibody Fab regions, each comprising:
  • LC antibody light chain
  • VL variable light
  • CL antibody light chain constant region
  • first portion and the second portion are on separate polypeptides; and wherein the first antigen binding domain binds to a first antigen.
  • a second antigen binding domain comprising an antibody Fv region comprising a VH region and an antibody variable light (VL) region, wherein the VH region and the VL region are on separate polypeptides; wherein the second antigen binding domain binds to a second antigen present on an immune cell,
  • first antigen and the second antigen are different antigens.
  • the first portion of one Fab region and the VH region of the Fv region are on the same polypeptide, and the portion of the other Fab region and the VL region of the Fv region are on the same polypeptide.
  • the binding molecule provided herein comprises:
  • a first antigen binding domain comprising a first antibody Fab region and a second antibody Fab region, each comprising:
  • antibody CH1 region wherein the first portion does not contain an antibody CH2 region and an antibody CH3 region;
  • VL antibody variable light
  • CL antibody light chain constant region
  • the first antigen binding domain binds to a first antigen
  • a second antigen binding domain comprising an antibody Fv region comprising a VH region and an antibody variable light (VL) region, wherein the second antigen binding domain binds to a second antigen present on an immune cell
  • first antigen and the second antigen are different antigens
  • the first antigen is a cancer antigen (e.g., PD-L1). In other embodiments, the first antigen is not a cancer antigen.
  • the second antigen is expressed on an immune cells including lymphocytes and monocytes. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a B cell. In other embodiments, the second antigen is expressed on a dendritic cell. In other embodiments, the second antigen is expressed on a granulocyte. In yet other embodiments, the second antigen is expressed on an innate lymphoid cell. In yet other embodiments, the second antigen is expressed on a
  • the second antigen is expressed on a monocyte. In yet other embodiments, the second antigen is expressed on a myeloid-derived suppressor cell. In yet other embodiments, the second antigen is expressed on a NK cell.
  • the second antigen is expressed on an effector cell. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a T helper cell, a regulatory T cell, or a cytotoxic T cell. In some embodiments, the second antigen is expressed on a T helper cell. In some embodiments, the second antigen is expressed on a regulatory T cell. In some embodiments, the second antigen is expressed on a cytotoxic T cell. In some embodiments, the second antigen is expressed on a CD8+ T cell. In some embodiments, the second antigen is expressed on a CD4+ T cell. In some embodiments, the second antigen comprises an extracellular domain.
  • the second antigen is CD3.
  • the first antigen is a cancer antigen and the second antigen is CD3.
  • the first antigen is PD-L1 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.: 11;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 4; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 8; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first antigen is CD20 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 31, SEQ ID NO.: 32, and SEQ ID NO.: 33;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 26; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 30; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first antigen is EGFR and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43;
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 45, SEQ ID NO.: 46, and SEQ ID NO.: 47;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15 and the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 40; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 44; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first antigen is Her2 and the second antigen is TNF alpha.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 51; the VF region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 52; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 53; and the VF region of the Fv region has an amino acid sequence of SEQ ID NO.: 54.
  • the binding molecules provided herein include four peptides (two antibody light chains and two heavy chain like chains), and their overall structure is similar to a traditional IgG except that the Fc region of the IgG is replaced with a Fv region. This structure can be further modified to generate variations that confer various properties. More specifically, in some embodiments, the binding molecule provided herein comprises:
  • a fourth polypeptide comprising a third VH region and a second CH1, and a VF region, wherein the first polypeptide and the first VH region and the first CH1 region of the third polypeptide form a first antigen binding Fab region;
  • the first polypeptide and the second polypeptide have the same amino acid sequence.
  • the binding molecules provided herein comprise two identical light chains (the first and second polypeptides) and two different heavy chain like chains (the third and fourth polypeptides).
  • the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region.
  • the flexible peptide region comprises an antibody hinge region.
  • the antibody is an IgG hinge region.
  • the IgG hinge region is of IgG 1 subtype.
  • the IgG hinge region is of IgG2 subtype.
  • the IgG hinge region is of IgG3 subtype.
  • the IgG hinge region is of IgG4 subtype.
  • the flexible peptide region provided herein comprises an amino acid sequence of SEQ ID NO: 55.
  • the flexible peptide region provided herein comprises an amino acid sequence of SEQ ID NO: 56. In other specific embodiments, the flexible peptide region provided herein comprises an amino acid sequence of SEQ ID NO: 57. In some specific embodiments, the flexible peptide region provided herein comprises an amino acid sequence of SEQ ID NO: 58.
  • the flexible peptide region comprise additional amino acids.
  • the flexible peptide region further comprises a linker (e.g ., G4S) between the antibody hinge region and the second Fv antigen binding domain.
  • a linker e.g ., G4S
  • Flexible linker between antibody hinge region and second Fv domain may influence binding affinity of the second Fv domain. Improved binding affinity of second Fv domain can lead to increased redirectional efficiency of immune cells (e.g., effector cells including T cells) to target cells (e.g., cancer cells).
  • the second Fv domain need to bend to be able to interact with and bind to a surface antigen presented on immune cells (e.g., effector cells including T cells), because the paratope of the second Fv domain of AFiCE is struructually masked by the first Fab domain of AFiCE. Therefore, to reduce streic hinderace and optimize the binidng of the second Fv domain to immune cells (e.g., effector cells including T cells), flexible liniker such as G4S can be introduced between the the antibody hinge region and the second Fv domain.
  • the linker comprises an amino acid sequence of GGGGS (G4S). In some embodiments, the linker comprises an amino acid sequence of (G4S)n where n is an integer.
  • the linker comprises an amino acid sequence of (G4S)i. In some more specific embodiments, the linker comprises an amino acid sequence of (G4S) 2 . In other more specific embodiments, the linker comprises an amino acid sequence of (G4S) 3 . In yet other more specific embodiments, the linker comprises an amino acid sequence of (G4S) 4 .
