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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • A—HUMAN NECESSITIES
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  • A61P35/00—Antineoplastic agents
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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  • C07—ORGANIC CHEMISTRY
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • 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
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70532—B7 molecules, e.g. CD80, CD86
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/66—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a swap of domains, e.g. CH3-CH2, VH-CL or VL-CH1
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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  • 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
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  • 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
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  • C07—ORGANIC CHEMISTRY
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

• TMDD target mediated drug disposition
• a fusion protein comprising a biologically functional protein, a ligand- receptor pair, a first peptidic linker and a second peptidic linker; wherein the biologically functional protein comprises at least a first polypeptide and a second polypeptide; and the ligand- receptor pair comprises an extracellular portion of an immunoglobulin superfamily (IgSF) receptor and its cognate ligand or a receptor-binding fragment thereof; wherein the ligand is fused to a terminus of the first polypeptide via the first peptidic linker; the receptor is fused to the same respective terminus of the second polypeptide via the second peptidic linker; and the first and second peptidic linkers are of sufficient length to allow pairing of the ligand and receptor.
• IgSF immunoglobulin superfamily
• At least one of the first and second peptidic linkers comprises a protease cleavage site.
• the ligand is fused to the N-terminus of the first polypeptide via the first peptidic linker, and the receptor is fused to the N-terminus of the second polypeptide via the second peptidic linker.
• the biologically functional protein comprises an antibody or antigen-binding antibody fragment.
• the biologically functional protein consists of a polypeptide scaffold.
• the polypeptide scaffold is a dimeric Fc region, wherein the first polypeptide consists of a first Fc polypeptide and the second polypeptide consists of a second Fc polypeptide, the first and second Fc polypeptides forming the dimeric Fc region.
• the biologically functional protein comprises a polypeptide scaffold.
• the polypeptide scaffold comprises a dimeric Fc region.
• the dimeric Fc region is a heterodimeric Fc.
• the at least one of the ligand or the receptor in the ligand-receptor pair is capable of binding to an immunomodulatory target.
• the ligand receptor pair is involved in a cellular response selected from the group consisting of: modulation of an immune checkpoint, modulation of immune cell activity, modulation of T-cell receptor signaling, modulation of T-cell dependent cytotoxicity (TDCC), modulation of antibody-dependent cellular phagocytosis (ADCP) and modulation of antibody-dependent cellular cytotoxicity (ADCC).
• the receptor comprises one or more mutations that increase or decrease binding affinity of the receptor for its cognate ligand as compared to a wild-type receptor.
• the ligand comprises one or more mutations that increase or decrease binding affinity of the ligand for its cognate receptor as compared to a wild-type ligand.
• the ligand-receptor pair is selected from the group consisting of: PD1- PDL1, PD1-PDL2, CTLA4-CD80, CD28-CD80, CD28-CD86, CTLA4-CD86, PDL1-CD80, ICOS-ICOSL, NCRSRLGl-NKp30 and CD47-SIRPa.
• the ligand- receptor pair is PD1-PDL1.
• the ligand PDL1 comprises an amino acid sequence according to SEQ ID NO: 8.
• the receptor PD1 comprises an amino acid sequence according to SEQ ID NO: 9.
• the ligand-receptor pair is CTLA4-CD80.
• the ligand CD80 comprises an amino acid sequence according to SEQ ID NO: 25, SEQ ID NO: 185, SEQ ID NO: 187 or SEQ ID NO: 189.
• the receptor CTLA4 comprises an amino acid sequence according to SEQ ID NO: 26.
• the receptor and the ligand are fused to the respective N- termini of the first and second polypeptides.
• the one of the first or second peptidic linkers comprises more than one protease cleavage site.
• the one of the peptidic linkers fused to the ligand or the receptor is engineered to comprise one or more additional protease cleavage sites, and wherein the one or more protease cleavage sites in the ligand or the receptor and the protease cleavage site in the first or second peptidic linker are cleavable by the same protease or a different protease.
• the protease is selected from the group consisting of: a serine protease, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18 (collagenase 4), MMP19, MMP20, MMP21, an adamalysin, a serralysin, an astacin, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin S, granzyme B, guanidinobenzoatase (GB), hepsin, guanidinobenzoata
• the peptidic linker is 3-50 or 5-20 amino acids in length. In certain embodiments, the one of the first or second peptidic linkers does not have a protease cleavage site. In certain embodiments, the peptidic linker is a (Gly n Ser) linker, wherein the (Gly n Ser) linker comprises an amino acid sequence selected from the group consisting of (Gly 3 Ser) n (Gly 4 Ser) 1 , (Gly 3 Ser) 1 (Gly 4 Ser) n , (Gly 3 Ser) n (Gly 4 Ser) n , and (Gly 4 Ser) n , wherein n is an integer of 1 to 5.
• the peptidic linker is an (EAAAK) n linker, wherein n is an integer between 1 and 5.
• the peptidic linker comprises the amino acid sequence EAAAKEAAAK (SEQ ID. NO: 38).
• the peptidic linker is a polyproline linker, optionally PPP or PPPP.
• the peptidic linker comprises an immunoglobulin hinge region sequence comprising an amino acid sequence having up to a 30 percent difference in amino acid sequence identity compared to a wild type immunoglobulin hinge region amino acid sequence.
• the peptidic linker comprises a protease cleavage site comprising the amino acid sequence MSGRSANA (SEQ ID NO: 28).
• a fusion protein comprising a Fab region and an Fc region; wherein the Fab region comprises a VH polypeptide and a VL polypeptide that form an antigen-binding domain, and a ligand-receptor pair comprising an extracellular portion of an immunoglobulin superfamily receptor and its cognate ligand or a receptor-binding fragment thereof; wherein the ligand is fused to the N-terminus of one of the VH or VL polypeptides via a first peptidic linker and the receptor is fused to the N-terminus of the other VH or VL polypeptide via a second peptidic linker; wherein first and second peptidic linkers are of sufficient length to allow pairing of the ligand and receptor; wherein at least one of the first and second peptidic linkers comprises a protease cleavage site; and wherein the ligand-receptor pair sterically hinders binding of the antigen-binding domain to its cognate
• the at least one of the first and second polypeptides comprise a first VH polypeptide and a first VL polypeptide, the first VH and VL polypeptides forming a first antigen-binding domain of the antibody, wherein the ligand is fused to one of the first VH or VL polypeptides via the first peptidic linker and the receptor is fused to the other of the first VH or VL polypeptides via the second peptidic linker, and wherein the ligand-receptor pair sterically hinders binding of the first antigen-binding domain to its cognate antigen.
• the first and second polypeptides further comprise a dimeric Fc. In certain embodiments, the dimeric Fc region is a heterodimeric Fc.
• the fusion protein comprises, from N terminus to C terminus, Ligand-Linker- VL, Receptor-Linker- VL, Ligand-Linker- VH, or Receptor-Linker- VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-cleavable Linker- VL, Receptor- cleavable Linker- VL, Ligand- cleavable Linker- VH, or Receptor- cleavable Linker- VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-linker (SEQ ID NO: 114)-VL, Receptor-linker (SEQ ID NO: 114)-VL, Ligand-linker (SEQ ID NO: 14)-VH, or Receptor-linker (SEQ ID NO: 14)-VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-linker (SEQ ID NO: 145)-VL, Receptor-linker (SEQ ID NO: 145)-VL, Ligand-linker (SEQ ID NO: 145)- VH, or Receptor-linker (SEQ ID NO: 145)-VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-linker (SEQ ID NO: 147)-VL, Receptor-linker (SEQ ID NO: 147)-VL, Ligand-linker (SEQ ID NO: 147)-VH, or Receptor-linker (SEQ ID NO: 147)-VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-linker (SEQ ID NO: 154)-VL, Receptor-linker (SEQ ID NO: 154)-VL, Ligand-linker (SEQ ID NO: 154)-VH, or Receptor-linker (SEQ ID NO: 154)-VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-linker (SEQ ID NO:203)-VL, Receptor-linker (SEQ ID NO:203)-VL, Ligand-linker (SEQ ID NO:203)-VH, or Receptor-linker (SEQ ID NO:203)-VH.
• the at least one of the ligand or the receptor of the ligand-receptor pair is capable of binding to an immunomodulatory target.
• the ligand receptor pair is involved in a cellular response selected from the group consisting of: modulation of an immune checkpoint, modulation of immune cell activity, modulation of T-cell receptor signaling, modulation of T-cell dependent cytotoxicity (TDCC), modulation of antibody- dependent cellular phagocytosis (ADCP) and modulation of antibody-dependent cellular cytotoxicity (ADCC).
• the receptor comprises one or more mutations that increase or decrease binding affinity of the receptor for its cognate ligand as compared to a wild-type receptor.
• the ligand comprises one or more mutations that increase or decrease binding affinity of the ligand for its cognate receptor as compared to a wild-type ligand.
• the ligand-receptor pair is selected from the group consisting of: PD1-PDL1, PD1- PDL2, CTLA4-CD80, CD28-CD80, CD28-CD86, CTLA4-CD86, PDL1-CD80, ICOS-ICOSL, NCRSRLGl-NKp30 and CD47-SIRPa.
• the ligand-receptor pair is PD1- PDL1.
• the ligand PDL1 comprises an amino acid sequence according to SEQ ID NO: 8.
• the receptor PD1 comprises an amino acid sequence according to SEQ ID NO: 9.
• the ligand-receptor pair is CTLA4-CD80.
• the ligand CD80 comprises an amino acid sequence according to SEQ ID NO: 25.
• the receptor CTLA4 comprises an amino acid sequence according to SEQ ID NO: 26.
• the receptor and the ligand are fused to the respective N- termini of the first and second polypeptides.
• one of the first or second peptidic linkers comprises more than one protease cleavage site.
• one of the ligand or the receptor is engineered to comprise one or more additional protease cleavage sites, and wherein the one or more protease cleavage sites in the ligand or the receptor and the protease cleavage site in the first or second peptidic linker are cleavable by the same protease or by different proteases.
• the protease is selected from the group consisting of: a serine protease, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18 (collagenase 4), MMP19, MMP20, MMP21, an adamalysin, a serralysin, an astacin, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin S, granzyme B, guanidinobenzoatase (GB), hepsin, guanidinobenzoata
• the protease is uPA or matriptase.
• the peptidic linker is 3-50 or 5-20 amino acids in length.
• the one of the first or second peptidic linkers does not have a protease cleavage site.
• the peptidic linker is a (Gly n Ser) linker, wherein the (Gly n Ser) linker comprises an amino acid sequence selected from the group consisting of (Gly 3 Ser) n (Gly 4 Ser) 1 , (Gly 3 Ser) 1 (Gly 4 Ser) n , (Gly 3 Ser) n (Gly 4 Ser) n , and (Gly 4 Ser) n , wherein n is an integer of 1 to 5.
• the peptidic linker an (EAAAK) n linker, wherein n is an integer between 1 and 5.
• the peptidic linker that does not have a protease cleavage site comprises the amino acid sequence EAAAKEAAAK (SEQ ID. NO: 38).
• the peptidic linker is a polyproline linker, optionally PPP or PPPP.
• the linker is glycine (G) proline (P) polypeptide linker, optionally GPPPG, GGPPPGG, GPPPPG or GGPPPGG.
• the peptidic linker comprises an immunoglobulin hinge region sequence comprising an amino acid sequence having up to a 30 percent difference in amino acid sequence identity compared to a wild type immunoglobulin hinge region amino acid sequence.
• the peptidic linker comprising a protease cleavage site comprises the amino acid sequence MSGRSANA (SEQ ID NO: 28).
• binding of the first antigen-binding domain to its cognate antigen is reduced by 10-fold or more as compared to a parental antigen-binding domain that is not fused to the ligand-receptor pair.
• cleavage of the protease cleavage site in a cellular environment releases one member of the ligand-receptor pair from the fusion protein, thereby allowing the antigen-binding domain to bind its cognate antigen.
• the first antigen-binding domain is a Fab. In certain embodiments, the first antigen-binding domain binds an antigen that is expressed on a cancer cell or an immune cell. In certain embodiments, the first antigen-binding domain binds an antigen that is expressed on a T-cell. In certain embodiments, the first antigen binding domain binds to a tumor-associated antigen (TAA).
• TAA tumor-associated antigen
• the first antigen-binding domain binds to an antigen selected from the group consisting of: Cluster of Differentiation 3 (CD3), Human Epidermal Growth Factor Receptor 2 (HER2), Epidermal Growth Factor Receptor (EGFR), Mesothelin (MSLN), Tissue Factor (TF), Cluster of Differentiation 19 (CD19), tyrosine-protein kinase Met (c-Met), Cluster of Differentiation 40 (CD40) and Cadherin 3 (CDH3).
• CD3 Cluster of Differentiation 3
• HER2 Human Epidermal Growth Factor Receptor 2
• EGFR Epidermal Growth Factor Receptor
• MSLN Mesothelin
• TF Tissue Factor
• CD19 Cluster of Differentiation 19
• CD19 tyrosine-protein kinase Met
• CD40 Cluster of Differentiation 40
• Cadherin 3 Cadherin 3
• the antibody or antibody fragment comprises a second antigen binding domain comprising a second VH polypeptide and a second VL polypeptide.
• the fusion protein comprises a second ligand-receptor pair, wherein the ligand of the second ligand-receptor pair is fused to one of the second VH or VL polypeptides via a third peptidic linker and the receptor of the second ligand-receptor pair is fused to the other of the second VH or VL polypeptides via a fourth peptidic linker, wherein at least one of the third and fourth peptidic linkers comprise a protease cleavage site, and wherein the ligand-receptor pair sterically hinders binding of the second antigen-binding domain to its cognate antigen.
• the fusion protein binds to two distinct antigens.
• one antigen is an antigen expressed by T cells and the other antigen is an antigen expressed by cancer cells.
• the fusion protein binds to CD3 and HER2.
• a fusion protein comprising an Fc region comprising a first Fc polypeptide and a second Fc polypeptide, and a ligand-receptor pair comprising an extracellular portion of an immunoglobulin superfamily receptor and its cognate ligand or a receptor -binding fragment thereof; wherein the ligand is fused to a terminus of the first Fc polypeptide via a first peptidic linker and the receptor is fused to the same respective terminus of the second Fc polypeptide via a second peptidic linker; wherein the first and second peptidic linkers are of sufficient length to allow pairing of the ligand and receptor; and wherein at least one of the first and second peptidic linkers comprises a protease cleavage site.
• FIG. 1(A) shows a schematic drawing of the structure of certain fusion proteins described herein.
• Figure 1(B) shows a schematic of an antibody with two Fab arms that are masked using IgSF domain pairs attached N-terminally with TME protease cleavable or uncleavable linkers.
• Fab paratopes a-TAA 1 and a-TAA 2 may be the same or different and IgSF pairs 1:2 and 3:4 may be the same or different.
• Figure 1(C) shows a schematic of a Fab x scFv construct with a Fab arm specific for target 1 and an scFv arm specific for target 2. The Fab arm and binding to target 1 is masked by a IgSF domain pair attached to the N-termini using TME protease cleavable or uncleavable linkers.
• Figure 2 shows a schematic drawing of a modified bispecific CD3 x Her2 Fab x scFv Fc fusion protein described herein.
• One arm of the antibody-like molecule contains the anti CD3 Fab that is blocked by a PD-1/PD-L1 mask, while the other arm contains an anti-Her2 scFv.
• Figure 3 shows UPLC-SEC chromatograms and non-reducing and reducing CE-SDS profiles for representative bispecific CD3 x Her2 Fab x scFv Fc variants.