  • the first Fab region and the second Fab region bind to different antigens. In other embodiments, the first Fab region and the second Fab region bind to the same antigen. In some embodiments, the first Fab region and the second Fab region bind to the same epitope of the same antigen. In other embodiments, the first Fab region and the second Fab region bind to different epitopes of the same antigen.
  • the first Fab region and the second Fab region form a first antigen binding domain, and the Fv region forms a second antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain bind to the same antigen.
  • the second antigen binding domain binds to the same epitope as at least one of the epitopes bound by the first antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain bind to different antigens, and wherein the first antigen binding domain binds to a first antigen and the second antigen binding domain binds to a second antigen.
  • the first antigen is a cancer antigen. In other embodiments, the first antigen is not a cancer antigen.
  • the second antigen is expressed on an immune cells including lymphocytes and monocytes. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a B cell. In other embodiments, the second antigen is expressed on a dendritic cell. In other embodiments, the second antigen is expressed on a granulocyte. In yet other embodiments, the second antigen is expressed on an innate lymphoid cell. In yet other embodiments, the second antigen is expressed on a megakaryocyte. In yet other embodiments, the second antigen is expressed on a monocyte. In yet other embodiments, the second antigen is expressed on a myeloid-derived suppressor cell. In yet other embodiments, the second antigen is expressed on a NK cell.
  • the second antigen is expressed on an effector cell. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a T helper cell, a regulatory T cell, or a cytotoxic T cell. In some embodiments, the second antigen is expressed on a T helper cell. In some embodiments, the second antigen is expressed on a regulatory T cell. In some embodiments, the second antigen is expressed on a cytotoxic T cell. In some embodiments, the second antigen is expressed on a CD8+ T cell. In some embodiments, the second antigen is expressed on a CD4+ T cell. In some embodiments, the second antigen comprises an extracellular domain. [00332] In some specific embodiments, the second antigen is CD3. In some embodiments, the first antigen is a cancer antigen and the second antigen is CD3.
  • the first antigen is PD-L1 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.: 11;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 16, SEQ ID NO.: 17, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 4; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 8; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 3; the third polypeptide has the amino acid sequence of SEQ ID NO.: 1; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.:2.
  • the first antigen is CD20 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, and SEQ ID NO.: 29;
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 31, SEQ ID NO.: 32, and SEQ ID NO.: 33;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 26; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 30; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 25; the third polypeptide has the amino acid sequence of SEQ ID NO.: 23; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 24.
  • the first antigen is EGFR and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 45, SEQ ID NO.: 46, and SEQ ID NO.: 47;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 40; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 44; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 39; the third polypeptide has the amino acid sequence of SEQ ID NO.: 37; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 38.
  • the first antigen is Her2 and the second antigen is TNF alpha.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 51; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 52; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 53; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 54.
  • the binding molecules provided herein comprise two identical light chains and two different heavy chain like chains.
  • Antibody is assembled and secreted as a tetramer H2L2 and the quality control machinery is very tightly regulated in ER by BiP and PDI. It was known that unfolded CH1 domain of heavy chain has a role of regulation of antibody assembly in BiP dependent manner.
  • antibody VH domain also has a role of antibody assembly, and shows that the heavy chain like chain of the binding molecule provided herein that contains two VH regions (one in a Fab region and one in a Fv region) contributes to the proper assembly of the binding molecule.
  • C-terminal Fv also has an important role of heterodimerization of two different heavy chain like chains (the third and fourth polypeptides). Because the interaction between VH and VL regions is much stronger than VL-VL interaction, the VH-VL interaction was selected to make heterodimerization between the two different heavy chain like chains (the third and fourth polypeptides). The efficiency of heterodimerization was found very high and most of the binding molecules expressed and purified in mammalian cells were heterodimerized form (close to 99% heterodimerization efficiency).
  • this structure provides for the optimal synaptic distance between target and effector cells.
  • the distance of N-terminal two Fab region and the C-terminal Fv region was estimated to be 40 A.
  • the binding molecule provided herein has more folding complexity (molecular size) than other known bispecific antibodies such as BiTE, DART and other ScFv based bispecific antibody formats and thus are expected to have improved thermodynamic stability.
  • the binding molecules provided herein are bispecific binding molecules, in which the two Fab regions (N-terminal F(ab’)2) bind to the first antigen (e.g., a cancer antigen) and the Fv region binds to an immune cell (e.g., T cell).
  • the binding molecules provided herein are designed and constructed in Y- shape to provide synergistic effect of antibody function and immune redirecting (e.g., T cell redirecting), for example, for the maximum anti-tumor activity.
  • AFiCE molecules (predominantly reside in Y -shape) is designed to confer optimal immunological synaptic distance between the two antigen binding domains (two target paratopes) and maximize functional redirection of a cell (e.g., T cell) to other cells (e.g., tumor cells).
  • a cell e.g., T cell
  • other cells e.g., tumor cells.
  • high affinity and bivalent N-terminal two Fab regions
  • unwanted target independent T cell activation is reduced due to the monovalent and low affinity of the Fv region to an immune cell antigen.
  • ALiCE molecules may have different configurations, for example ALiCE molecules may reside in a Y -shape, or in a T-shape. In certain embodiments, the different configurations of ALiCE molecules may contribute to different distance between N-terminal two Fab region and the C-terminal Fv region in the ALiCE molecules. In certain embodiments, the distance between N-terminal two Fab region and the C- terminal Fv region in the binding molecules provided herein may be estimated to be in a range of between around 40 A and around 70 A. In certain embodiments, the distance between N- terminal two Fab region and the C-terminal Fv region in the binding molecules provided herein may be estimated to be around 42 A. In some other embodiments, the distance between N- terminal two Fab region and the C-terminal Fv region in the binding molecules provided herein may be estimated to be around 60 A.
  • the binding affinity of the first antigen binding domain to the first antigen is higher than the binding affinity of the second antigen binding domain to the second antigen.