• A UPLC-SEC chromatogram of unmasked variant 30421
• B non-reducing (left) and reducing (right) CE-SDS profiles of unmasked variant 30421
• C UPLC-SEC chromatogram of masked, uncleavable variant 30423
• D non-reducing (left) and reducing (right) CE-SDS profiles of masked, uncleavable variant 30423
• E UPLC-SEC chromatogram of masked, light-chain-cleavable variant 30430
• F non-reducing (left) and reducing (right) CE-SDS profiles of masked, light- chain-cleavable variant 30430
• G UPLC-SEC chromatogram of masked, heavy-chain-cleavable variant
• Figure 4 shows an overlay of DSC thermograms for unmodified (30421) and PD-1:PD-L1 masked variants (30430, 30436) of the investigated CD3 x Her2 Fab x scFv Fc system.
• Figure 5 shows reducing CE-SDS profiles of representative variants without (-uPa) and with uPa treatment (+uPa) for 24 h at 37 °C at a 1:50 uPa: variant ratio. Profiles for unmasked (30421), masked but uncleavable (30423), and masked cleavable variants (30430, 30436, 31934) are shown.
• Figure 6 shows native binding results of CD3 targeted variants to Jurkat cells as determined by ELISA. Results are shown for an unmasked variant (30421), constructs with only the PD-L1 or PD-1 moiety attached (31929, 31931), and variants with a full, uncleavable mask (30423) or with a full mask and a cleavable PD-L1 or PD-1 moiety (30430, 30436). For samples of variants 30423, 30430, 30436, uPa untreated (-uPa) and treated (+uPa) samples were tested.
• Figure 7 shows cell killing of JIMT-1 tumor cells by Pan T-cells as determined in a TDCC assay after treatment with engineered variants cross-linking T-cells and tumor cells. Results are shown for an unmasked variant (30421), a variant with only the PD-1 moiety attached to the heavy chain (31929), and variants with a full, uncleavable mask (30423) or with a full mask and a cleavable PD-L1 moiety on the light chain (30430). For variant 30430 uPa untreated (-uPa) and treated (+uPa) samples were tested. An irrelevant anti-RSV antibody (22277) was used as a negative control.
• Figure 8 shows results of a native binding study by flow cytometry of select CD3 targeted variants to (A) PD-L1 transfected and (B) PD-1 transfected CHO-S cells. Results are shown for an unmasked variant (30421), constructs with only the PD-L1 or PD-1 moiety attached (31929, 31931), and variants with a full, uncleavable mask (30423, 30426) or with a full mask and a cleavable PD-L1 or PD-1 moiety (30430, 30436). An Fc-fusion of the affinity-matured PD-1 moiety is also included (31829). For samples of variants 30423, 30426, 30430, 30436, uPa untreated (-uPa) and treated (+uPa) samples were tested.
• Figure 9 shows a schematic of a hybrid PD-1/PD-L1 Reporter Gene Assay probing cross- linking of T-cells and JIMT-1 cells and blockade of the PD-1:PD-L1 checkpoint engagement (A) as well as the analysis of the same (B). Results are shown for an unmasked variant (30421) and the same unmasked variant in combination with an excess of anti-PD-L1 antibody (30421 + 150 nM anti-PD-L1). A construct with only the PD-1 moiety attached to the heavy chain (31929), and variants with a full, uncleavable mask (30423) or with a full mask and a cleavable PD-L1 moiety on the light chain (30430) were also investigated. For variant 30430, uPa untreated (-uPa) and treated (+uPa) samples were tested. An irrelevant anti-RSV antibody (22277) was used as a negative control. Measurements were performed in triplicate and error bars reflecting standard deviation are shown.
• Figure 10 is a drawing representative of a modified monospecific, bivalent fusion protein targeted against tumor associated antigens (TAA).
• TAA tumor associated antigens
• Figure 11 shows UPLC-SEC chromatograms (A-J) and non-reducing SDS-PAGE (K) or non-reducing and reducing CE-SDS profiles (L) of masked fusion proteins targeted against EGFR, MSLN, TF, CD 19, cMet, CDH3.
• A-J UPLC-SEC chromatograms
• K non-reducing SDS-PAGE
• L non-reducing and reducing CE-SDS profiles
• Figure 12 shows reducing SDS-PAGE profiles of representative fusion proteins targeted against (A) EGFR, (B) MSLN, (C) TF, (D) CD 19.
• Untreated (-uPa) and uPa-treated (+uPa) samples are investigated.
• data for a uPa-uncleavable variant (31722, 31728, 31736, 31732) and a variant with a u-Pa cleavage sequence between the VL and the PD-L1 moiety (31723, 31729, 31737, 31733) is shown.
• Figure 13 Shows flow cytometry native binding results for select fusion proteins targeted against different antigens to the following cell lines expressing that antigen: (A) EGFR on MDA- MB-468, (B) MSLN on OVCAR3, (C) TF on MDA-MB-231, (D) CD19 on Raji, (E) cMet on EBC1, (F) CDH3 on JIMT1.
• Figure 14 shows results from a growth inhibition study of NCI-H292 cells treated with EGFR-targeted variants. Data is shown for unmasked (32474) and PD-EPD-L masked variants.
• the masked variants include an uncleavable form (31722) as well as one with a cleavable PD-L1 moiety on the light chain (31723).
• An irrelevant control (22277) is also included.
• samples are tested with (-uPa) and without (+uPa) treatment.
• the error bars reflect the standard deviation of triplicate measurements.
• Figure 15 shows a schematic drawing of a modified bispecific CD3 x Her2 Fab x scFv Fc variant that was investigated here.
• One arm of the fusion protein contains the anti CD3 Fab that is blocked by a CD80/CTLA4 mask, while the other arm contains an anti-Her2 scFv.
• Figure 16 shows ETPLC-SEC chromatogram and non-reducing and reducing CE-SDS profiles of variant 30444.
• A ETPLC-SEC chromatogram of masked, light-chain-cleavable variant 30444
• B non-reducing (left) and reducing
• C non-reducing (left) and reducing
• D-F UPLC-SEC chromatograms of masked, light-chain- cleavable variants 33525, 33526, 33527 after protein A purification.
• Figure 17 shows reducing CE-SDS profiles of variant 30444 without (-uPa) and with uPa treatment (+uPa).
• Figure 18 shows native binding results of CD3 targeted variants to Jurkat cells as determined by ELISA. Results are shown for an unmasked variant (30421), a variant with a full PD- 1/PD-L1 -based mask and a cleavable PD-L1 moiety (30430) and a variant with a full CD80/CTLA4-based mask and a cleavable CTLA4 moiety (30444). For samples of variants 30430 and 30444, uPa untreated (-uPa) and treated (+uPa) samples were tested.
• FIG 19 shows a schematic of IgVs of an immunomodulator pair (e.g. PD-1:PD-L1) fused via the hinge to a heterodimeric IgG Fc.
• Cleavage of one of the two linkers by a TME- associated protease such as uPa releases one moiety (e.g. PD-L1) and leaves the one with the desired function (e.g. PD-1) still attached to the Fc and available to bind to its partner on cells.
• PD-1 it is able to bind PD-L1 on target cells and inhibit checkpoint function.
• Figure 20 shows (A-C) UPLC-SEC chromatograms and (D) non-reducing and reducing CE-SDS profiles of CD40-targeted variants.
• E Reducing CE-SDS
• F flow cytometry binding data
• G results from a CD40 RGA assay are also shown for the same variants without (-uPa) and with (+uPa) treatment with uPa.
• Test articles include an unmasked variant (32477), a variant with an uncleavable PD- 1/PD-L1 -based mask (32478) and one with a PD-l/PD-L1-based mask in which the PD-L1 moiety can be removed by cleavage with uPa (32479).
• the native CD40 binding partner CD40L and an irrelevant control are also included.
• Data for the CD40 RGA assay is summarized in the table in (H).
• Figure 21 (A) PD1 and PDL1 are comprised of immunoglobulin domains that form a complex.
• the binding Fab is docked with the PD1-PDL1 complex on the paratope end.
• Linking the PD1 and PDL1 to the VH and VL chains with appropriate linker could block antigen binding.
• Figure 22 shows native binding results of CD3 targeted variants to Pan T-cells as determined by flow cytometry. Results are shown for an unmasked variant (30421), an anti-CD3 one-armed antibody (18560), a construct with only the PD-1 moiety attached (31929), and variants with a full, uncleavable mask (30423) or with a full mask and a cleavable PD-L1 moiety (30430, 30436). For samples of variants 30423, 30430, uPa untreated (-uPa) and treated (+uPa) samples were tested. Data is also shown for an irrelevant control (22277).
• Figures 23 A and B show cell killing of HCC1954, JIMT-1, HCC827 and MCF-7 tumor cells by Pan T-cells as determined in two repeats of a TDCC assay after treatment with engineered variants cross-linking T-cells and tumor cells. Results are shown for an unmasked variant (30421) as well as a combination of unmasked variant with saturating amounts of an anti-PD-L1 antibody (30421 + 120 nM atezolizumab), a variant with only the PD-1 moiety attached to the heavy chain (31929), and variants with a full, uncleavable mask (30423) or with a full mask and a cleavable PD-L1 moiety on the light chain (30430). For variants 30430 and 30423, uPa untreated (-uPa) and treated (+uPa) samples were tested. An irrelevant anti-RSV antibody (22277) was used as a negative control.
• Figure 24 shows IFN ⁇ release of Pan T-cells as determined in two repeats of a TDCC assay with HCC1954, JIMT-1, HCC827 and MCF-7cancer cells after treatment with engineered variants cross-linking T-cells and tumor cells.
• Results are shown for an unmasked variant (30421) as well as a combination of unmasked variant with saturating amounts of an anti-PD-L1 antibody (30421 + 120 nM atezolizumab), a variant with only the PD-1 moiety attached to the heavy chain (31929), and variants with a full, uncleavable mask (30423) or with a full mask and a cleavable PD-L1 moiety on the light chain (30430).
• variants 30430 and 30423 uPa untreated (-uPa) and treated (+uPa) samples were tested.
• An irrelevant anti-RSV antibody (22277) was used as a negative control.
• Figure 25 shows the receptor number per cell of Her2 and PD-L1 for a set of cancer cell lines used in TDCC and RGA assays as determined by flow cytometry.
• Figures 26 A to D show results from a hybrid PD-1/PD-L1 Reporter Gene Assay probing cross-linking of T-cells four different cancer cell lines (HCC1954, JIMT-1, HCC827, MCF-7) and blockade of the PD-1 :PD-L1 checkpoint engagement.
• results are shown for an unmasked variant (30421) as well as a combination of unmasked variant with saturating amounts of an anti-PD-L1 antibody (30421 + 150 nM atezolizumab), a variant with only the PD-1 moiety attached to the heavy chain (31929), and variants with a full, uncleavable mask (30423) or with a full mask and a cleavable PD-L1 moiety on the light chain (30430).
• variant 30430 uPa untreated (-uPa) and treated (+uPa) samples were tested.
• An irrelevant anti-RSV antibody (22277) was used as a negative control.
• Figure 27 is a drawing representative of a modified monospecific, bivalent fusion protein targeting EGFR (a-EGFR).
• the paratope of the Fab is sterically blocked by the SIRP ⁇ /CD47 mask.
• Figure 28 shows (A) a UPLC-SEC chromatogram and (B) non-reducing and reducing CE- SDS profiles of an EGFR-targeted, SIRP ⁇ /CD47-masked, fully cleavable variant (34164). (C) Reducing CE-SDS are also shown for the same variant without (-uPa) and with (+uPa) treatment with uPa.
• Figure 29 shows results from a native binding assay by high content analysis to EGFR positive H292 cells.
• Test articles include an unmasked, EGFR-targeted control (v32474), an EGFR-targeted, SIRP ⁇ /CD47-masked, fully cleavable variant (34164) without (-uPa) and with (+uPa) treatment with uPa and an irrelevant control (v22277).
• Figure 30 shows (A) data from a single titration point (1 nM) in a flow cytometry binding experiment to Her2+/PD-L1+ JIMT-1 cells as well as (B) data from a bridging experiment of human Pan T-cells and Her2+/PD-L1+ JIMT-1 cells. Data is shown for a trispecific variant with only the PD-1 moiety attached to the heavy chain (v31929) as well as bispecific variants in the same format but incapable of binding to either PD-L1 or Her2 (v32497 and v33551, respectively). Data for an irrelevant control (v22277) is included in the bridging assay (B).
• Figure 31 shows the mechanism of T-cell recruitment and activation of a PD-EPD-L1 masked CD3 x Her2 Fab x scFv Fc variant.
• the therapeutic antibody gets directed to the tumor microenvironment (TME) via TAA binding.
• TAA tumor microenvironment
• B The PD-L1 moiety of the mask gets released via cleavage of a TME specific protease.
• C The activated therapeutic engages and activates a T-cell for tumor cell killing via the unmasked a-CD3 paratope and inhibits checkpoint activity by binding to PD-L1 on the tumor cell.
• Figure 32 shows native binding results of CD3 targeted variants to Pan T-cells as determined by flow cytometry. Results are shown for an unmasked variant (30421), a construct with only the PD-1 moiety attached (31929) and a variant with a non-functional PD-1 domain appended to the heavy chain (32497). Data is also shown for an irrelevant control (22277).
• Figure 33 shows cell killing of JIMT-1 tumor cells by Pan T-cells as determined in a TDCC assay after treatment with engineered variants cross-linking T-cells and tumor cells. Results are shown for an unmasked variant (30421), a variant with only the PD-1 moiety attached to the heavy chain (31929) and a variant with a non-functional PD-1 domain appended to the heavy chain (32497).
• Fusion protein refers to a protein that comprises more than one polypeptide region or domain linked to each other, e.g., by peptide bonds. Accordingly, “fused” as used herein, refers to polypeptide sequences linked to one another through a peptide bond. Examples include antibodies or scaffolds fused to immunomodulatory ligand/receptor pairs. Fusion proteins described herein are sometimes referred to as “variants” or “constructs”.
• Bioly functional protein broadly refers to a polypeptide or protein that has a biological function, e.g., an antibody, e.g., a dimeric Fc.
• Ligand-receptor pairs refers to a receptor polypeptide and a ligand polypeptide that specifically bind to one another. Examples include PD-1-PD-L1, CTLA4-CD80 or CD28-CD80.
• Receptor-binding fragment refers to any polypeptide that binds specifically to the receptor of the ligand-receptor pair. A receptor binding fragment can be naturally occurring or non-naturally occurring.
• an “immunomodulatory” molecule refers to a molecule having the ability either directly or indirectly to modulate an immune response, e.g., upregulation or downregulation of an immune response, and/or immune cell activity.
• Peptidic linker refers to a peptide that joins or links other peptides or polypeptides.
• Fc region Fc region
• Fc domain Fc domain
• Bispecific refers to a biologically functional protein that can bind specifically two distinct epitopes.
• Multispecific refers to a biologically functional protein that can bind specifically to at two or more distinct target molecules or epitopes.
• Mask refers to a polypeptide domain, e.g., an antigen-binding domain of an antibody, that is sterically hindered from binding to a target sequence, or a ligand that is sterically hindered from binding to its cognate binding partner, e.g., its receptor.
• protease-activated or “protease-cleaved” or “cleaved” refers to a fusion protein comprising a protease cleavage site after it has been cleaved by a protease.
• protease cleavage site refers to an amino acid sequence within a fusion protein that contains a protease recognition sequence and is cleaved by a protease.
• Immuno checkpoint refers to a regulatory pathway of the immune system that regulates the immune system activation.
• binds (and grammatical variations thereof) when referring to binding of a particular antigen, epitope, ligand or receptor, means binding that is measurably different from a non-specific interaction.
• mammal includes both humans and non-humans and include, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
• Abbreviations used in this application include the following: PD-1 (Programmed Cell Death Protein 1); PDL-1 (Programmed death-ligand 1); CD3 (Cluster of Differentiation 3); CTLA4 (Cytotoxic T-lymphocyte-Associated Protein 4 or Cluster of Differentiation 152); CD80 (Cluster of Differentiation 80); CD28 (Cluster of Differentiation 28); CD86 (Cluster of Differentiation 86); ICOS (Inducible T Cell Costimulator); ICOSL (Inducible T Cell Costimulator Ligand); CD47 (Cluster of Differentiation 47); SIRPA (Signal-Regulatory Protein Alpha), HHLA2 (Human endogenous retro virus-H Long repeat-associating 2), NKp30 (Natural Killer cell Receptor 3), NCR3 LG 1 (Natural Killer Cell Cytotoxicity Receptor 3 Ligand 1), HHLA2 (HERV-H LTR-associating 2), VISTA (V-domain Ig S
• the term “about” refers to an approximately +/ - 10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
• the terms “comprising,” “having,” “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and/or method steps.