  • the binding kinetics of ACE-05 to human PD-L1 was comparable to the parental anti-PD- Ll antibody (i.e., YBL-007 from Y-Biologics, Inc.) (see FIGs. 12A-12C).
  • the binding affinity of ACE-05 to CD3 was much lower than the parental anti-CD3 antibody (UCHT1 from BioLegend, USA) (see FIGs. 12A-12C).
  • antigen-antibody interactions are non-covalent and reversible, formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic and van der Waals forces.
  • affinity and/or avidity are usually mentioned.
  • affinity and/or avidity are usually mentioned.
  • the binding of an antibody to its antigen is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (KD).
  • KD is the ratio of an antibody dissociation rate (k 0ff or kd) (how quickly it dissociates from its antigen) to the antibody association rate (k on or k a ) (how quickly it binds to its antigen).
  • KD values are determined by measuring the k on and k off rates of a specific antibody/antigen interaction and then using a ratio of these values to calculate the KD value.
  • KD values may be used to evaluate and rank order the strength of individual antibody/antigen interactions. The lower the KD of an antibody, the higher the affinity of the antibody for its target. Avidity gives a measure of the overall strength of an antibody- antigen complex. It is dependent on three major parameters: (i) affinity of the antibody for the epitope, (ii) valency of both the antibody and antigen, and (iii) structural arrangement of the parts that interact.
  • the binding molecule provided herein binds one or more targets, antigens, or epitopes with a dissociation constant (K D ) of about 1 mM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less.
  • the binding molecule provided herein binds a target, antigen, or epitope with a K D of about 20 nM or less.
  • the binding molecule binds a target, antigen, or epitope with a K D of about 10 nM or less. In some embodiments, the binding molecule binds a target, antigen, or epitope with a K D of about 1 nM or less. In some embodiments, the binding molecule binds a target, antigen, or epitope with a K D of about 0.5 nM or less. In some embodiments, the binding molecule provided herein binds a target, antigen, or epitope with a K D of about 0.1 nM or less.
  • the binding molecule provided herein binds a target, antigen, or epitope with a K D of about 50 pM or less. In some embodiments, the binding molecule provided herein binds a target, antigen, or epitope with a K D of about 25 pM or less. In some embodiments, the binding molecule provided herein binds a target, antigen, or epitope with a K D of about 10 pM or less. In some embodiments, the binding molecule provided herein binds a target, antigen, or epitope with a K D of about 1 pM or less.
  • the dissociation constant of a binding molecule provided herein to a target or an antigen is the dissociation constant determined using a fusion protein comprising at least a portion of the target protein immobilized on an Octet ® chip. In some embodiments, the dissociation constant of a binding molecule provided herein to a target or an antigen is the dissociation constant determined using the binding agent captured by an anti-human IgG antibody on an Octet ® chip and a soluble target protein.
  • the binding molecule provided herein binds a target, antigen, or epitope with a half maximal effective concentration (EC50) of about 1 mM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.
  • a binding molecule provided herein binds a target, antigen, or epitope with an EC50 of about 1 mM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.
  • the K D for the binding molecule to the first antigen is about 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 50 fold or more of the K D for the binding molecule to the second antigen.
  • the K D for the binding molecule to the first antigen is about 10, 10 2 , 10 3 , or 10 4 fold of the K D for the binding molecule to the second antigen.
  • the binding molecule provided herein comprises at least a portion of one or more“parental” antibodies.
  • a parental antibody is a recombinant antibody.
  • a parental antibody is a monoclonal antibody.
  • the parental antibody is a polyclonal antibody.
  • a parental antibody is a chimeric antibody.
  • a parental antibody is a humanized antibody. In some embodiments, a parental antibody is a human antibody or fully human antibody. In some embodiments, a parental antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In certain embodiments, a parental antibody is an IgGl antibody. In certain embodiments, a parental antibody is an IgG2 antibody. In some embodiments, the parental antibody is an IgG3 antibody. In some embodiments, a parental antibody is an IgG4 antibody.
  • the binding molecule provided herein e.g., a bispecific binding molecule
  • the binding molecule provided herein is isolated.
  • the binding molecule provided herein e.g., a bispecific binding molecule
  • the binding molecule provided herein e.g., a bispecific binding molecule
  • a portion thereof is derived from at least one monoclonal antibody.
  • a monoclonal antibody is prepared using hybridoma methods known to one of skill in the art. For example, using the hybridoma method, a mouse, rat, rabbit, hamster, or other appropriate host animal, is immunized as described above to elicit the production of antibodies that specifically bind the immunizing antigen.
  • lymphocytes are immunized in vitro.
  • the immunizing antigen is a human protein or a fragment thereof.
  • the immunizing antigen is a mouse protein or a fragment thereof.
  • lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol.
  • the hybridoma cells are selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process.
  • Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen can be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, and radioimmunoassay).
  • the clones may be subcloned by limiting dilution techniques.
  • the hybridomas can be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal.
  • the monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.
  • monoclonal antibodies can be made using recombinant DNA techniques as known to one skilled in the art.
  • polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using standard techniques.
  • the isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.
  • suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.
  • recombinant monoclonal antibodies, or fragments thereof can be isolated from phage display libraries expressing variable domains or CDRs of a desired species. Screening of phage libraries can be accomplished by various techniques known in the art.
  • a monoclonal antibody is modified, for example, by using recombinant DNA technology to generate alternative antibodies.
  • the constant domains of the light chain and heavy chain of, for example, a mouse monoclonal antibody can be substituted for constant regions of, for example, a human antibody to generate a chimeric antibody, or for a non-immunoglobulin polypeptide to generate a fusion antibody.
  • the constant regions are truncated or removed to generate a desired antibody fragment of a monoclonal antibody.
  • site-directed or high-density mutagenesis of the variable region(s) is used to optimize specificity and/or affinity of a monoclonal antibody.
  • the binding molecule provided herein e.g., a bispecific binding molecule
  • a portin thereof is derived from a humanized antibody.
  • humanization is performed by substituting one or more non-human CDR sequences for the corresponding CDR sequences of a human antibody.
  • humanized antibodies are generated by substituting all six CDRs of a parent non-human antibody (e.g., rodent) for the corresponding CDR sequences of a human antibody.
  • variable region and light chain variable region are used in generating humanized antibodies based on a variety of factors and by a variety of methods.
  • the“best-fit” method is used where the sequence of the variable region of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable region sequences. The human sequence that is most similar to that of the non-human sequence is selected as the human variable region backbone for the humanized antibody.
  • a method is used wherein a particular variable region backbone derived from a consensus sequence of all human antibodies of a particular subgroup of light or heavy chains is selected.
  • the framework is derived from the consensus sequences of the most abundant human subclasses.
  • human germline genes are used as the source of the variable region framework sequences.
  • binding molecule e.g., a bispecific binding molecule
  • Human antibodies can be directly prepared using various techniques known in the art.
  • human antibodies are generated from immortalized human B lymphocytes immunized in vitro.
  • human antibodies are generated from lymphocytes isolated from an immunized individual.
  • cells that produce an antibody directed against a target antigen can be generated and isolated.
  • a human antibody is selected from a phage library, where that phage library expresses human antibodies.
  • phage display technology may be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable region gene repertoires from unimmunized donors. Techniques for the generation and use of antibody phage libraries are well known in the art. Once antibodies are identified, affinity maturation strategies known in the art, including but not limited to, chain shuffling and site-directed mutagenesis, may be employed to generate higher affinity human antibodies.
  • human antibodies are produced in transgenic mice that contain human immunoglobulin loci. Upon immunization, these mice are capable of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • the binding molecule provided herein e.g., a bispecific binding molecule or a portion thereof described herein are derived from antibodies (e.g., full-length antibodies or fragments thereof) that comprise modifications in at least one or more of the constant regions.
  • the antibodies comprise modifications to one or more of the three heavy chain constant regions (e.g., CH1) and/or to the light chain constant region (CL).
  • the heavy chain constant region of the modified antibodies comprises at least one human constant region.
  • the heavy chain constant region of the modified antibodies comprises more than one human constant region.
  • modifications to the constant region comprise additions, deletions, or substitutions of one or more amino acids in one or more regions.
  • one or more regions are partially or entirely deleted from the constant regions of the modified antibodies.
  • the entire CH2 domain has been removed from an antibody (ACH2 constructs).
  • the entire CH3 domain has been removed from an antibody (ACH3 constructs).
  • an omitted constant region is replaced by a short amino acid spacer (e.g ., 10 amino acid residues) that provides some of the molecular flexibility typically imparted by the absent constant region.
  • Modifications to the constant region of antibodies (e.g., parental antibody) and/or the binding molecule provided herein (e.g., a bispecific antibody) described herein may be made using well known biochemical or molecular engineering techniques.
  • variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by direct synthesis of the desired polypeptide or agent. In this respect it may be possible to disrupt the activity or effector function provided by a specific sequence or region while substantially maintaining the structure, binding activity, and other desired characteristics of the modified binding agent.
  • the present disclosure further embraces additional variants and equivalents which are substantially homologous to the binding molecules described herein.
  • it is desirable to improve the binding affinity and/or other biological properties of the binding molecules including but not limited to, specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity, or solubility.
  • amino acid changes may alter post-translational processes of a polypeptide.
  • Variations may be a substitution, deletion, or insertion of one or more nucleotides encoding a multispecific binding agent that results in a change in the amino acid sequence as compared with the sequence of the parental binding agent.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements.
  • insertions or deletions are in the range of about 1 to 5 amino acids.
  • the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule.
  • Variations in the amino acid sequence that are biologically useful and/or relevant may be determined by systematically making insertions, deletions, or substitutions in the sequence and testing the resulting variant proteins for activity as compared to the parental protein.
  • variants may include the addition of amino acid residues at the amino- and/or carboxyl-terminal end of one or more polypeptides that make up the binding molecules provided herein. The length of additional amino acids residues may range from one residue to a hundred or more residues.
  • a variant comprises an N-terminal methionyl residue.
  • polypeptide/protein i.e., a fusion protein.
  • a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g ., an enzyme).
  • a cysteine residue not involved in maintaining the proper conformation of the binding molecule provided herein is substituted or deleted to modulate the agent’s characteristics, for example, to improve oxidative stability and/or prevent aberrant disulfide crosslinking.
  • one or more cysteine residues are added to create disulfide bond(s) to improve stability.
  • the binding molecule of the present disclosure is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • deimmunized The deimmunization of agents such as antibodies generally consists of introducing specific mutations to remove T cell epitopes without significantly reducing the binding affinity or other desired activities of the agent.
  • variant binding molecules or polypeptides described herein may be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.
  • a binding molecule described herein is chemically modified.
  • a binding molecule is a bispecific antibody that has been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications may be carried out by known techniques.
  • polypeptides that make up the multispecific binding agents described herein can be produced by any suitable method known in the art and described in more detail in Section III and Section IV below.
  • the present disclosure also provides conjugates comprising any one of the binding molecules (e.g., bispecific antibodies) described herein.
  • the binding molecule provided herein is attached to an additional molecule.
  • the binding molecule provided herein is conjugated to a cytotoxic agent or moiety.
  • the binding molecule provided herein is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate).
  • the cytotoxic moiety is a
  • the cytotoxic moiety is a microtubule inhibitor including, but not limited to, auristatins, maytansinoids (e.g ., DMI and DM4), and tubulysins.
  • the cytotoxic moiety is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes.
  • diphtheria A chain non-binding active fragments of diphtheria toxin
  • exotoxin A chain exotoxin A chain
  • ricin A chain abrin A chain
  • modeccin A chain
  • the binding molecule provided herein is conjugated to one or more small molecule toxins, such as calicheamicins, maytansinoids, trichothenes, and CC1065.