• the term “consisting essentially of’ when used herein in connection with a composition, use or method, denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions.
• compositions, use or method excludes the presence of additional elements and/or method steps.
• a composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in other embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.
• fusion proteins comprising a biologically functional protein, e.g., an antibody or a polypeptide scaffold, fused to a ligand-receptor pair.
• the biologically functional protein comprises at least a first polypeptide and a second polypeptide and the ligand is fused to a terminus of one of the polypeptides via a first peptidic linker and the receptor is fused to the same respective terminus of the other polypeptide via a second peptidic linker.
• At least one of the first and second peptidic linkers comprises a cleavage site for a protease that naturally occurs in a target cellular environment, e.g., in a tumor microenvironment. Also disclosed are methods of using the fusion proteins disclosed herein.
• the fusion proteins according to the present disclosure are masked to decrease any on- target off-tissue (e.g., off-tumor) action (i.e., toxicity) associated with target engagements. Cleavage of the peptidic linker(s) comprising the protease cleavage site in the target cellular environment results in unmasking of the fusion protein.
• the fusion proteins according to the present disclosure comprise a polypeptide scaffold fused to the ligand- receptor pair.
• the fusion protein is masked in that each of the ligand and receptor of the ligand-receptor pair are hindered from engaging a native cognate receptor or ligand through their association with each other.
• Cleavage of the peptidic linker(s) comprising the protease cleavage site in the target cellular environment results in unmasking of the fusion protein by releasing one member of the ligand-receptor pair from the fusion protein, thereby allowing the other member of the ligand-receptor pair to bind its cognate partner.
• the present disclosure provides a biologic design for programmed checkpoint or costimulatory receptor targeting.
• the fusion proteins according to the present disclosure comprise an antibody or antigen-binding antibody fragment comprising an antigen-binding domain fused to the ligand-receptor pair.
• the fusion protein is masked in that the ligand-receptor pair sterically hinders the antigen-binding domain from binding to its cognate antigen.
• the fusion protein is further masked in that each of the ligand and receptor of the ligand-receptor pair are hindered from engaging a native cognate receptor or ligand through their association with each other.
• the present disclosure provides a multifunctional biologic design for programmed target antigen engagement and concurrent checkpoint or costimulatory receptor targeting.
• the design of the fusion proteins described herein decreases target mediated drug disposition.
• the fusion proteins provide a masked antigen binding domain, e.g., a biologically functional protein, as well as a masked immunomodulatory target binding domain, e.g., a ligand-receptor pair, such that the programmed activation of one binding functionality results in the activation of the other binding functionality as well, thereby yielding a bifunctional molecule.
• a masked antigen binding domain e.g., a biologically functional protein
• a masked immunomodulatory target binding domain e.g., a ligand-receptor pair
• fusion proteins each comprising a ligand-receptor pair.
• the ligand receptor pair is an immunomodulatory pair of ligand-receptor domains belonging to the Immunoglobulin Superfamily (IgSF) (Natarajan, Kannan; Mage, Michael G; and Margulies, David H (April 2015) Immunoglobulin Superfamily.
• IgSF Immunoglobulin Superfamily
• the Immunoglobulin Superfamily classifies a commonly found domain in proteins that is based on the core Immunoglobulin (Ig) fold.
• This Ig-fold consists of a beta-sandwich that is made up of a total of 7 antiparallel beta-strands that are arranged in two beta-sheets of 3 and 4 strands ( Figure 34A).
• the two beta-sandwiches are interconnected via a disulfide bridge between strands B and F.
• a structural motif commonly identified in Ig-folds is the “Greek Key” motif.
• Common sub-groups of the IgSF are IgV, IgCl and IgC2 domains.
• IgC domains comprise 7 beta-strands arranged in two sheets of 3 and 4 strands ( Figure 34B), IgV domains comprise 9 beta-strands arranged in two sheets of 4 and 5 strands ( Figure 34C,D).
• IgCl and IgC2 differ in the structural arrangement of the strands.
• IgSF domains can be found in a wide variety of biologically important proteins including antigen receptors, immunoglobulins and immunomodulatory receptors. Surface exposed residues of the core beta sandwich as well as the loops connecting the beta strands can serve as interaction interfaces for antigen recognition, other structural domains in a tertiary/quarternary assembly or a receptor/ligand pair.
• the antigen recognition site of immunoglobulins comprises a dimer of two IgV domains
• a dimer of either IgSF or IgV domains is structurally compatible to form a steric mask for that antigen recognition site if attached covalently to the N-termini of the antibody ( Figure 21).
• the ligand-receptor pair is immunomodulatory, e.g., is an immune checkpoint, causes immune cell effector function modulation, modulation of T-cell receptor signaling, modulates interactions between antigen-presenting cells and effector cells or combinations thereof.
• the ligand-receptor pair comprises an extracellular portion of an IgSF receptor and its cognate ligand, or a receptor -binding fragment thereof.
• a receptor-binding fragment refers to any polypeptide that binds specifically to the receptor of the ligand-receptor pair, and can be naturally occurring or non-naturally occurring. “Naturally occurring,” as used herein and as applied to an object, refers to the fact that an object can be found in nature.
• the ligand-receptor pairs may be two interacting protein domains that belong to the immunoglobulin domain superfamily.
• Non- naturally occurring refers to an engineered polypeptide sequence with structural similarity to IgSF such as a mutant of a naturally occurring protein.
• the disclosure herein relates to the use of an immunomodulatory pair of ligand-receptor domains belonging to the IgSF as a mask of an antibody or antibody fragment, thereby hindering target antigen binding.
• immunomodulatory pairs of ligand-receptor domains belonging to the Immunoglobulin Superfamily include, but are not limited to, pairs of the B7/CD28 families (such as PD1-PDL1, PD1-PDL2, CTLA4-CD80, CD28- CD80, CD28-CD86, CTLA4-CD86, PDL1-CD80, and ICOS-ICOSL, NCR3LGl-NKp30, HHLA2-CD28H and CD47-SIRP ⁇ .
• CD80 also known as B7-1
• CD86 B7-2
• PDL1 B7- Hl
• ICOSL B7-H2
• PDL2 B7-DC
• CD276 B7-H3
• VTCN1 B7-H4
• VISTA B7- H5
• NCR3LG1 B7-H6
• HHLA2 B7-H7
• the B7 family of proteins is typically considered the ligand and pair with members of the CD28 family which comprises CD28, CTLA4, CD28H, NKp30, PD1 and ICOS. (S.M.West and X.A. Deng. Considering B7-CD28 as a family through sequence and structure. Exp Biol Med (Maywood) 2019; 244(17): 1577-1583; doi: 10.1177/1535370219855970).
• the ligand-receptor pair comprises a member of the IgSF B7/CD28 family.
• the ligand and the receptor comprise an extracellular portion of an immunoglobulin superfamily (IgSF) polypeptide.
• the ligand and the receptor comprise extracellular portions of an IgSF immunoglobulin variable (IgV) polypeptide.
• the ligand is a member of the IgSF B7 family and the receptor is a member of the IgSF CD28 family.
• the ligand-receptor pair comprises a leukocyte costimulatory receptor.
• leukocyte costimulatory receptors that belong to the B7/CD28 family include ICOS (also known as CD278) and CD28.
• co-stimulatory ligand-receptor pairs include CD80:CD28, CD86:CD28 and ICOS:ICOSL (ICOS ligand).
• co- inhibitory ligand-receptor pairs include PD1-PDL1, PD1-PDL2, CTLA4-CD80, CTLA4-CD86, PDL1-CD80 and CD47-SIRP ⁇ .
• Figure 2 IB shows a representation of known structures of known B7-CD28 members.
• the size and orientation of the domains of the other pairs is quite similar to that of PD- 1 and PD-L1, and hence they may be used for binding or functional blockade similar to the PD- 1/PD-L1 receptor-ligand pair.
• figure 2 IB shows a representation of the structure of SIRP ⁇ /CD47, another ligand receptor pair with domains belonging to the IgSF, which shows good spatial compatibility to be situated at the N-terminus of a Fab and block binding.
• a number of therapeutic candidates are evaluating the use of antagonists in this axis to increase phagocytosis of cancer cells, making them good candidates for functional masks. (Murata Y, Saito Y, Kotani T, Matozaki T. (2018) CD47-signal regulatory protein a signaling system and its application to cancer immunotherapy. Cancer Sci. 2018 Aug;109(8):2349-2357).
• the affinity of the ligand-receptor domains in the ligand-receptor pair of the fusion protein is altered as compared to the wild-type ligand and receptor.
• one or both of the ligand-receptor domains in the masking pair is engineered, so as the ligand and receptor comprise sequences that are distinct from the wild-type ligand or receptor.
• the ligand comprises one or more mutations that increase binding affinity of the ligand for its cognate receptor.
• the relative binding affinity of the ligand of the ligand-receptor pair compared to a wild-type ligand is greater than 1, 1.5, 2, 2.5 3, 5, 10, 20, 30, 40, 50, 100, 500, 1000, 5,000, 10,000, 50,000 or 100,000 -fold that of the wild-type ligand to its naturally occurring, cognate receptor.
• the receptor comprises one or more mutations that increase binding affinity of the receptor for its cognate ligand.
• the relative binding affinity of the receptor of the ligand-receptor pair compared to a wild-type receptor is greater than 1, 1.5, 2, 2.5 3, 5, 10, 20, 30, 40, 50, 100, 500, 1000, 5,000, 10,000, or 100,000 -fold that of the wild-type receptor to its naturally occurring, cognate ligand.
• the ligand comprises one or more mutations that decrease binding affinity of the ligand for its cognate receptor.
• the relative binding affinity of the ligand of the ligand-receptor pair compared to a wild-type ligand is greater than 1, 1.5, 2, 2.5 3, 5, 10, 20, 30, 40, 50, 100, 500, 1000, 5,000, 10,000, 50,000 or 100,000 -fold lower than that of the wild-type ligand to its naturally occurring, cognate receptor.
• the receptor comprises one or more mutations that decrease binding affinity of the receptor for its cognate ligand.
• the relative binding affinity of the receptor of the ligand-receptor pair compared to a wild-type receptor is greater than 1, 1.5, 2, 2.5 3, 5, 10, 20, 30, 40, 50, 100, 500, 1000, 5,000, 10,000, or 100,000 -fold less than that of the wild-type receptor to its naturally occurring, cognate ligand.
• the ligand-receptor pair can be, e.g., the IgV domains of PD-L1 (Uniprot ID Q9NZQ7, 33-146) and PD-1 (Uniprot ID Q 15116, 18-132)
• the ligand is PD-L1 and has, e.g., an amino acid sequence corresponding to SEQ ID NO: 8 or SEQ ID NO: 10.
• the PD-L1 has an amino acid sequence that is substantially identical to SEQ ID NO:
• the PD-L1 has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 8. In certain embodiments, the PD-L1 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
• Any PD- L1 variant, e.g. high affinity variants, known in the art can be used, for example those provided in Z. Laing et al., High-affinity human PD-L1 variants attenuate the suppression of T cell activation; Oncotarget 8, 88360-88375 (2017) or W02018/170021A1.
• the receptor is a high affinity PD-L1 variant. In some embodiments, the receptor is a high affinity PD-L1 variant having an amino acid sequence corresponding to SEQ ID NO: 10 or an amino acid sequence substantially identical to SEQ ID NO: 10.
• the receptor is PD-1 and has, e.g., an amino acid sequence corresponding to SEQ ID NO: 7 or 11.
• the PD-1 has an amino acid sequence that is substantially identical to SEQ ID NO: 7 or 11.
• the PD-1 has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 7 or 11.
• the PD-1 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or 11.
• Any PD-1 variant, e.g. high affinity variants, known in the art can be used, for example those provided in R. L.
• the receptor is a high affinity PD-1 variant.
• the receptor is a high affinity PD-1 variant having an amino acid sequence corresponding to SEQ ID NO: 9 or an amino acid sequence substantially identical to SEQ ID NO:
• the ligand is CD80 and has, e.g., an amino acid sequence corresponding to SEQ ID NO: 25.
• the CD80 has an amino acid sequence that is substantially identical to SEQ ID NO: 25.
• the CD80 has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 25.
• the CD80 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25.
• the CD80 has an amino acid sequence that is substantially identical to SEQ ID NO: 185, SEQ ID NO: 187 or SEQ ID NO: 189.
• the CD80 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 185, SEQ ID NO: 187 or SEQ ID NO: 189. In certain embodiments, the CD80 has mutations that increase its affinity for its receptor or decrease its propensity to form homodimers during preparation.
• the CD80 has an amino acid sequence corresponding to SEQ ID NO: 25 with one of the following sets of mutations: (a) H18Y, A26E, E35D, M47S, 16 IS and D90G; (b) E35D, M47S, N48K, 16 IS, K89N; (c) E35D, D46V, M47S, 16 IS, D90G, K93E; or (d) H18Y, A26E, E35D, M47S, 16 IS, V68M, A71G, D90G.
• the ligand is PD-L2 and has, e.g. an amino acid sequence corresponding to SEQ ID NO: 250.
• the PD-L2 has an amino acid sequence that is substantially identical to SEQ ID NO: 250.
• the PD-L2 has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 250.
• the PD-L2 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 250.
• the ligand is CD86 and has, e.g. an amino acid sequence corresponding to SEQ ID NO: 248.
• the CD86 has an amino acid sequence that is substantially identical to SEQ ID NO: 248.
• the CD86 has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 248.
• the CD86 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 248.
• the ligand is ICOSL and has, e.g. an amino acid sequence corresponding to SEQ ID NO: 256.
• the ICOSL has an amino acid sequence that is substantially identical to SEQ ID NO: 256.
• the ICOSL has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 256.
• the ICOSL has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 256.
• the ligand is CD276 and has, e.g. an amino acid sequence corresponding to SEQ ID NO: 258.
• the CD276 has an amino acid sequence that is substantially identical to SEQ ID NO: 258.
• the CD276 has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 258.
• the CD276 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 258.
• the ligand is VTCN1 and has, e.g. an amino acid sequence corresponding to SEQ ID NO: 259.
• the VTCN1 has an amino acid sequence that is substantially identical to SEQ ID NO: 259.
• the VTCN1 has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 259.
• the VTCN1 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 259.
• the ligand is VISTA and has, e.g. an amino acid sequence corresponding to SEQ ID NO: 260.
• the VISTA has an amino acid sequence that is substantially identical to SEQ ID NO: 260.
• the VISTA has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 260.
• the VISTA has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 260.
• the ligand is HHLA2 and has, e.g. an amino acid sequence corresponding to SEQ ID NO: 262.
• the HHLA2 has an amino acid sequence that is substantially identical to SEQ ID NO: 262.
• the HHLA2 has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 262.
• the HHLA2 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 262.
• the ligand is SIRP ⁇ and has, e.g. an amino acid sequence corresponding to SEQ ID NO: 255.
• the SIRP ⁇ has an amino acid sequence that is substantially identical to SEQ ID NO: 255. In certain embodiments, the SIRP ⁇ has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 255. In certain embodiments, the SIRP ⁇ has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 255.
• the receptor is CTLA4 and has, e.g., an amino acid sequence corresponding to SEQ ID NO: 26.
• the CTLA4 has an amino acid sequence that is substantially identical to SEQ ID NO: 26.
• the CTLA4 has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 26.
• the CTLA4 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26.