  • small molecule toxins such as calicheamicins, maytansinoids, trichothenes, and CC1065.
  • the derivatives of any one of these toxins can be used in a conjugate as long as the derivative retains the cytotoxic activity.
  • Conjugates comprising the binding molecule provided herein may be made using any suitable methods as known in the art.
  • conjugates are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyidi thiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-di
  • the binding molecule (e.g., a bispecific antibody) described herein is conjugated to detectable substances or molecules that allow the antibodies to be used for diagnosis and/or detection.
  • the detectable substances may include but not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine(s); fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine,
  • TRITC tetramethylrhodamine isothiocyanate
  • dichlorotriazinylamine fluorescein dansyl chloride
  • cyanine (Cy3) cyanine
  • bioluminescent materials such as luciferase
  • radioactive materials such as 212 Bi, 14 C, 57 Co, 51 Cr, 67 Cu, 18 F, 68 Ga, 67 Ga, 153 Gd, 159 Gd, 68 Ge, 3 H, 166 HO, 131 I, 125 I, 123 I, 121 I, 115 In, 113 In, 112 In, In, 140 La, 177 Lu, 54 Mn, "Mo, 32 P, 103 Pd, 149 Pm,
  • the binding molecule provided herein described herein is attached to a solid support, that are particularly useful for immunoassays or purification of a target antigen(s).
  • solid supports include, but are not limited to, glass, cellulose,
  • polyacrylamide nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • the binding molecules provided herein is formulated in a pharmaceutical composition. Therefore, in yet another aspect, provided herein is a pharmaceutical composition.
  • composition comprising a therapeutically effective amount of the binding molecule provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition provided herein is described in more detail in Section V below.
  • the pharmaceutical composition is for use in treating a disease or condition in a subject.
  • the disease or condition is a cancer.
  • the cancer is a PD-L1 positive cancer.
  • the cancer is a lung cancer.
  • the cancer is a Non Small Cell Lung Carcinoma (NSCLC).
  • NSCLC Non Small Cell Lung Carcinoma
  • the cancer is a Diffuse Large B cell Lymphoma (DLBCL). In some embodiments, the cancer is a colorectal cancer. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is a lymphoma. In some embodiments, the cancer is a melanoma. In some embodiments, the cancer is ovarian cancer.
  • DLBCL Diffuse Large B cell Lymphoma
  • the cancer is a colorectal cancer.
  • the cancer is a breast cancer.
  • the cancer is a lymphoma. In some embodiments, the cancer is a melanoma. In some embodiments, the cancer is ovarian cancer.
  • the binding molecules provided herein is used for treating a disease or condition. Therefore, in yet another aspect, provided herein is a method of treating a disease or condition in a subject comprising administering a therapeutically effective amount of the binding molecule provided herein to the subject.
  • the disease or condition is a cancer.
  • the cancer is a PD-L1 positive cancer.
  • the cancer is a lung cancer.
  • the cancer is a Non Small Cell Lung Carcinoma (NSCLC).
  • the cancer is a Diffuse Large B cell Lymphoma (DLBCL).
  • the cancer is a colorectal cancer.
  • the cancer is a breast cancer. In some embodiments, the cancer is a lymphoma. In some embodiments, the cancer is a melanoma. In some embodiments, the cancer is ovarian cancer. A more detail description of methods for administering the present binding molecules is in Section VI below.
  • the disclosure encompasses polynucleotides that encode the binding molecule described herein.
  • polynucleotides that encode a polypeptide encompasses a polynucleotide that includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences.
  • the polynucleotides of the disclosure can be in the form of RNA or in the form of DNA.
  • DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single- stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.
  • a polynucleotide comprises the coding sequence for a polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide).
  • a host cell e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide.
  • the polypeptide can have the leader sequence cleaved by the host cell to form a“mature” form of the
  • a polynucleotide comprises the coding sequence for a polypeptide fused in the same reading frame to a marker or tag sequence.
  • a marker sequence is a hexa-histidine tag supplied by a vector that allows efficient purification of the polypeptide fused to the marker in the case of a bacterial host.
  • a marker is used in conjunction with other affinity tags.
  • the present disclosure further relates to variants of the polynucleotides described herein, wherein the variant encodes, for example, fragments, analogs, and/or derivatives of a polypeptide.
  • the present disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98% or 99% identical to a polynucleotide encoding a polypeptide comprising a binding molecule described herein.
  • the phrase“a polynucleotide having a nucleotide sequence at least, for example, 95%“identical” to a reference nucleotide sequence” is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence.
  • These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both.
  • a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide.
  • a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code).
  • Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (i.e., change codons in the human mRNA to those preferred by a bacterial host such as E. coli ).
  • a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.
  • a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
  • a polynucleotide is isolated. In certain embodiments, a polynucleotide is substantially pure.
  • an expression vector comprises a polynucleotide molecule.
  • a host cell comprises an expression vector comprising the polynucleotide molecule.
  • a host cell comprises one or more expression vectors comprising polynucleotide molecules.
  • a host cell comprises a polynucleotide molecule.
  • a host cell comprises one or more
  • provided herein are methods for making the various binding molecules provided herein.
  • a method of making a binding molecule comprising transfecting one or more vectors into a host cell, wherein the one or more vectors comprise:
  • first polypeptide and the first VH region and the first CH1 region of the third polypeptide can form a first antigen binding Fab region
  • the first Fab region and the second Fab region are linked to the Fv region via a flexible peptide region.
  • the flexible peptide region comprises an antibody hinge region.
  • the antibody hinge region is an IgG hinge region.
  • the IgG hinge region is of IgGl subtype.
  • the IgG hinge region is of IgG2 subtype.
  • the IgG hinge region is of IgG3 subtype.
  • the IgG hinge region is of IgG4 subtype.