• the receptor is CD28 and has, e.g., an amino acid sequence corresponding to SEQ ID NO: 253.
• the CD28 has an amino acid sequence that is substantially identical to SEQ ID NO: 253.
• the CD28 has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 253.
• the CD28 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 253.
• the receptor is CD28H and has, e.g., an amino acid sequence corresponding to SEQ ID NO: 263.
• the CD28H has an amino acid sequence that is substantially identical to SEQ ID NO: 263.
• the CD28H has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 263.
• the CD28H has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 263.
• the receptor is NKp30 and has, e.g., an amino acid sequence corresponding to SEQ ID NO: 264.
• the NKp30 has an amino acid sequence that is substantially identical to SEQ ID NO: 264.
• the NKp30 has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 264.
• the NKp30 has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 264.
• the receptor is ICOS and has, e.g., an amino acid sequence corresponding to SEQ ID NO: 257.
• the ICOS has an amino acid sequence that is substantially identical to SEQ ID NO: 257.
• the ICOS has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 257.
• the ICOS has an amino acid sequence that is about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 257.
• the IgSF ligand and/or receptor has an immunoglobulin variable domain (IgV) like structure.
• IgV immunoglobulin variable domain
• engineered non-naturally occurring but pairing ligands and/or receptors of the ligand-receptor pairs comprise an immunoglobulin domain with at least one of the domains with affinity for a naturally occurring immunomodulatory receptor.
• the immunomodulatory ligand-receptor pairs are selected to function as antagonists or agonists of their cognate target pair. In certain embodiments, the immunomodulatory ligand-receptor pairs are selected to function as antagonist or agonist of their cognate target pair in a tumor environment. In certain embodiments, one or both the ligand or receptor of the ligand-receptor pair are designed to play a functional role following activation by protease cleavage.
• the fusion proteins described herein can be in a number of different formats.
• the fusion proteins can be considered to have a modular architecture that includes at least a ligand receptor pair, wherein each of the ligand and receptor are fused to a biologically functional protein via peptidic linkers.
• the biologically functional protein comprises at least a first and a second polypeptide.
• either the N-terminus or C-terminus of the ligand or receptor of the ligand-receptor pair can be fused to the first and second polypeptides of the biologically functional protein, e.g., via a peptidic linker.
• the ligand is fused to the first polypeptide and the receptor is fused to the same respective terminus of the second polypeptide.
• the ligand is fused to the N-terminus of a first polypeptide via a first peptidic linker
• the receptor is fused to the N-terminus of a second polypeptide via a second peptidic linker.
• the ligand is fused to the C-terminus of a first polypeptide via a first peptidic linker
• the receptor is fused to the C-terminus of a second polypeptide via a second peptidic linker.
• the ligand and receptor may be fused via their C-termini or their N-termini. Both the ligand and receptor may be fused via their N- or C-termini or one of the ligand or receptor may be fused via its N-terminus while the other of the ligand or receptor is fused via its C-terminus.
• the N-terminus of the ligand is fused to the N-terminus of a first polypeptide via a first peptidic linker, and the N-terminus of the receptor is fused to the N- terminus of a second polypeptide via a second peptidic linker.
• the C- terminus of the ligand is fused to the C-terminus of a first polypeptide via a first peptidic linker, and the C-terminus of the receptor is fused to a second polypeptide via a second peptidic linker.
• the ligand is fused to a terminus of the first polypeptide of the biologically functional protein via a first peptidic linker that comprises a protease cleavage site.
• the receptor is fused to a terminus of the second polypeptide of the biologically functional protein via a second peptidic linker that comprises a protease cleavage site.
• the ligand is fused to a terminus of the first polypeptide of the biologically functional protein via a first peptidic linker that comprises a protease cleavage site
• the receptor is fused to a terminus of the second polypeptide of the biologically functional protein via a second peptidic linker that comprises a protease cleavage site.
• the protease cleavage sites may be cleavable by the same protease or they may be cleavable by different proteases.
• the ligand is fused to a terminus of the first polypeptide of the biologically functional protein via a first peptidic linker that comprises a protease cleavage site and the ligand is engineered to include an internal protease cleavage site which may be the same or different to the cleavage site in the first peptidic linker.
• the receptor is fused to a terminus of the second polypeptide of the biologically functional protein via a second peptidic linker that comprises a protease cleavage site and the receptor is engineered to include an internal protease cleavage site which may be the same or different to the cleavage site in the first peptidic linker.
• Including protease cleavage sites in both the peptidic linker and the member of the ligand-receptor pair that is joined to the biologically functional protein by the linker allows for cleavage and inactivation of that member of the ligand-receptor pair in the target cellular environment, while the member of the ligand-receptor pair that is still fused to the biologically active protein is unmasked (i.e., conditionally activated).
• the fusion protein is conjugated to another therapeutic and/or diagnostic moiety, for example, a chemotherapeutic agent, or a radioisotope.
• the biologically functional protein can function as a scaffold and/or comprise a binding domain.
• polypeptide scaffolds include immunoglobulin Fc regions, albumin, albumin analogs and derivatives, toxins, cytokines, chemokines, growth factors and protein pairs such as leucine zipper domains.
• the biologically functional protein comprises a label, a drug, or combinations thereof. Any label known in the art suitable for detection of the fusion proteins described herein can be used.
• the biologically functional protein can comprise any drug, toxin or chemical known in the art to be capable of conjugation to a protein and to achieve a desired biological result.
• the biologically functional proteins of the fusion proteins described herein comprise at least one antigen-binding domain.
• the binding domains can be, for example, immunoglobulin-based binding domains or non-immunoglobulin-based antibody mimetics, or other polypeptides or small molecules capable of specifically binding to their target, for example, a natural or engineered ligand.
• Non-immunoglobulin-based antibody mimetic formats include, for example, anticalins, fynomers, affimers, alphabodies, DARPins, and avimers.
• the fusion proteins described herein include a biologically functional protein.
• biologically functional proteins include but are not limited to antibodies, e.g., polypeptides with antigen binding domains, and polypeptide scaffolds, e.g., a dimeric Fc.
• the first and second polypeptides of the biologically functional proteins are polypeptides comprising variable and/or constant domains of antibodies, or other domains conferring an antigen binding function or a scaffolding function to the fusion protein.
• the biologically functional protein is an antibody, i.e., immunoglobin.
• Antibodies according to the present disclosure can take on various formats as described herein, including antibody fragments.
• the biologically functional protein is an antibody fragment.
• antibody and immunoglobulin are used interchangeably herein to refer to a polypeptide encoded by an immunoglobulin gene or genes, or a modified version of an immunoglobulin gene, which polypeptide specifically binds to an antigen.
• Specific binding can be measured, for example, through an enzyme-linked immunosorbent assay (ELISA), a surface plasmon resonance (SPR) technique (employing, for example, a BIAcore instrument) (Liljeblad et al ., 2000, Glyco J, 17:323-329), or a traditional binding assay (Heeley, 2002, Endocr Res, 28:217-229).
• ELISA enzyme-linked immunosorbent assay
• SPR surface plasmon resonance
• specific binding is defined as the extent of binding to an unrelated protein being less than about 10% of the binding to the target antigen as measured by SPR, for example.
• specific binding of an antibody or antibody fragment for a particular antigen or an epitope is defined by a dissociation constant (KD) of ⁇ 1 ⁇ M, for example, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM.
• KD dissociation constant
• specific binding of an antibody or antibody fragment for a particular antigen or an epitope is defined by a dissociation constant (KD) of 10 -6 M or less, for example, 10 -7 M or less, or 10 -8 M or less.
• specific binding of an antibody or antibody fragment for a particular antigen or an epitope is defined by a dissociation constant (KD) between 10 -6 M and 10 -13 M, for example, between 10 -7 M and 10 -13 M, between 10 -8 M and 10 -13 M, or between 10 -9 M and 10 -13 M.
• KD dissociation constant
• a traditional immunoglobulin structural unit is typically composed of two pairs of polypeptide chains, each pair having one “light” chain (about 25kD) and one “heavy” chain (about 50-70kD). Light chains are classified as either kappa or lambda.
• the “class” of an immunoglobulin refers to the type of constant domain possessed by its heavy chain.
• IgA immunoglobulin
• IgD immunoglobulin
• IgE immunoglobulin
• IgG immunoglobulin
• IgM immunoglobulins
• IgG1, IgG2, IgG3, IgG4, IgAl and IgA2 immunoglobulins
• the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ), gamma ( ⁇ ) and mu ( ⁇ ), respectively.
• the antibodies described herein are based on an IgG class immunoglobulin, for example, an IgGl, IgG2, IgG3 or IgG4 immunoglobulin. In some embodiments, the antibodies described herein are based on an IgGl, IgG2 or IgG4 immunoglobulin. In some embodiments, the antibodies described herein are based on an IgG1 immunoglobulin. In the context of the present disclosure, when an antibody is based on a specified immunoglobulin isotype, it is meant that the antibody comprises all or a portion of the constant region of the specified immunoglobulin isotype. It is to be understood that the antibody can also comprise hybrids of isotypes and/or subclasses in some embodiments.
• variable light chain (VL) and variable heavy chain (VH) refer to these domains in the light and heavy chain respectively.
• immunoglobulins comprise different domains within the heavy and light chains.
• Such domains can be overlapping and include, the Fc domain (or Fc region), the CHI domain, the CH2 domain, the CH3 domain, the hinge domain, the heavy constant domain (CHl-hinge-Fc or CHl-hinge-CH2-CH3), the variable heavy domain (VH), the variable light domain (VL) and the light constant domain (CL).
• the “Fc domain” includes the CH2 and CH3 domains, and optionally a hinge domain (or hinge region).
• In each of the VH and VL domains of an immunoglobulin are three loops which are hypervariable in sequence and form an antigen-binding site.
• hypervariable region HVR
• CDR complementarity determining region
• the VH and VL domains consist of relatively invariant stretches called framework regions (FRs) of between about 15 to 30 amino acids in length separated by the shorter CDRs, which are each typically between about 5 and 15 amino acids in length, although can occasionally be longer or shorter.
• FRs framework regions
• the three CDRs and four FRs that make up each VH and VL domain are arranged from N- to C-terminus as follows: FR 1 -CDR1 -FR2-CDR2-FR3 -CDR3 -FR4.
• CDR regions are in common use, including those described by Rabat et al. (1983, Sequences of Proteins of Immunological Interest , NIH Publication No. 369-847, Bethesda, MD), by Chothia et al. (1987, J Mol Biol , 196:901-917), as well as the IMGT, AbM and Contact definitions. These different definitions include overlapping or subsets of amino acid residues when compared against each other. By way of example, CDR definitions according to Rabat, Chothia, IMGT, AbM and Contact are provided in Table 1 below.
• variable heavy domain includes the disclosure of the associated (inherent) heavy chain CDRs (HCDRs) as defined by any of the known numbering systems.
• variable light domain includes the disclosure of the associated (inherent) heavy chain CDRs (HCDRs) as defined by any of the known numbering systems.
• the EGFR binding domain comprised by the fusion protein comprises a set of CDRs (i.e., heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3) that have 90% or greater, 95% or greater, 98% or greater, 99% or greater, or 100% sequence identity to a set of CDRs from cetuximab or panitumumab, wherein the binding domain retains the ability to bind EGFR.
• CDRs i.e., heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3
• the EGFR binding domain comprised by the fusion protein comprises a variant of these CDR sequences comprising between 1 and 10 amino acid substitutions across the three CDRs (that is, the CDRs can be modified by including up to 10 amino acid substitutions with any combination of CDRs being modified), for example, between 1 and 7 amino acid substitutions, between 1 and 5 amino acid substitutions, between 1 and 4 amino acid substitutions, between 1 and 3 amino acid substitutions, between 1 and 2 amino acid substitutions, or 1 amino acid substitution, across the CDRs, wherein the variant retains the ability to bind EGFR.
• amino acid substitutions will be conservative amino acid substitutions such as those outlined in Column 1 or Column 2 of Table 4 below.
• the antibodies described herein comprise at least one immunoglobulin domain from a mammalian immunoglobulin, such as a bovine immunoglobulin, a human immunoglobulin, a camelid immunoglobulin, a rat immunoglobulin or a mouse immunoglobulin.
• a biologically functional protein can be a chimeric antibody and comprises two or more immunoglobulin domains, in which at least one domain is from a first mammalian immunoglobulin, for example a human immunoglobulin, and at least a second domain is from a second mammalian immunoglobulin, for example, a mouse or rat immunoglobulin.
• the biologically functional protein comprises at least one immunoglobulin constant domain from a human immunoglobulin.
• the antibodies of the biologically functional proteins described herein can have different valencies.
• the biologically functional protein comprises a single antigen binding domain.
• the biologically functional protein comprises two or more antigen binding domains.
• the biologically functional protein comprises an antibody that has different valencies and specificities.
• a “bispecific antibody” as used herein comprises two binding domains.
• each of the two binding domains has a unique binding specificity.
• a “multispecific antibody” as used herein, comprises two or more binding domains.
• each of the two or more binding domains has a unique binding specificity.
• at least two of the two or more binding domains have unique binding specificities.
• the antibody can be bivalent and bispecific, or can be bivalent and have a single specificity.
• the antibody can be trivalent and bispecific, that is the antibody comprises three binding domains.
• the antibody can also be bispecific and tetravalent, that is the antibody comprises four binding domains. Other valencies are also possible.
• the binding domains can bind to the same epitope on the target molecule or they can bind to different epitopes on the target molecule. In some embodiments, the antibody comprises two binding domains that bind to different epitopes on the target molecule.
• the term “biparatopic” can be used to refer to an antibody which comprises two binding domains that bind to different epitopes on the same target molecule (antigen).
• a biparatopic antibody can bind to a single antigen molecule through the two different epitopes, or it can bind to two separate antigen molecules, each through a different epitope.
• the antibody is biparatopic and bispecific in that it comprises a first binding domain and a second binding domain, each of which binds to a different epitope on the first target molecule, and a third binding domain that binds to the second target molecule.
• a bispecific biparatopic antibody can comprise a first binding domain and a second binding domain, each binding to a different epitope on the first target molecule, and a third binding domain and a fourth binding domain, each binding to a different epitope on the second target molecule.
• the antibody further comprises a scaffold and the binding domains are operably linked to the scaffold.
• “Operably linked,” as used herein, means that the components described are in a relationship permitting each of them to function in their intended manner.
• the binding domains can be directly or indirectly linked to the scaffold. By indirectly linked, it is meant that a given binding domain is linked to the scaffold via another component, for example, a linker or one of the other binding domains.
• indirectly linked it is meant that a given binding domain is linked to the scaffold via another component, for example, a linker or one of the other binding domains.
• Various formats for fusion proteins that comprise a scaffold are described in more detail below. Antigen binding domain formats
• the fusion proteins described herein include an antibody having at least one antigen binding domain that is an antibody fragment, such as a Fab, a Fab’, a single chain Fab (scFab), a single chain Fv (scFv) or a single domain antibody (sdAb).
• an antibody fragment such as a Fab, a Fab’, a single chain Fab (scFab), a single chain Fv (scFv) or a single domain antibody (sdAb).
• a “Fab” or “Fab fragment” contains the constant domain (CL) of the light chain and the first constant domain (CHI) of the heavy chain along with the variable domains VL and VH on the light and heavy chains, respectively, which comprise the CDRs.
• a Fab’ or Fab’ fragment differs from a Fab fragment by the addition of a few amino acid residues at the C-terminus of the heavy chain CHI domain, including one or more cysteine residues from the hinge region.
• a Fab fragment can comprise two separate polypeptide chains (a light chain and a heavy chain) or it can be a single chain Fab.
• a single chain Fab is a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain.
• the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule, however, other formats are also possible.
• An “scFv” includes a heavy chain variable domain (VH) and a light chain variable domain (VL) of an antibody in a single polypeptide chain.
• the scFv can optionally comprise a polypeptide linker between the VH and VL domains which can assist the scFv in forming a desired structure for antigen binding.
• An scFv can include a VL connected from its C-terminus to the N- terminus of a VH by a linker, i.e., VL-Linker-VH, or alternately, an scFv can comprise a VH connected through its C-terminus to the N-terminus of a VL by a linker, i.e., VH-Linker-VL.
• sdAb refers to a single immunoglobulin domain.
• An sdAb can be, for example, of camelid origin. Camelid antibodies lack light chains and their antigen-binding sites consist of a single domain, termed a “VHH.”
• An sdAb comprises three CDR/hypervariable loops that form the antigen-binding site: CDR1, CDR2 and CDR3.
• sdAbs are fairly stable and easy to express, for example, as a fusion with the Fc chain of an antibody (see, for example, Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol. 77(1): 13-22).
• one or more of the binding domains comprised by the antibody can be a natural or engineered ligand for the target receptor, or a functional fragment of such a ligand, i.e., a fragment capable of specifically binding to the target receptor.
• the antigen binding domains can be in the form of combinations of individual scFvs, Fabs, sdAbs.
• binding domains are in the form of scFvs
• formats such as tandem scFv ((scFv)2 or taFv) or triplebody (3 scFvs) can be constructed, in which the scFvs are connected together by a flexible linker.
• scFvs can also be used to construct diabody, triabody and tetrabody (tandem diabodies or TandAbs) formats, which comprise 2, 3 and 4 scFvs, respectively, connected by a short linker.
• the restricted length of the linker results in dimerization of the scFvs in a head-to-tail manner.
• the scFvs can be further stabilized by inclusion of an interdomain disulfide bond.
• a disulfide bond can be introduced between VL and VH through introduction of an additional cysteine residue in each chain (for example, at position 44 in VH and 100 in VL) (see, for example, Fitzgerald et al., 1997, Protein Engineering , 10: 1221-1225) or a disulfide bond can be introduced between two VHs to provide an antigen binding domain having a DART format (see, for example, Johnson et al, 2010, JMol. Biol., 399:436-449).
• formats comprising two or more sdAbs, such as VHs or VHHs, connected together through a suitable linker can be used for the biologically functional protein.
• suitable linker such as VHs or VHHs
• Other examples of antibody formats that lack a scaffold include those based on Fab fragments, for example, Fab2, F(ab’)2 and F(ab’) 3 formats, in which the Fab fragments are connected through a linker or an IgG hinge region.
• an scFv or a sdAb can be fused to the C-terminus of either or both of the light and heavy chain of a Fab fragment resulting in a bivalent (Fab-scFv) or (Fab-sdAb) or trivalent (Fab-(scFv)2 or Fab-(sdAb)2).
• one or two scFvs or sdAbs can be fused at the hinge region of a F(ab’) fragment to produce a tri-or tetravalent F(ab’)2-scFv/sdAb.
• the binding domains can be in one or a combination of the forms described above (for example, scFvs, Fabs and/or sdAbs, or ligand-based binding domains).
• the biologically functional protein comprises a bi- specific antibody that binds an immune cell antigen, e.g., CD3, and a tumor associated antigen (TAA), e.g., HER2.
• the biologically functional protein comprises a bi-specific antibody with a Fab-scFv format wherein the Fab binds an immune cell antigen and the scFv binds a TAA.
• the biologically functional protein comprises a bi-specific antibody with a Fab-scFv format wherein the Fab binds CD3 and the scFv binds HER2.
• the biologically functional protein comprises a bi-specific antibody with a Fab-Fab format wherein one Fab binds CD3 and the other Fab binds HER2.
• the biologically functional protein comprises two or more antigen binding domains operably linked to a heterodimeric Fc.
• the biologically functional protein can be bivalent, trivalent or tetravalent.
• formats are described below. Other configurations are known in the art (see, for example, Spiess et al, 2015, Mol Immunol., 67:95-106).
• Exemplary configurations for a biologically functional protein comprising two binding domains operably linked to a heterodimeric Fc include, but are not limited to: a) mAb format in which the first binding domain is a Fab that is operably linked to the N- terminus of the first Fc polypeptide of the heterodimeric Fc and the second binding domain is a Fab that is operably linked to the N-terminus of the second Fc polypeptide; b) hybrid format in which the first binding domain is an scFv that is operably linked to the N-terminus of one Fc polypeptide of the heterodimeric Fc and the second binding domain is a Fab that is operably linked to the N-terminus of the other Fc polypeptide, and c) dual scFv format in which the first binding domain is an scFv that is operably linked to the N-terminus of the first Fc polypeptide of the heterodimeric Fc and
• antibodies comprising one binding domain (either first or second) as a Fab or an scFv operably linked to the N-terminus of the first Fc polypeptide and the other binding domain as a Fab or an scFv operably linked to the C-terminus of the second Fc polypeptide.
• Exemplary configurations for a multispecific antibody comprising three binding domains operably linked to a heterodimeric Fc include, but are not limited to:
• the first binding domain is a Fab that is operably linked to the N-terminus of the first Fc polypeptide of the heterodimeric Fc
• the second binding domain is a Fab that is operably linked to the N-terminus of the second Fc polypeptide
• the third binding domain is an scFv operably linked to the C-terminus of either the first or the second Fc polypeptide
• the first binding domain is an scFv that is operably linked to the N-terminus of one Fc polypeptide of the heterodimeric Fc
• the second binding domain is a Fab that is operably linked to the N-terminus of the other Fc polypeptide
• the third binding domain is a Fab that is operably linked to the C-terminus of either the first or the second Fc polypeptide
• the first binding domain is a Fab that is operably linked to the N-terminus of the first Fc polypeptide of the heterodimeric Fc
• the second binding domain is a Fab that is operably linked to the N-terminus of the second Fc polypeptide
• the third binding domain is a Fab operably linked to the N-terminus of either the first or the second binding domain.
• Exemplary configurations for a multispecific antibody comprising four binding domains operably linked to a heterodimeric Fc include, but are not limited to: i) central-scFv2 format in which the first binding domain is an scFv that is operably linked to the N-terminus of one Fc polypeptide of the heterodimeric Fc, the second binding domain is an scFv that is operably linked to the N-terminus of the other Fc polypeptide, the third binding domain is a Fab that is operably linked to one of the scFvs and the fourth binding domain is a Fab that is operably linked to the other scFv, and ii) dual variable domain format in which the first binding domain is a Fab that is operably linked to the N-terminus of one Fc polypeptide of the heterodimeric Fc, the second binding domain is a Fab that is operably linked to the N-terminus of the
• the antibodies of the biologically functional proteins described herein can comprise a label, a drug, or combinations thereof. Any label known in the art suitable for detection of the fusion proteins described herein can be used. Antibody drug conjugates are described in more detail below.
• the antigen binding domains of the antibodies of the biologically functional protein described herein bind to the same antigen on the same cell. In certain embodiments, the antigen binding domains bind to more than one antigen on the same cell. In certain embodiments, the antigen binding domains bind to more than one antigen, wherein at least one antigen is on a different cell than another antigen. In certain embodiments, the antigen binding domain(s) of the antibody bind to a tumor cell or an immune cell. In certain embodiments, the antigen binding domains of the antibody bind to a tumor cell and an immune cell.
• the antibodies can be derived from immunoglobulins that are from different species, for example, the antibody can be a chimeric antibody or a humanized antibody.
• a “chimeric antibody” refers to an antibody that typically comprises at least one variable domain from a rodent antibody (usually a murine antibody) and at least one constant domain from a human antibody.
• a “humanized antibody” is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody.
• human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain it replaces.
• Chimeric antibodies are discussed, for example, in Morrison et al ., 1984, Proc. Natl. Acad. Sci. USA , 81:6851-55, and U.S. Patent No. 4,816,567.
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity and affinity for a target antigen.
• donor antibody such as mouse, rat, rabbit, or non-human primate
• framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues, or the humanized antibodies can comprise residues that are not found in either the recipient antibody or the donor antibody.
• a variable domain in a humanized antibody will comprise all or substantially all of the hypervariable regions from a non-human immunoglobulin and all or substantially all of the FRs from a human immunoglobulin sequence.
• Humanized antibodies are described in more detail in Jones, et al., 1986, Nature, 321:522-525; Riechmann, et al., 1988, Nature, 332:323-329 and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596, for example.
• a number of approaches are known in the art for selecting the most appropriate human frameworks in which to graft the non-human CDRs.
• Early approaches used a limited subset of well-characterized human antibodies, irrespective of the sequence identity to the non-human antibody providing the CDRs (the “fixed frameworks” approach).
• More recent approaches have employed variable regions with high amino acid sequence identity to the variable regions of the non-human antibody providing the CDRs (“homology matching” or “best-fit” approach).
• An alternative approach is to select fragments of the framework sequences within each light or heavy chain variable region from several different human antibodies. CDR-grafting can in some cases result in a partial or complete loss of affinity of the grafted molecule for its target antigen.
• the antibody comprises humanized antibody sequences, for example, one or more humanized variable domains. In some embodiments, the antibody is a humanized antibody.
• an antigen binding domain comprised by the fusion protein is a substitutional variant of a known antibody that comprises one or more amino acid substitutions in the CDRs of the parent antibody.
• the substitution variant has modifications (for example, improvements) in certain biological properties relative to the parent antibody.
• the substitution variant can have increased affinity for the target protein or it can have reduced immunogenicity.
• the substitution variant substantially retains certain biological properties of the parent antibody.
• CDR hotspots are residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, for example, Chowdhury, 2008, Methods Mol. Biol., 207:179-196).
• Methods of affinity maturation are well known in the art. For example, diversity can be introduced into the variable genes chosen for maturation by various techniques including, for example, error-prone PCR, chain shuffling or oligonucleotide-directed mutagenesis. A secondary library is then created, and this library is screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (for example, 2, 3, 4 or more residues at a time) are randomized. CDR3 of either or both of the heavy or light chain is often targeted for CDR-directed approaches.
• CDR residues involved in antigen binding can be identified for example using alanine scanning mutagenesis (see, for example, Cunningham and Wells, 1989, Science , 244:1081-1085) or by computer modeling using a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen.
• a substitution variant comprises one or more substitutions within one or more CDRs provided that the substitutions do not substantially reduce the ability of the binding domain to bind its target antigen.
• a substitution variant can comprise one or more conservative substitutions as described herein within one or more CDRs that do not substantially reduce binding affinity.
• a substitution variant comprises one or more amino acid substitutions within the CDRs that do not involve the antigen-contacting amino acids.
• a substitution variant comprises a variant VH or VL sequence in which each CDR either is unaltered or contains no more than one, two or three amino acid substitutions.
• the fusion proteins described herein comprise a biologically functional protein based on an IgG Fc in which native glycosylation has been modified.
• glycosylation of an Fc can be modified to increase or decrease effector function.
• mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine or histidine i.e. N297A, Q, K or H results in an aglycoslated Fc that lacks all effector function (Bolt et al., 1993, Eur. ./. Immunol., 23:403-411; Tao & Morrison, 1989, ./. Immunol., 143:2595-2601).
• glycosylation variants include those with bisected oligosaccharides, for example, variants in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc).
• GlcNAc N-acetylglucosamine
• Such glycosylation variants can have reduced fucosylation and/or improved ADCC function. See, for example, International Publication No. WO 2003/011878, U.S. Patent No. 6,602,684 and US Patent Application Publication No. US 2005/0123546.
• Useful glycosylation variants also include those having at least one galactose residue in the oligosaccharide attached to the Fc region, which can have improved CDC function (see, for example, International Publication Nos. WO 1997/030087, WO 1998/58964 and WO 1999/22764).
• the biologically functional protein of the fusion proteins described herein is a polypeptide scaffold, which can function, e.g., to stabilize or extend the in vivo half-life of the ligand receptor pair.
• the biologically functional protein consists of a dimeric Fc region.
• the first and second polypeptide of the biologically functional protein consists of a dimeric Fc, wherein the first polypeptide consists of a first Fc polypeptide and the second polypeptide consists of a second Fc polypeptide, the first and second Fc polypeptides forming a dimeric Fc region.
• the dimeric Fc region is a heterodimeric Fc. Heterodimeric Fc regions are described in more detail herein.
• the polypeptide scaffolds are comprised of a first and second polypeptide.
• the ligand of the ligand receptor pair is fused via a peptidic linker to the first polypeptide and the receptor is fused via a peptidic linker to the same respective terminus of the second polypeptide.
• the ligand is fused to the N-terminus of a first polypeptide via a peptidic linker, and the receptor is fused to the N-terminus of a second polypeptide via a second peptidic linker.
• the ligand is fused to the C-terminus of a first polypeptide via a peptidic linker, and the receptor is fused to a second polypeptide via a second peptidic linker.
• the biologically functional protein comprises a polypeptide scaffold that consists of a dimeric Fc region and a ligand-receptor pair that is PDL-1 and PD-1.
• the fusion protein comprises a biologically functional protein that consists of a dimeric Fc region and a ligand-receptor pair that is CD80 and CTLA4.
• an Fc domain of the polypeptide scaffold comprises an amino acid sequence corresponding to SEQ ID NOs: 4 and 5, and optionally SEQ ID NO: 6.
• the polypeptide scaffold consists of a heterodimeric Fc comprising SEQ ID NO: 4 and SEQ ID NO: 5; wherein a first Fc polypeptide comprises SEQ ID NO: 4 and a second Fc polypeptide comprises SEQ ID NO: 5.
• the polypeptide scaffold consisting of a heterodimeric Fc comprises a modified CH3 and/or CH2 domain of Table 2 and Table 3, respectively.
• the fusion proteins described herein include biologically functional proteins, e.g., antibodies or polypeptide scaffolds, comprising a dimeric immunoglobulin Fc region.
• Fc region includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat el al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
• an “Fc polypeptide” of a dimeric Fc refers to one of the two polypeptides forming the dimeric Fc region, that is a polypeptide comprising C- terminal constant regions of an immunoglobulin heavy chain that is capable of stable self- association.
• An Fc region can comprise either a CH3 domain or a CH3 and a CH2 domain.
• the CH3 domain comprises two CH3 sequences, each comprised by one of the two Fc polypeptides of the dimeric Fc.
• the CH2 domain comprises two CH2 sequences, each comprised by one of the two Fc polypeptides of the dimeric Fc.
• the fusion protein comprises an Fc based on a human IgG Fc. In some embodiments, the fusion protein comprises an Fc based on a human IgG1 Fc. In some embodiments, the fusion protein comprises an Fc based on a heterodimeric Fc comprising two different Fc polypeptides.
• the fusion protein comprises an Fc based on a modified IgG Fc in which the CH3 domain comprises one or more amino acid modifications. In some embodiments, the fusion protein comprises an Fc based on a modified IgG Fc in which the CH2 domain comprises one or more amino acid modifications. In some embodiments, the fusion protein comprises an Fc based on a modified IgG Fc in which the CH3 domain comprises one or more amino acid modifications and the CH2 domain comprises one or more amino acid modifications.
• the fusion protein comprises a heterodimeric immunoglobulin Fc comprising a modified CH3 domain, wherein the modified CH3 domain comprises one or more asymmetric amino acid modifications.
• an “asymmetric amino acid modification” refers to a modification in which an amino acid at a specific position on the first Fc polypeptide is different to the amino acid at the corresponding position on the second Fc polypeptide.
• These asymmetric amino acid modifications can comprise modification of only one of the two amino acids at the corresponding position on each Fc polypeptide, or they can comprise modifications of both amino acids at the corresponding positions on each of the first and second Fc polypeptides.
• the fusion protein comprises a heterodimeric Fc comprising a modified CH3 domain, wherein the modified CH3 domain comprises one or more asymmetric amino acid modifications that promote formation of the heterodimeric Fc over formation of a homodimeric Fc.
• Amino acid modifications that can be made to the CH3 domain of an Fc in order to promote formation of a heterodimeric Fc are known in the art and include, for example, those described in International Publication No.