  • the flexible peptide region further comprises a linker between the antibody hinge region and the second antigen binding domain.
  • the linker comprises an amino acid sequence of GGGGS (G4S). In some embodiments, the linker comprises an amino acid sequence of (G4S)n where n is an integer. In some specific embodiments, the linker comprises an amino acid sequence of (G4S)i. In some more specific embodiments, the linker comprises an amino acid sequence of (G4S) 2 . In other more specific embodiments, the linker comprises an amino acid sequence of (G4S) 3 . In yet other more specific embodiments, the linker comprises an amino acid sequence of (G4S) 4 .
  • the first Fab region and the second Fab region bind to different antigens. In other embodiments, the first Fab region and the second Fab region bind to the same antigen. In some embodiments, the first Fab region and the second Fab region bind to the same epitope of the same antigen. In other embodiments, the first Fab region and the second Fab region bind to different epitopes of the same antigen.
  • the first Fab region and the second Fab region form a first antigen binding domain, and the Fv region forms a second antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain bind to the same antigen.
  • the second antigen binding domain binds to the same epitope as at least one of the epitopes bound by the first antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain bind to different antigens, and wherein the first antigen binding domain binds to a first antigen and the second antigen binding domain binds to a second antigen.
  • the first antigen is a cancer antigen. In other embodiments, the first antigen is not a cancer antigen.
  • the second antigen is expressed on an immune cells including lymphocytes and monocytes. In some embodiments, the second antigen is expressed on a T cell. In some embodiments, the second antigen is expressed on a B cell. In other embodiments, the second antigen is expressed on a dendritic cell. In other embodiments, the second antigen is expressed on a granulocyte. In yet other embodiments, the second antigen is expressed on an innate lymphoid cell. In yet other embodiments, the second antigen is expressed on a
  • the second antigen is expressed on a monocyte. In yet other embodiments, the second antigen is expressed on a myeloid-derived suppressor cell. In yet other embodiments, the second antigen is expressed on a NK cell.
  • the second antigen is CD3.
  • the first antigen is a cancer antigen and the second antigen is CD3.
  • the first antigen is PD-L1 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 5, SEQ ID NO.: 6, and SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.: 11;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 4; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 8; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 3; the third polypeptide has the amino acid sequence of SEQ ID NO.: 1; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 2.
  • the first nucleic acid has a nucleotide sequence of SEQ ID NO.: 22; the second nucleic acid has a nucleotide sequence of SEQ ID NO.: 20; and the third nucleic acid has a nucleotide sequence of SEQ ID NO.: 21.
  • the first antigen is CD20 and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 27, SEQ ID NO.: 28, SEQ ID NO.:
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 31, SEQ ID NO.: 32, and SEQ ID NO.: 33;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 26; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 30; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 25; the third polypeptide has the amino acid sequence of SEQ ID NO.: 23; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 24.
  • the first nucleic acid has a nucleotide sequence of SEQ ID NO.: 36; the second nucleic acid has a nucleotide sequence of SEQ ID NO.: 34; and the third nucleic acid has a nucleotide sequence of SEQ ID NO.: 35.
  • the first antigen is EGFR and the second antigen is CD3.
  • the VH region of the first portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 41, SEQ ID NO.: 42, and SEQ ID NO.: 43;
  • the VL region of the second portion of each Fab region comprises three CDRs having amino acid sequences of SEQ ID NO.: 45, SEQ ID NO.: 46, and SEQ ID NO.: 47;
  • the VH region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15;
  • the VL region of the Fv region comprises three CDRs having amino acid sequences of SEQ ID NO.: 17, SEQ ID NO.: 18, and SEQ ID NO.: 19.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 40; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 44; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 12; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 16.
  • the first polypeptide and the second polypeptide each have the amino acid sequence of SEQ ID NO.: 39; the third polypeptide has the amino acid sequence of SEQ ID NO.: 37; and the fourth polypeptide has the amino acid sequence of SEQ ID NO.: 38.
  • the first nucleic acid has a nucleotide sequence of SEQ ID NO.: 50; the second nucleic acid has a nucleotide sequence of SEQ ID NO.: 48; and the third nucleic acid has a nucleotide sequence of SEQ ID NO.: 49.
  • the first antigen is Her2 and the second antigen is TNF alpha.
  • the VH region of the first portion of each Fab region has an amino acid sequence of SEQ ID NO.: 51; the VL region of the second portion of each Fab region has an amino acid sequence of SEQ ID NO.: 52; the VH region of the Fv region has an amino acid sequence of SEQ ID NO.: 53; and the VL region of the Fv region has an amino acid sequence of SEQ ID NO.: 54.
  • Recombinant expression of a binding molecule provided herein may require construction of an expression vector containing a polynucleotide that encodes the binding molecule or a fragment thereof.
  • a polynucleotide encoding a binding molecule, an antibody heavy or light chain, or fragment thereof (such as, but not necessarily, containing the heavy and/or light chain variable domain) provided herein has been obtained, the vector for the production of the binding molecule may be produced by recombinant DNA technology using techniques well-known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an encoding nucleotide sequence are described herein.
  • vectors containing coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding a binding molecule provided herein, or a fragment thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of an antibody molecule (see, e.g.,
  • variable domain of an antibody may be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce a binding molecule provided herein.
  • host cells containing a polynucleotide encoding a binding molecule provided herein or fragments thereof, or a heavy or light chain thereof, or fragment thereof, operably linked to a heterologous promoter.
  • multiple vectors comprising polynucleotides encoding different portions of a binding molecule provided herein may be co-expressed in the host cell for expression of the entire binding molecule, as detailed below.
  • host-expression vector systems may be utilized to express the binding molecules provided herein (see, e.g., U.S. Patent No. 5,807,715).
  • host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express a binding molecule provided herein in situ.
  • microorganisms such as bacteria (e.g., E. coli and B.