• WO 96/027011 (“knobs into holes”), Gunasekaran et al, 2010, J Biol Chem, 285, 19637-46 (“electrostatic steering”), Davis et al, 2010, Prot Eng Des Sel , 23(4): 195-202 (strand exchange engineered domain (SEED) technology) and Labrijn et al, 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab-arm exchange).
• SEED strand exchange engineered domain
• the fusion protein comprises a heterodimeric Fc having a modified CH3 domain as described in International Publication No. WO 2012/058768 or International Patent Publication No. WO 2013/063702.
• the fusion protein comprises a heterodimeric human IgG1 Fc having a modified CH3 domain.
• Table 2 below provides the amino acid sequence of the human IgG1 Fc sequence, corresponding to amino acids 231 to 447 of the full-length human IgG1 heavy chain.
• the CH2 domain is typically defined as comprising amino acids 231-340 of the full-length human IgG1 heavy chain and the CH3 domain is typically defined as comprising amino acids 341- 447 of the full-length human IgG1 heavy chain.
• the fusion protein comprises a heterodimeric Fc having a modified CH3 domain comprising one or more asymmetric amino acid modifications that promote formation of the heterodimeric Fc over formation of a homodimeric Fc, in which the modified CH3 domain comprises a first Fc polypeptide including amino acid modifications at positions F405 and Y407, and a second Fc polypeptide including amino acid modifications at positions T366 and T394.
• the amino acid modification at position F405 of the first Fc polypeptide of the modified CH3 domain is F405A, F405I, F405M, F405S, F405T or F405V.
• the amino acid modification at position Y407 of the first Fc polypeptide of the modified CH3 domain is Y407I or Y407V.
• the amino acid modification at position T366 of the second Fc polypeptide of the modified CH3 domain is T366I, T366L or T366M.
• the amino acid modification at position T394 of the second Fc polypeptide of the modified CH3 domain is T394W.
• the first Fc polypeptide of the modified CH3 domain further includes an amino acid modification at position L351.
• the amino acid modification at position L351 in the first Fc polypeptide of the modified CH3 domain is L351Y.
• the second Fc polypeptide of the modified CH3 domain further includes an amino acid modification at position K392.
• the amino acid modification at position K392 in the second Fc polypeptide of the modified CH3 domain is K392F, K392L or K392M.
• one or both of the first and second Fc polypeptides of the modified CH3 domain further comprises the amino acid modification T350V.
• the fusion protein comprises a heterodimeric Fc having a modified CH3 domain comprising one or more asymmetric amino acid modifications that promote formation of the heterodimeric Fc over formation of a homodimeric Fc, in which the modified CH3 domain comprises a first Fc polypeptide including the amino acid modification F405A, F405I, F405M, F405S, F405T or F405V together with the amino acid modification Y407I or Y407V, and a second Fc polypeptide including the amino acid modification T366I, T366L or T366M, together with the amino acid modification T394W.
• the modified CH3 domain comprises a first Fc polypeptide including the amino acid modification F405A, F405I, F405M, F405S, F405T or F405V together with the amino acid modification Y407I or Y407V, and a second Fc polypeptide including the amino acid modification T366I, T366L or
• the first Fc polypeptide of the modified CH3 domain further includes the amino acid modification L351Y.
• the second Fc polypeptide of the modified CH3 domain further includes the amino acid modification K392F, K392L or K392M.
• one or both of the first and second Fc polypeptides of the modified CH3 domain further comprises the amino acid modification T350V.
• the fusion protein comprises a heterodimeric Fc comprising a modified CH3 domain having a first Fc polypeptide that comprises amino acid modifications at positions F405 and Y407, and optionally further comprises an amino acid modification at position L351, and a second Fc polypeptide that comprises amino acid modifications at positions T366 and T394, and optionally further comprises an amino acid modification at position K392, as described above, and the first Fc polypeptide further comprises an amino acid modification at one or both of positions S400 or Q347 and/or the second Fc polypeptide further comprises an amino acid modification at one or both of positions K360 or N390, where the amino acid modification at position S400 is S400E, S400D, S400R or S400K; the amino acid modification at position Q347 is Q347R, Q347E or Q347K; the amino acid modification at position K360 is K360D or K360E, and the amino acid modification at position N390 is N390R, N390K or N
• the fusion protein comprises a heterodimeric Fc comprising a modified CH3 domain comprising the modifications of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 2.
• the CH3 domain has an amino acid sequence corresponding to SEQ ID NO: 4 or SEQ ID NO: 5.
• the CH3 has an amino acid sequence that is substantially identical to SEQ ID NO: 4 or SEQ ID NO: 5.
• the CH3 domain has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 4 or SEQ ID NO: 5.
• Table 2 Human IgG1 Fc Sequences and Variants
• the fusion protein comprises an Fc based on an IgG Fc having a modified CH2 domain. In some embodiments, the fusion protein comprises an Fc based on an IgG Fc having a modified CH2 domain, wherein the modification of the CH2 domain results in altered binding to one or more Fc receptors (FcRs) such as receptors of the Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII subclasses.
• FcRs Fc receptors
• a number of amino acid modifications to the CH2 domain that selectively alter the affinity of the Fc for different Fey receptors are known in the art.
• Amino acid modifications that result in increased binding and amino acid modifications that result in decreased binding can both be useful in certain indications.
• increasing binding affinity of an Fc for Fc ⁇ RIIIa results in increased antibody dependent cell-mediated cytotoxicity (ADCC), which in turn results in increased lysis of the target cell.
• ADCC antibody dependent cell-mediated cytotoxicity
• Decreased binding to Fc ⁇ RIIb an inhibitory receptor
• CDC complement-mediated cytotoxicity
• modified CH2 domains comprising amino acid modifications that result in increased binding to Fc ⁇ RIIb or amino acid modifications that decrease or eliminate binding of the Fc region to all of the Fey receptors (“knock-out” variants) can be useful.
• Examples of amino acid modifications to the CH2 domain that alter binding of the Fc by Fey receptors include, but are not limited to, the following: S298A/E333A/K334A and S298A/E333A/K334A/K326A (increased affinity for Fc ⁇ RIIIa) (Lu, et al, 2011, J Immunol Methods , 365(1-2): 132-41); F243L/R292P/Y300L/V305I/P396L (increased affinity for Fc ⁇ RIIIa) (Stavenhagen, et al, 2007, Cancer Res, 67(18):8882-90); F243L/R292P/Y300L/L235V/P396L (increased affinity for Fc ⁇ RIIIa) (Nordstrom JL, et al, 2011, Breast Cancer Res, 13(6):R123); F243L (increased affinity for Fc ⁇ RIIIa) (Stewart, et al,
• the fusion protein comprises an Fc based on an IgG Fc having a modified CH2 domain, in which the modified CH2 domain comprises one or more amino acid modifications that result in decreased or eliminated binding of the Fc region to all of the Fey receptors (i.e., a “knock-out” variant).
• Additional examples include Fc regions engineered to include the amino acid modifications L235A/L236A/D265S.
• asymmetric amino acid modifications in the CH2 domain that decrease binding of the Fc to all Fey receptors are described in International Publication No. WO 2014/190441.
• the CH2 domain has an amino acid sequence corresponding to SEQ ID NO: 6.
• the CH2 has an amino acid sequence that is substantially identical to SEQ ID NO: 6.
• the CH2 domain has an amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 6
• Certain embodiments of the fusion proteins described herein comprise biologically functional proteins that are an antibody conjugated to a drug, i.e., an antibody drug conjugate (ADC).
• ADC antibody drug conjugate
• the drug of an ADC can be any therapeutic molecule, e.g., a toxin, a chemotherapeutic agent, a small molecule inhibitor.
• the ADC can be conjugated to the drug via a linker, which may be a cleavable linker or a non-cleavable linker.
• a cleavable linker can be susceptible to cleavage under intracellular conditions, for example, through lysosomal processes.
• cleavable linkers include linkers that are protease-sensitive, acid-sensitive, reduction-sensitive or photolabile. Conjugation of the drug can be performed by any method known in the art including, but not limited to, lysine or cysteine conjugation, bis-thiol linkers, conjugation using glycosylation sites of antibodies, ultraviolet light conjugation, and use of unnatural amino acids.
• the fusion proteins described herein comprise at least a first and a second peptidic linker.
• a peptidic linker is a peptide that joins or links other peptides or polypeptides.
• the peptidic linker fuses a polypeptide of the biologically functional protein, e.g., the antibody or dimeric Fc scaffold, to the ligand and/or receptor of the ligand-receptor pair.
• an Fc polypeptide is fused to a ligand or receptor of the ligand-receptor pair, or a linker can join an Fc polypeptide to a ligand or receptor of the ligand-receptor pair.
• the ligand is fused to a terminus of the first polypeptide via the first peptidic linker; the receptor is fused to the same respective terminus of the second polypeptide via the second peptidic linker.
• the receptor and the ligand are both fused to the respective N-termini of the first and second polypeptides via the peptidic linkers.
• the receptor and the ligand are both fused to the respective C-termini of the first and second polypeptides via the peptidic linkers.
• the peptidic linker is of sufficient length to allow pairing of ligand and receptor.
• a peptidic linker can provide flexibility or rigidity suitable for properly orienting the one or more domains of the fusion proteins herein, both within the fusion protein and between or among the fusion proteins and their target(s).
• a peptidic linker can support expression of a full-length fusion protein and stability of the purified protein both in vitro and in vivo following administration to a subject in need thereof, such as a human, and is preferably non-immunogenic or poorly immunogenic in those same subjects.
• a peptidic linker can comprises part or all of a human immunoglobulin hinge, a stalk region of C- type lectins, a family of type II membrane proteins, or combinations thereof.
• the peptidic linker is of sufficient length to allow pairing of ligand and receptor and is of about 2 to about 150 amino acids. In certain embodiments, peptidic linkers range in length from about 3 to about 50 amino acids, or about 5 to about 20 amino acids, or about 10 to about 50 amino acids, or about 2 to about 40 amino acids, or about 8 to about 20 amino acids, about 10 to about 60 amino acids, about 10 to about 30 amino acids, or about 15 to about 25 amino acids. In some embodiments, the peptidic linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
• At least one of the peptidic linkers of the fusion proteins described herein comprise a protease cleavage site, also referred to as cleavage sequences.
• the fusion protein comprises at least one peptidic linker comprising a protease cleavage site and at least one peptidic linker that does not comprise a protease cleavage site.
• the protease cleavage sites are positioned within the peptidic linkers so as to maximize recognition and cleavage by the desired protease or proteases and minimize recognition and non-specific cleavage by other proteases.
• the peptidic linker can comprise one or more cleavage sites.
• a fusion protein can be cleaved by 1, 2, 3, 4, 5 or more proteases.
• the protease cleavage site or sites can be positioned within the peptidic linkers (or said differently, can be surrounded by linkers) and are positioned within the fusion protein as a whole so as to achieve the best desired cleavage and release of fusion protein fragments (e.g., the ligand of the receptor ligand pair, the receptor of the ligand receptor pair or both the ligand and the receptor) post-cleavage.
• CM cleavable moiety
• the protease cleavage site or cleavage sequence can be selected based on a protease that is co-localized in tissue where the activity of the fusion protein or biologically functional protein is desired.
• a cleavage site can serve as a substrate for multiple proteases, e.g., a substrate for a serine protease and a second different protease, e.g., a matrix metalloproteinase (an MMP).
• a cleavage site can serve as a substrate for more than one serine protease, e.g., a matriptase and a urokinase-type plasminogen activator (uPA).
• a peptidic linker can serve as a substrate for more than one MMP, e.g, an MMP9 and an MMP 14.
• the peptidic linker is specifically cleaved by a protease at a rate of about 0.001-1500 x 10 4 M -1 S -1 or at least 0.001, 0 005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 10 4 M -1 S -1 .
• the fusion protein comprises at least a first peptidic linker and is in the presence of sufficient enzyme activity
• the peptidic linker is cleaved.
• Sufficient enzyme activity can refer to the ability of the enzyme to make contact with the peptidic linker and effect cleavage. It can readily be envisioned that an enzyme can be in the vicinity of the peptidic linker but is unable to cleave because of other cellular factors or protein modification of the enzyme.
• the peptidic linker comprises a protease cleavage site of 5-10 amino acids, or 7-10 amino acids, or 8-10 amino acids in length.
• the peptidic linker consists of a protease cleavage site of 5-10 amino acids, or 7-10 amino acids, or 8- 10 amino acids in length.
• the protease cleavage site is preceded on the N- terminus by a linker sequence of about 1-20 amino acids, 2-5 amino acids, 5-10 amino acids, 10- 15 amino acids, 10-20 amino acids, 12-16 amino acids, or about 5 or about 10 amino acids in length.
• the protease cleavage site is followed on the C-terminus by a linker sequence of about 1-20 amino acids, 2 -5 amino acids, 5-10 amino acids, 10- 15 amino acids, 10- 20 amino acids, 12-16 amino acids amino acids, or in some cases, about 5 or about 10 amino acids in length.
• the protease cleavage site is preceded by a linker sequence on the N-terminus and followed by a linker sequence on the C-terminus.
• the protease cleavage site is situated between two linkers.
• the linkers on either the N or C-terminal end of the protease cleavage site can be of varying lengths, for example, between about 2-20, 6-20, 8-15, 8-10, 10-18, or 12-16 amino acids in length. In certain embodiments, the N- or C-terminal linker sequence is about 3 or about 5 amino acids in length.
• Exemplary peptidic linkers of the disclosure comprise one or more protease cleavage sites recognized by any of a variety of proteases, such as, but not limited to, serine proteases, MMPs (MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18 (collagenase 4), MMP19, MMP20, MMP21, etc), adamalysins, serralysins, astacins, caspases (e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14), cathepsins, (e.g., cathepsin A, cathepsin B, catheps
• a peptidic linker comprises a cleavage site that is cleaved by more than one protease.
• an individual cleavage site can be cleaved by 1, 2, 3, 4, 5 or more proteases.
• a peptidic linker can comprise a cleavage site that is substantially cleaved by one enzyme but not by others.
• a peptidic linker comprises a cleavage site that has high specificity.
• high specificity is meant >90% cleavage observed by a particular protease and less than 50% cleavage observed by other proteases.
• a peptidic linker comprises a cleavage site that demonstrates >80% cleavage by one protease but less than 50% cleavage by other proteases. In certain embodiments, a peptidic linker comprises a cleavage site that demonstrates >70%, 75%, 76%, 77%, 78%, or 79%, cleavage by one protease but less than 65%, 60%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, or 45% cleavage by other proteases.
• the cleavage site can be >90% cleaved by matriptase and -75% cleaved by uPa and plasmin.
• the cleavage site can be cleaved by uPa and matriptase but no specific cleavage by plasmin is observed.
• the cleavage site can be cleaved by uPa and not by matriptase or plasmin.
• a cleavage site can demonstrate some level of resistance to non-specific protease cleavage, e.g., cleavage by plasmin or other non-specific proteases.
• a protease cleavage site can have “high non-specific protease resistance” ( ⁇ 25% cleavage by plasmin or an equivalent non-specific protease), “moderate non-specific protease resistance” ( ⁇ 75% cleavage by plasmin or an equivalent non-specific protease), or “low non-specific protease resistance” (up to about 90% cleavage by plasmin or an equivalent non-specific protease).
• Such cleavage activity can be measured using assays known in the art, such as by incubation with the appropriate protease followed by SDS-PAGE or other analysis.
• a protease cleavage site may display up to complete resistance to protease cleavage to 24 hours contact with protease.
• a protease cleavage sequence may display up to complete resistance to non-specific protease cleavage after 0.5 hour to 36 hours contact with protease.
• a protease cleavage sequence displays up to complete resistance to non-specific protease cleavage after 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 20, 24, 36, 48, or 72 hours contact with an appropriate protease.
• the cleavage sites are selected based on preferences for various desired proteases.