  • subtili transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences; yeast (e.g., Saccharomyces Pichid) transformed with recombinant yeast expression vectors containing coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
  • Bacterial cells such as Escherichia coli, or, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, can be used for the expression of a recombinant binding molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO)
  • CHO Chinese hamster ovary cells
  • a vector such as the major intermediate early gene promoter element from human cytomegalovirus
  • antibodies provided herein are produced in CHO cells.
  • the expression of nucleotide sequences encoding binding molecules provided herein is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the binding molecule being expressed. For example, when a large quantity of such a binding molecule is to be produced, for the generation of
  • compositions of a binding molecule which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione.
  • GST glutathione 5-transferase
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • a number of viral-based expression systems may be utilized.
  • the coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the binding molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc.
  • Specific initiation signals may also be required for efficient translation of inserted coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et ah, 1987, Methods in Enzymol. 153:51-544).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T,
  • HTB2, BT20 and T47D NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030 and HsS78Bst cells.
  • stable expression For long-term, high-yield production of recombinant proteins, stable expression can be utilized.
  • cell lines which stably express the binding molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the binding molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the binding molecule.
  • a number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977 , Cell 11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, 1980, Natl. Acad. Sci. USA 77:357; O’Hare et al, 1981, Proc. Natl. Acad. Sci. USA
  • the expression levels of a binding molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)).
  • a marker in the vector system expressing a binding molecule is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the binding molecule gene, production of the binding molecule will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
  • the host cell may be co-transfected with multiple expression vectors provided herein.
  • the vectors may contain identical selectable markers which enable equal expression of respective encoding polypeptides.
  • a single vector may be used which encodes, and is capable of expressing multiple polypeptides.
  • the coding sequences may comprise cDNA or genomic DNA.
  • binding molecule provided herein has been produced by recombinant expression, it may be purified by any method known in the art for purification of an
  • immunoglobulin molecule for example, by chromatography (e.g ., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, sizing column chromatography, and kappa-select affinity chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • kappa-select e.g., KappaSelect developed by GE Healthcare Life Science
  • the binding molecules provided herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • the present disclosure further provides pharmaceutical compositions comprising at least one binding molecule of the present disclosure.
  • a pharmaceutical composition comprises therapeutically effective amount of a binding molecule provided herein and a pharmaceutically acceptable carrier.
  • compositions comprising a binding molecule are prepared for storage by mixing the binding molecule having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (see, e.g., Remington, Remington’s Pharmaceutical Sciences ( 18th ed. 1980)) in the form of aqueous solutions or lyophilized or other dried forms.
  • the binding molecule of the present disclosure may be formulated in any suitable form for delivery to a target cell/tissue, e.g., as microcapsules or macroemulsions (Remington, supra ⁇ , Park et al, 2005, Molecules 10: 146-61 ; Malik et al, 2007, Curr. Drug. Deliv. 4: 141-51), as sustained release formulations (Putney and Burke, 1998, Nature Biotechnol. 16: 153-57), or in liposomes (Maclean et al., 1997, Int. J. Oncol. 11 :325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8:39-45).
  • a binding molecule provided herein can also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
  • macroemulsions for example, in Remington, supra.
  • compositions and delivery systems are known and can be used with a binding molecule as described herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the binding molecule, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-32), construction of a nucleic acid as part of a retroviral or other vector, etc.
  • a composition can be provided as a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release (see, e.g., Langer, supra Sefton, 1987, Crit.
  • polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., a binding molecule as described herein) or a composition provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126; Levy et al., 1985, Science 228: 190-92; During et al, 1989, Ann. Neurol. 25:351-56; Howard et al., 1989, J.
  • a prophylactic or therapeutic agent e.g., a binding molecule as described herein
  • a composition provided herein see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavail
  • polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly( ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co- glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of a particular target tissue, for example, the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer, 1990, Science 249: 1527-33. Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more binding molecule as described herein (see, e.g., U.S. Pat. No. 4,526,938, PCT publication Nos. WO 91/05548 and WO
  • provided herein is a composition for use in the prevention, management, treatment and/or amelioration of a disease or condition comprising a binding molecule provided herein.
  • a composition for use in the prevention of a disease or condition wherein the composition comprises a binding molecule provided herein.
  • provided herein is a composition for use in the composition for use in the
  • the composition comprises a binding molecule provided herein.
  • a composition for use in the treatment of a disease or condition wherein the composition comprises a binding molecule provided herein.
  • the disease or condition is a cancer.
  • the cancer is a PD-L1 positive cancer.
  • the cancer is a lung cancer.
  • the cancer is a Non Small Cell Lung Carcinoma (NSCLC).
  • NSCLC Non Small Cell Lung Carcinoma
  • the cancer is a Diffuse Large B cell Lymphoma (DLBCL). In some embodiments, the cancer is a colorectal cancer. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is a lymphoma. In some embodiments, the cancer is a melanoma. In some embodiments, the cancer is ovarian cancer. In certain embodiments, the subject is a subject in need thereof. In some embodiments, the subject has the disease or condition. In other embodiments, the subject is at risk of having the disease or condition. In some embodiments, the administration results in the prevention, management, treatment or amelioration of the disease or condition.
  • DLBCL Diffuse Large B cell Lymphoma
  • the cancer is a colorectal cancer. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is a lymphoma. In some embodiments, the cancer is a melanoma. In some embodiments, the cancer is ovarian cancer. In certain embodiments, the subject
  • provided herein is a composition for use in the prevention, management, treatment and/or amelioration of a symptom of a disease or condition, wherein the composition comprises a binding molecule provided herein.
  • a composition for use in the prevention of a symptom of a disease or condition wherein the composition comprises a binding molecule provided herein.
  • a composition for use in the management of a symptom of a disease or condition wherein the composition comprises a binding molecule provided herein.
  • provided herein is a composition for use in the treatment of a symptom of a disease or condition, wherein the composition comprises an a binding molecule provided herein.
  • a composition for use in the amelioration of a symptom of a disease or condition wherein the composition comprises a binding molecule provided herein.