• a desired cleavage profile for a particular peptidic linker comprising a cleavage site can be selected for a desired purpose (e.g., high specific cleavage in particular tumor microenvironments or specific organs) where a particular protease or set of proteases can demonstrate high, specific, elevated, efficient, moderate, low or no cleavage of a particular cleavage site within a peptidic linker.
• a desired purpose e.g., high specific cleavage in particular tumor microenvironments or specific organs
• Methods for determining cleavage are known in the art.
• a peptidic linker can comprise one or more cleavage sites arranged in tandem, with or without additional linkers in between each cleavage site.
• a peptidic linker comprises a first cleavage site and a second cleavage site where the first cleavage site is cleaved by a first protease and the second cleavage site is cleaved by a second protease.
• a peptidic linker can comprise a first cleavage site cleaved by matriptase and uPa and a second cleavage site cleaved by an MMP.
• a peptidic linker comprises a first cleavage site, a second cleavage site and a third cleavage site where the first cleavage site is cleaved by a first protease, the second cleavage site is cleaved by a second protease and the third cleavage site is cleaved by a third protease.
• Illustrative proteolytic enzymes and their recognition sequences useful in the fusion proteins herein can be identified by one of skill and are known in the art, such as those described in MEROPS database (see e.g., Rawlings, et al. Nucleic Acids Research, Volume 46, Issue Dl, 4 January 2018, Pages D624-D632), and elsewhere (Hoadley et al, Cell, 2018; GTEX Consortium, Nature, 2017; Robinson et al, Nature, 2017).
• an embodiment of the present disclosure provides a fusion protein that comprises at least two peptidic linkers wherein at least one of the peptidic linkers comprises one or more of the cleavage sites set forth herein.
• the present disclosure provides a fusion protein that comprises a peptidic linker wherein the peptidic linker comprises a protease cleavage site and is cleavable by uPA.
• the present disclosure provides a fusion protein that comprises a peptidic linker wherein the peptidic linker comprises the amino acid sequence MSGRSANA (SEQ ID NO: NO:28).
• the peptidic linker sequence comprises at least one protease cleavage site selected from TSGRSANP, LSGRSDNH, GSGRSAQV, GSSRNADV, GTARSDNV, GTARSDNV. GGGRVNNV, MSARILQV or GKGRSANA (SEQ ID NOS: 30-37 respectively).
• the fusion protein comprising the peptidic linker described herein comprises two heterologous polypeptides, a first polypeptide located amino (N) terminally to the peptidic linker and a second polypeptide located carboxyl (C) terminally to the peptidic linker, the two heterologous polypeptides thus separated by the peptidic linker.
• the fusion protein comprises at least one peptidic linker that does not comprise a protease cleavage site.
• the peptidic linker comprises an amino acid sequence (EAAAK)n where n is an integer of 1 to 5.
• the peptidic linker is EAAAK (SEQ ID NO:39).
• the peptidic linker EAAAKEAAAK (SEQ ID NO:38).
• the peptidic linker comprises a polyproline linker, optionally having an amino acid sequence of PPP (SEQ ID NO: 41) or PPPP (SEQ ID NO: 40).
• the linker is glycine (G)-proline (P) polypeptide linker, optionally GPPPG, GGPPPGG, GPPPPG or GGPPPGG.
• the peptidic linker is a Gly n Ser linker.
• the peptidic linker comprises an amino acid sequence of (Gly 3 Ser) n (Gly 4 Ser) 1 , (Gly 3 Ser) 1 (Gly 4 Ser) n , (Gly 3 Ser) n (Gly 4 Ser) n , or (Gly 4 Ser) n , wherein n is an integer of 1 to 5.
• the peptidic linkers are suitable for connecting the different domains include sequences comprising glycine-serine linkers, for example, but not limited to, (G m S) n -GG, (SGn)m, (SEGn)m, wherein m and n are between 0-20.
• a peptidic linker is an amino acid sequence obtained, derived, or designed from an antibody hinge region sequence, a sequence linking a binding domain to a receptor, or a sequence linking a binding domain to a cell surface transmembrane region or membrane anchor.
• a peptidic linker has at least one cysteine capable of participating in at least one disulfide bond under physiological conditions or other standard peptide conditions (e.g., peptide purification conditions, conditions for peptide storage).
• a peptidic linker corresponding or similar to an immunoglobulin hinge peptide retains a cysteine that corresponds to the hinge cysteine disposed toward the amino-terminus of that hinge.
• a peptidic linker is from an IgG1 hinge and has been modified to remove any cysteine residues or is an IgG1 hinge that has one cysteine or two cysteines corresponding to hinge cysteines.
• a peptidic linker for use herein can comprise an “altered wild type immunoglobulin hinge region” or “altered immunoglobulin hinge region”.
• altered hinge regions refers to (a) a wild type immunoglobulin hinge region with up to 30 percent amino acid changes (e.g., up to 25 percent, 20 percent, 15 percent, 10 percent, or 5 percent amino acid substitutions or deletions), (b) a portion of a wild type immunoglobulin hinge region that is at least 10 amino acids (e.g., at least 12, 13, 14 or 15 amino acids) in length with up to 30 percent amino acid changes (e.g., up to 25 percent, 20 percent, 15 percent, 10 percent, or 5 percent amino acid substitutions or deletions), or (c) a portion of a wild type immunoglobulin hinge region that comprises the core hinge region (which portion can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length).
• one or more cysteine residues in a wild type immunoglobulin hinge region can be substituted by one or more other amino acid residues (e.g., one or more serine residues).
• An altered immunoglobulin hinge region can alternatively or additionally have a proline residue of a wild type immunoglobulin hinge region, such as an IgG1 hinge comprising the upper and core regions, substituted by another amino acid residue (e.g., a serine residue).
• hinge and linker sequences that can be used as connecting regions can be crafted from portions of cell surface receptors that connect Ig V -like or IgC-like domains. Regions between IgV-like domains where the cell surface receptor contains multiple Ig V -like domains in tandem and between IgC-like domains where the cell surface receptor contains multiple tandem IgC-like regions could also be used as connecting regions or linker peptides.
• hinge and linker sequences are from 5 to 60 amino acids long, and can be primarily flexible, but can also provide more rigid characteristics, can contain primarily a helical structure with minimal beta sheet structure.
• proteases described herein are expressed at higher amounts near a particular target cell of interest, e.g., the tumor microenvironment of target tumor cell, in vivo.
• a target of interest such as a particular tumor type, a particular tumor that expresses a particular tumor associated antigen
• the target tissue can be a cancerous tissue, particularly cancerous tissue of a solid tumor.
• the fusion protein comprises, from N terminus to C terminus, Ligand-Linker- VL, Receptor-Linker- VL, Ligand-Linker- VH, or Receptor-Linker- VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-cleavable Linker- VL, Receptor- cleavable Linker- VL, Ligand- cleavable Linker- VH, or Receptor- cleavable Linker- VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-linker (SEQ ID NO: 114)-VL, Receptor-linker (SEQ ID NO: 114)-VL, Ligand-linker (SEQ ID NO: 14)-VH, or Receptor-linker (SEQ ID NO: 14)-VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-linker (SEQ ID NO: 145)-VL, Receptor-linker (SEQ ID NO: 145)-VL, Ligand-linker (SEQ ID NO: 145)- VH, or Receptor-linker (SEQ ID NO: 145)-VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-linker (SEQ ID NO: 147)-VL, Receptor-linker (SEQ ID NO: 147)-VL, Ligand-linker (SEQ ID NO: 147)-VH, or Receptor-linker (SEQ ID NO: 147)-VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-linker (SEQ ID NO:154)-VL, Receptor-linker (SEQ ID NO:154)-VL, Ligand-linker (SEQ ID NO: 154)-VH, or Receptor-linker (SEQ ID NO: 154)-VH.
• the fusion protein comprises from N terminus to C terminus, Ligand-linker (SEQ ID NO:203)-VL, Receptor-linker (SEQ ID NO:203)-VL, Ligand-linker (SEQ ID NO:203)-VH, or Receptor-linker (SEQ ID NO:203)-VH.
• the fusion protein comprises, from N terminus to C terminus, Ligand-linker-Fc or Receptor-linker-Fc.
• the fusion protein comprises, from N terminus to C terminus, Ligand-cleavable linker-Fc or Receptor- cleavable linker-Fc.
• the fusion protein comprises, from N terminus to C terminus, Ligand-cleavable linker (SEQ ID NO:28)-Fc or Receptor- cleavable linker (SEQ ID NO:28)-Fc. [00242] In certain embodiments, the fusion protein comprises, from N terminus to C terminus, Ligand-linker-Fcl or Receptor-linker-Fc 1.
• the fusion protein comprises, from N terminus to C terminus, Ligand-cleavable Iinker-Fc2 or Receptor-cleavable Iinker-Fc2.
• Fcl and Fc2 can form heterodimers.
• Fcl is linked to a Ligand and Fc2 is linked to a Receptor.
• the linker connecting the Ligand with Fcl is cleavable and the linker connecting the Receptor with Fc2 is non- cleavable.
• the linker connecting the Ligand with Fcl is non- cleavable and the linker connecting the Receptor with Fc2 is cleavable.
• the linker connecting the Ligand with Fcl is cleavable and the linker connecting the Receptor with Fc2 is cleavable.
• the linker connecting the Ligand with Fcl is non- cleavable and the linker connecting the Receptor with Fc2 is non-cleavable.
• an antigen-binding domain of the fusion protein described herein specifically binds to a cell surface molecule.
• an antigen-binding domain of the fusion protein specifically binds to a tumor-associated antigen (TAA).
• TAA is any antigenic substance expressed on a tumor cell surface.
• an antigen- binding domain specifically and binds to a TAA selected from Fibroblast activation protein alpha (FAPa), Trophoblast glycoprotein (5T4), Tumor-associated calcium signal transducer 2 (Trop2), Fibronectin EDB (EDB-FN), fibronectin F.IIIB domain, CGS-2, EpCAM, EGER, HER-2, HER- 3, cMet, CEA, and FOLR1, EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, PSMA, CD38, BCMA, and CEA.
• FAPa Fibroblast activation protein alpha
• T4 Tumor-associated calcium signal transducer 2
• Trop2 Tumor-associated calcium signal transducer 2
• EDB-FN Fibronectin EDB
• F.IIIB domain CGS-2, EpCAM, EGER, HER-2, HER- 3, cMet, CEA
• an antigen-binding domain specifically binds to an immune checkpoint protein.
• immune checkpoint proteins include but are not limited to CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, 0X40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD80, CD40, CEACAMl, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, ID02, TDO, KIR, LAG-3, TIM-3, VISTA, CD47, or SIRP ⁇ .
• an antigen-binding domain specifically binds to an antigen expressed on a virally infected cell, bacterially infected cell, damaged red blood cell, arterial plaque cell, inflamed or fibrotic tissue cell.
• an antigen-binding domain specifically binds a cytokine receptor.
• cytokine receptors include, but are not limited to, Type I cytokine receptors, such as GM-CSF receptor, G-CSF receptor, Type I IL receptors, Epo receptor, LIF receptor, CNTF receptor, TPO receptor; Type II Cytokine receptors, such as IFN-alpha receptor (IFNARl, IFNAR2), IFB-beta receptor, IFN-gamma receptor (IFNGRl, IFNGR2), Type II IF receptors; chemokine receptors, such as CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, XC chemokine receptors; tumor necrosis receptor superfamily receptors, such as TNFRSF5/CD40, TNFRSF8/CD30, TNFRSF7/CD27, TNFRSF1A/TNFR1/CD 120a
• the antigen-binding domains of the fusion proteins described herein specifically bind to at least one molecule or target of interest in vivo.
• the target of interest is: Cluster of Differentiation 3 (CD3), Human Epidermal Growth Factor Receptor 2 (HER2), Epidermal Growth Factor Receptor (EGFR), Mesothelin (MSLN), Tissue Factor (TF), Cluster of Differentiation 19 (CD19), tyrosine-protein kinase Met (c-Met), Cluster of Differentiation 40 (CD40), Cadherin 3 (CDH3), or combinations thereof.
• the fusion protein comprises an antibody and at least one antigen binding domain of the antibody binds to an epitope on CD3, HER2, EGFR, MSLN, TF, CD 19, c-Met, CD40, CDH3, or combinations thereof.
• the target of interest is HER2
• the anti-HER2 paratope of the fusion protein has a VH having an amino acid sequence corresponding to SEQ ID NO: 120 and a VL having an amino acid sequence corresponding to SEQ ID NO: 124.
• the anti-HER2 paratope has a VH amino acid sequence that is substantially identical to SEQ ID NO: 120 and a VL amino acid sequence that is substantially identical to SEQ ID NO: 124.
• the anti-HER2 paratope has a VH amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 120 and a VL amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 124. In certain embodiments, the anti-HER2 paratope has a VH amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 120 and a VL amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 124.
• the anti-HER2 paratope comprises an scFv having an amino acid sequence corresponding to SEQ ID NO: 3.
• the anti- HER2 has a VH having 3 CDRS, HCDR1, HDR2 and HCDR3 having amino acid sequences corresponding to SEQ ID NOS: 121, 122 and 123 respectively, and a VL having 3 CDRs LCDR1, LCDR2 and LCDR3 having amino acid sequences corresponding to SEQ ID NOS: 125, 126 and 127 respectively.
• the target of interest is EGFR
• the anti-EGFR paratope of the fusion protein has a VH having an amino acid sequence corresponding to SEQ ID NO: 14 and a VL having an amino acid sequence corresponding to SEQ ID NO: 13.
• the anti- EGFR paratope has a VH amino acid sequence that is substantially identical to SEQ ID NO: 14 and a VL amino acid sequence that is substantially identical to SEQ ID NO: 13.
• the anti-EGFR paratope has a VH amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 14 and a VL amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 13. In certain embodiments, the anti-EGFR paratope has a VH amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 14 and a VL amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 13.
• the anti-EGFR has a VH having 3 CDRS, HCDR1, HDR2 and HCDR3 having amino acid sequences corresponding to SEQ ID NOS: 84, 85 and 86 respectively, and a VL having 3 CDRs LCDR1, LCDR2 and LCDR3 having amino acid sequences corresponding to SEQ ID NOS: 59, 60 and 61 respectively.
• the target of interest is MSLN
• the anti- MSLN paratope of the fusion protein has a VH having an amino acid sequence corresponding to SEQ ID NO: 16 and a VL having an amino acid sequence corresponding to SEQ ID NO: 15.
• the anti- MSLN paratope has a VH amino acid sequence that is substantially identical to SEQ ID NO: 16 and a VL amino acid sequence that is substantially identical to SEQ ID NO: 15.
• the anti-MSLN paratope has a VH amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 16 and a VL amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 15. In certain embodiments, the anti- MSLN paratope has a VH amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 16 and a VL amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 15.
• the anti-MSLN has a VH having 3 CDRS, HCDR1, HDR2 and HCDR3 having amino acid sequences corresponding to SEQ ID NOS: 69, 70 and 71 respectively, and a VL having 3 CDRs LCDR1, LCDR2 and LCDR3 having amino acid sequences corresponding to SEQ ID NOS: 74, 75 and 76 respectively.
• the target of interest is TF (Tissue Factor)
• the anti- TF paratope of the fusion protein has a VH having an amino acid sequence corresponding to SEQ ID NO: 18 and a VL having an amino acid sequence corresponding to SEQ ID NO: 17.
• the anti- TF paratope has a VH amino acid sequence that is substantially identical to SEQ ID NO: 18 and a VL amino acid sequence that is substantially identical to SEQ ID NO: 17. In certain embodiments, the anti-TF paratope has a VH amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 18 and a VL amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 17.
• the anti- TF paratope has a VH amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 18 and a VL amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 17.
• the anti-TF has a VH having 3 CDRS, HCDR1, HDR2 and HCDR3 having amino acid sequences corresponding to SEQ ID NOS: 54, 55 and 56 respectively, and a VL having 3 CDRs LCDR1, LCDR2 and LCDR3 having amino acid sequences corresponding to SEQ ID NOS: 48, 49 and 50 respectively.