  • the disease is cancer.
  • the subject is a subject in need thereof.
  • the subject has the disease or condition.
  • the subject is at risk of having the disease or condition.
  • the administration results in the prevention, management, treatment or amelioration of the symptom of the disease or condition.
  • provided herein is a method of preventing, managing, treating and/or ameliorating a disease or condition in a subject, comprising administering an effective amount of a binding molecule provided herein.
  • a method of preventing a disease or condition in a subject comprising administering an effective amount of a binding molecule provided herein.
  • a method of managing a disease or condition in a subject comprising administering an effective amount of a binding molecule provided herein.
  • provided herein is a method of treating a disease or condition in a subject, comprising administering an effective amount of a binding molecule provided herein.
  • provided herein is a method of ameliorating a disease or condition in a subject, comprising administering an effective amount of a binding molecule provided herein.
  • the disease or condition is cancer.
  • the subject is a subject in need thereof.
  • the subject has the disease or condition.
  • the subject is at risk of having the disease or condition.
  • the administration results in the prevention, management, treatment or amelioration of the disease or condition.
  • provided herein is a method of preventing, managing, treating and/or ameliorating a symptom of a disease or condition in a subject, comprising administering an effective amount of a binding molecule provided herein.
  • provided herein is a method of preventing a symptom of a disease or condition in a subject, comprising administering an effective amount of a binding molecule provided herein. In one embodiment, provided herein is a method of managing a symptom of a disease or condition in a subject, comprising administering an effective amount of a binding molecule provided herein. In one embodiment, provided herein is a method of treating a symptom of a disease or condition in a subject, comprising administering an effective amount of a binding molecule provided herein. In one embodiment, provided herein is a method of ameliorating a disease or condition in a subject, comprising administering an effective amount of a binding molecule provided herein. In one embodiment, the disease or condition is cancer.
  • the subject is a subject in need thereof. In some embodiments, the subject has the disease or condition. In other embodiments, the subject is at risk of having the disease or condition. In some embodiments, the administration results in the prevention, management, treatment or amelioration of the symptom of the disease or condition.
  • the binding molecule is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side effects).
  • the binding molecule is derived from one or more fully human monoclonal antibodies.
  • the subject administered a therapy can be a mammal such as non-primate (e.g ., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., a monkey, such as a cynomolgous monkey, or a human).
  • the subject is a human.
  • the subject is a human with a disease or condition, e.g., cancer.
  • a prophylactic or therapeutic agent e.g., a binding molecule provided herein
  • a prophylactic or therapeutic agent e.g., a binding molecule provided herein
  • encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the binding molecule, receptor-mediated endocytosis see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)
  • construction of a nucleic acid as part of a retroviral or other vector etc.
  • Methods of administering a prophylactic or therapeutic agent include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes).
  • parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous
  • epidural e.g., epidural and mucosal
  • mucosal e.g., intranasal and oral routes.
  • a prophylactic or therapeutic agent e.g., a binding molecule provided herein
  • a pharmaceutical composition is administered intranasally, intramuscularly, intravenously, or subcutaneously.
  • the prophylactic or therapeutic agents, or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, intranasal mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • Administration can be systemic or local.
  • pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • a prophylactic or therapeutic agent or a pharmaceutical composition provided herein locally to the area in need of treatment.
  • This may be achieved by, for example, and not by way of limitation, local infusion, by topical administration (e.g., by intranasal spray), by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • care when administering an antibody provided herein, care must be taken to use materials to which the antibody does not absorb.
  • a prophylactic or therapeutic agent, or a composition provided herein can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science
  • a prophylactic or therapeutic agent, or a composition provided herein can be delivered in a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release (see Langer, supra ⁇ , Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al, 1980, Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321 :574).
  • polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g ., an antibody provided herein) or a composition provided herein (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug
  • polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly (methyl methacrylate), poly( acrylic acid), poly (ethylene -co- vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), poly anhydrides, poly(N-vinyl pyrrolidone), poly( vinyl alcohol), polyacrylamide, poly( ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of the therapeutic target, i.e., the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115- 138 (1984)). Controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more binding molecule provided herein.
  • the composition provided herein is a nucleic acid encoding a prophylactic or therapeutic agent (e.g ., a binding molecule provided herein)
  • the nucleic acid can be administered in vivo to promote expression of its encoded prophylactic or therapeutic agent, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No.

Abstract

La présente invention se rapporte d'une manière générale au domaine de l'immunothérapie du cancer. La présente invention concerne d'une manière générale une molécule de liaison, donnée à titre d'exemple, comprenant des régions légères variables d'anticorps (VL), des régions lourdes variables (VH), des régions lourdes constantes 1 (CH1), et des régions constantes à chaîne légère (CL) qui sont configurées pour former deux régions Fab de liaison à l'antigène et une région Fv de liaison à l'antigène de telle sorte que la molécule de liaison se lie à deux antigènes différents.
PCT/IB2019/052896 2018-04-10 2019-04-09 Molécules de liaison impliquant des cellules WO2019197979A1 (fr)

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JP2020555789A JP7076571B2 (ja) 2018-04-10 2019-04-09 細胞エンゲージ結合分子
RU2020136600A RU2770620C1 (ru) 2018-04-10 2019-04-09 Связывающие молекулы, вызывающие клеточную активацию
CN201980030028.0A CN112074540B (zh) 2018-04-10 2019-04-09 细胞接合的结合分子
KR1020207032356A KR102567489B1 (ko) 2018-04-10 2019-04-09 세포 연계 결합 분자
AU2019251422A AU2019251422A1 (en) 2018-04-10 2019-04-09 Cell engaging binding molecules
CA3096791A CA3096791C (fr) 2018-04-10 2019-04-09 Molecules de liaison impliquant des cellules
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US16/372,196 US10654944B2 (en) 2018-04-10 2019-04-01 Cell engaging binding molecules
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