• the target of interest is CD 19 and the anti-CD 19 paratope of the fusion protein has a VH having an amino acid sequence corresponding to SEQ ID NO: 20 and a VL having an amino acid sequence corresponding to SEQ ID NO: 19
• the anti- CD 19 paratope has a VH amino acid sequence that is substantially identical to SEQ ID NO: 20 and a VL amino acid sequence that is substantially identical to SEQ ID NO: 19.
• the anti-CD 19 paratope has a VH amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 20 and a VL amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 19.
• the anti-CD 19 paratope has a VH amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 20 and a VL amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 19.
• the anti-CD 19 has a VH having 3 CDRS, HCDR1, HDR2 and HCDR3 having amino acid sequences corresponding to SEQ ID NOS: 64, 65 and 66 respectively, and a VL having 3 CDRs LCDR1, LCDR2 and LCDR3 having amino acid sequences corresponding to SEQ ID NOS: 74, 75 and 165 respectively.
• the target of interest is c-Met and the anti-c-Met paratope of the fusion protein has a VH having an amino acid sequence corresponding to SEQ ID NO: 22 and a VL having an amino acid sequence corresponding to SEQ ID NO: 21.
• the anti- c-Met paratope has a VH amino acid sequence that is substantially identical to SEQ ID NO: 22 and a VL amino acid sequence that is substantially identical to SEQ ID NO: 21.
• the anti-c-Met paratope has a VH amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 22 and a VL amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 21.
• the anti-c-Met paratope has a VH amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 22 and a VL amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 21.
• the anti- c-Met has a VH having 3 CDRS, HCDR1, HDR2 and HCDR3 having amino acid sequences corresponding to SEQ ID NOS: 99,100 and 101 respectively, and a VL having 3 CDRs LCDR1, LCDR2 and LCDR3 having amino acid sequences corresponding to SEQ ID NOS: 94, 95 and 96 respectively.
• the target of interest is CDH3 and the anti-CDH3 paratope of the fusion protein has a VH having an amino acid sequence corresponding to SEQ ID NO: 24 and a VL having an amino acid sequence corresponding to SEQ ID NO: 23.
• the anti- CDH3 paratope has a VH amino acid sequence that is substantially identical to SEQ ID NO: 24 and a VL amino acid sequence that is substantially identical to SEQ ID NO: 23.
• the anti- CDH3 paratope has a VH amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 24 and a VL amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 23.
• the anti- CDH3 paratope has a VH amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 24 and a VL amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 23.
• the anti-CDH3 has a VH having 3 CDRS, HCDR1, HDR2 and HCDR3 having amino acid sequences corresponding to SEQ ID NOS: 89, 90 and 91 respectively, and a VL having 3 CDRs LCDR1, LCDR2 and LCDR3 having amino acid sequences corresponding to SEQ ID NOS: 94, 95 and 96 respectively.
• the target of interest is CD40 and the anti-CD40 paratope of the fusion protein has a VH having an amino acid sequence corresponding to SEQ ID NO: 172 and a VL having an amino acid sequence corresponding to SEQ ID NO: 177.
• the anti-CD40 paratope has a VH amino acid sequence that is substantially identical to SEQ ID NO: 172 and a VL amino acid sequence that is substantially identical to SEQ ID NO: 177.
• the anti-CD40 paratope has a VH amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 172 and a VL amino acid sequence that is about 80%, about 85%, about 90%, or about 95% identical to SEQ ID NO: 177. In certain embodiments, the anti-CD40 paratope has a VH amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 172 and a VL amino acid sequence that is about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 177.
• the anti-CD40 has a VH having 3 CDRS, HCDR1, HDR2 and HCDR3 having amino acid sequences corresponding to SEQ ID NOS: 173, 174 and 175 respectively, and a VL having 3 CDRs LCDR1, LCDR2 and LCDR3 having amino acid sequences corresponding to SEQ ID NOS: 178, 179 and 180 respectively.
• an antigen-binding domain of the fusion protein binds specifically to a molecule, e.g., a polypeptide, on an immune cell.
• the fusion protein comprises an antigen-binding domain that binds specifically to both a TAA and an antigen-binding domain that specifically binds to a molecule, e.g., a polypeptide, on an immune cell.
• the fusion protein binds to both a tumor cell and an immune cell.
• the immune cell is a T cell.
• the immune cell is a macrophage, a dendritic cell, a neutrophil, a B-cell or an NK cell.
• the fusion protein binds to a CD3 antigen on a T cell and one or more TAAs on a tumor cell.
• a T cell engager is a polypeptide construct, often a bispecific antibody, that simultaneously binds a TAA on a tumor cell and CD3 epitope on a T-cell to form a TCR- independent artificial immune synapse. This causes the T cell to become activated and to exert a cytotoxic effect on the tumor cell.
• Bi-specific antibodies capable of targeting T cells to tumor cells have been identified and tested for their efficacy in the treatment of cancers.
• Blinatumomab is an example of a bi-specific anti-CD3-CD19 antibody in a format called BiTETM (Bi-specific T-cell Engager) that has been identified for the treatment of B-cell diseases such as relapsed B-cell non- Hodgkin lymphoma and chronic lymphocytic leukemia (Baeuerle et al (2009) Cancer Research 12:4941-4944) and is FDA approved.
• BiTETM Bi-specific T-cell Engager
• T cell engagers directed against other tumor- associated target antigens have also been made, and several have entered clinical trials: AMG110/MT110 EpCAM for lung cancer, gastric cancer and colorectal cancer; AMG21 1/MEDI565 CEA for gastrointestinal adenocarcinoma; and AMG 212 / BAY2010112 PSMA for prostate cancer (see Surubowa, C. M. et al, Oncoimmunology. 2015 Jun; 4(6): el008339). While these studies showed promising clinical efficacy, they were also hampered by severe dose-limiting toxicities primarily due to cytokine release syndrome (CRS). This resulted in narrow therapeutic windows. The use of masked T cell-binding paratopes which are activated primarily in a tumor microenvironment might reduce the toxicity of TCEs.
• CRS cytokine release syndrome
• the fusion protein binds a CD3 antigen on a T cell and a TAA on a tumor cell. In certain embodiments the fusion protein binds a CD3 antigen on a T cell, a TAA on a tumor cell and an IgSF extracellular domain on a tumor cell. In certain embodiments, the fusion protein binds a CD3 antigen on a T cell, a TAA on a tumor cell and an IgSF extracellular domain on the T cell.
• a fusion protein becomes unmasked by a protease in a tumor microenvironment, and binds to a TAA on a tumor cell and a CD3 antigen on a T cell, causing bridging of the T cell and the tumor cell, as is demonstrated in Example 20.
• an unmasked fusion protein binds a CD3 antigen on a T cell, and both a TAA and a IgSF ligand on a tumor cell, as is illustrated in Figure 31.
• the binding of the IgSF ligand (e.g. PD-L1) on the tumor cell prevents the binding of its IgSF receptor (e.g. PD-1) on the T cell, thus blocking checkpoint inhibition (Figure 31 C).
• the fusion proteins comprise an anti-CD3 paratope VH and a VL substantially identical to those of the paratopes shown in Table BB.
• the CD3 paratope comprises VH and VL amino acid sequences of:
• VH comprising an amino acid sequence corresponding to SEQ ID NO: 2 and a
• VL comprising an amino acid sequence according to SEQ ID NO: 1;
• VH comprising an amino acid sequence corresponding to SEQ ID NO: 206 and a
• VL comprising an amino acid sequence according to SEQ ID NO: 210;
• VH comprising an amino acid sequence corresponding to SEQ ID NO: 215 and a
• VL comprising an amino acid sequence according to SEQ ID NO: 219;
• VH comprising an amino acid sequence corresponding to SEQ ID NO: 223 and a
• VL comprising an amino acid sequence according to SEQ ID NO: 227;
• VH comprising an amino acid sequence corresponding to SEQ ID NO: 231 and a
• VL comprising an amino acid sequence according to SEQ ID NO: 235; or
• VH comprising an amino acid sequence corresponding to SEQ ID NO: 239 and a
• VL comprising an amino acid sequence according to SEQ ID NO: 243.
• the CD3 paratope comprises VH and VL that are about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to:
• VH comprising an amino acid sequence corresponding to SEQ ID NO: 2 and a
• VL comprising an amino acid sequence according to SEQ ID NO: 1; [00272] (b) a VH comprising an amino acid sequence corresponding to SEQ ID NO: 206 and a
• VL comprising an amino acid sequence according to SEQ ID NO: 210;
• VH comprising an amino acid sequence corresponding to SEQ ID NO: 215 and a
• VL comprising an amino acid sequence according to SEQ ID NO: 219;
• VH comprising an amino acid sequence corresponding to SEQ ID NO: 223 and a VL comprising an amino acid sequence according to SEQ ID NO: 227;
• VH comprising an amino acid sequence corresponding to SEQ ID NO: 231 and a VL comprising an amino acid sequence according to SEQ ID NO: 235; or
• VH comprising an amino acid sequence corresponding to SEQ ID NO: 239 and a VL comprising an amino acid sequence according to SEQ ID NO: 243.
• the anti-CD3 paratope comprises a VH comprising 3 heavy chain CDRs HCDR1, HCDR2 and HCDR3 comprising amino acid sequences corresponding to SEQ ID NOS: 207, 208 and 209, and a VL comprising 3 light chain CDRs LCDR1, LCDR2 and LCDR3 comprising amino acid sequences corresponding to SEQ ID NOS: 211, 212 and 214.
• the anti-CD3 paratope comprises a VH comprising 3 heavy chain CDRs HCDR1, HCDR2 and HCDR3 comprising amino acid sequences corresponding to SEQ ID NOS: 224, 225 and 226, and a VL comprising 3 light chain CDRS LCDR1, LCDR2 and LCDR3 comprising amino acid sequences corresponding to SEQ ID NOS: 228, 229 and 230.
• the anti-CD3 paratope comprises a VH comprising 3 heavy chain CDRs HCDR1, HCDR2 and HCDR3 comprising amino acid sequences corresponding to SEQ ID NOS: 232, 233 and 234, and a VL comprising 3 light chain CDRS LCDR1, LCDR2 and LCDR3 comprising amino acid sequences corresponding to SEQ ID NOS: 236, 237 and 238.
• the anti-CD3 paratope comprises a VH comprising 3 heavy chain CDRs HCDR1, HCDR2 and HCDR3 comprising amino acid sequences corresponding to SEQ ID NOS: 240, 241 and 242, and a VL comprising 3 light chain CDRS LCDR1, LCDR2 and LCDR3 comprising amino acid sequences corresponding to SEQ ID NOS: 244, 245 and 246.
• VH comprising 3 heavy chain CDRs HCDR1, HCDR2 and HCDR3 comprising amino acid sequences corresponding to SEQ ID NOS: 240, 241 and 242
• VL comprising 3 light chain CDRS LCDR1, LCDR2 and LCDR3 comprising amino acid sequences corresponding to SEQ ID NOS: 244, 245 and 246.
• the fusion protein can be included in a chimeric antigen receptor (CAR) or a CAR fragment.
• a CAR can comprise one or more extracellular ligand binding domains, optionally a hinge region, a transmembrane region, and an intracellular signaling region.
• the one or more extracellular ligand binding domains can include one or more fusion proteins.
• the extracellular ligand binding domain can typically comprises a single-chain immunoglobulin variable fragment (scFv) or other ligand binding domain, such as a Fab or a natural protein ligand.
• the hinge region can generally comprise a polypeptide hinge of variable length such as one or more amino acids, a CD8 alpha hinge region or an IgG4 region (or others), and combinations thereof.
• the transmembrane domain can typically include a transmembrane region derived from CD8 alpha, CD28, or other transmembrane proteins such as DAP 10, DAP 12, or NKG2D, and combinations thereof.
• the intracellular signaling region can include one or more intracellular signaling domains such as CD28, 4- IBB, CD3 zeta, 0X40, 2B4, or other intracellular signaling domains, and combinations thereof.
• the one or more intracellular signaling domains can include CD28 and CD3 zeta, 4- IBB and CD3 zeta, or CD3 zeta.
• Lymphocytes such as T cells and NK cells can be modified to produce chimeric antigen receptor cells (e.g., CAR-Ts).
• CAR-T cells can recognize specific soluble antigens or antigens on a target cell surface, such as a tumor cell surface, or on cells in the tumor microenvironment.
• the extracellular ligand binding domain binds to a cognate ligand, the intracellular signaling domain of the CAR can activate the lymphocyte. See, e.g., Brudno et al., Nature Rev. Clin. Oncol.
• a CAR construct that comprises a ligand receptor pair construct as described herein.
• the CAR construct comprises an scFv that can be fused to the ligand receptor pair construct.
• the ligand receptor pair construct is a single chain ligand receptor pair construct that can be fused to the N-terminus of the scFv with or without a linker.
• the single chain ligand receptor pair construct comprises a protease cleavable linker.
• the receptor is fused with or without a first linker to the N-terminus of the scFv and the ligand is internally fused to a second linker connecting the heavy chain and the light chain of the scFv.
• the linkers comprise a protease cleavage site cleavable by a protease.
• the ligand is fused with or without a first linker to the N-terminus of the scFv and the receptor is internally fused to a second linker connecting the heavy chain and the light chain of the scFv.
• the first linker is cleavable and the second linker is uncleavable by a protease.
• a T-cell can be modified to express a ligand receptor pair CAR.
• Certain embodiments of the present disclosure relate to an isolated polynucleotide or a set of polynucleotides encoding a fusion protein described herein.
• a polynucleotide in this context can encode all or part of a fusion protein.
• nucleic acid refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
• polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
• a polynucleotide that “encodes” a given polypeptide is a polynucleotide that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences.
• the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
• a transcription termination sequence can be located 3' to the coding sequence.
• the present disclosure relates to polynucleotide and polypeptide sequences that are identical or substantially identical to a polypeptide encoding at least a portion of a fusion protein described herein, e.g., a first or second polypeptide of a biologically functional protein.
• a polypeptide encoding at least a portion of a fusion protein described herein e.g., a first or second polypeptide of a biologically functional protein.
• the term “identical” in the context of two or more polynucleotide or polypeptide sequences refers to two or more sequences or subsequences that are the same.
• Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (for example, about 80%, about 85%, about 90%, or about 95% identity over a specified region) when compared and aligned for maximum correspondence over a comparison window or over a designated region as measured using one of the commonly used sequence comparison algorithms as known to persons of ordinary skill in the art or by manual alignment and visual inspection. This definition also refers to the complement of a test polynucleotide sequence.
• the identity can exist over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 75-100 amino acids or nucleotides in length, or, where not specified, across the entire sequence of a polypeptide or polynucleotide.
• sequence comparison typically test sequences are compared to a designated reference sequence.
• test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
• the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
• Comparison window refers to a segment of a sequence comprising contiguous amino acid or nucleotide positions which can be from 20 to 1000 contiguous amino acid or nucleotide positions, for example from about 50 to about 600 or from about 100 to about 300 or from about 150 to about 200 contiguous amino acid or nucleotide positions over which a test sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Longer segments up to and including the full-length sequence may also be used as a comparison window in certain embodiments. Methods of alignment of sequences for comparison are known to those of ordinary skill in the art.
• Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c; by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci.
• amino acid substitutions are conservative substitutions.
• a “conservative substitution” is considered to be a substitution of one amino acid with another amino acid having similar physical, chemical and/or structural properties. Common conservative substitutions are listed under Column 1 of Table 4.
• One skilled in the art will appreciate that the main factors in determining what constitutes a conservative substitution are usually the size of the amino acid side chain and its physical/chemical properties, but that certain environments allow for substitution of a given amino acid with a broader range of amino acids than those listed in Column 1.
• fusion proteins described herein can be produced using standard recombinant methods known in the art (see, for example, U.S. Patent No. 4,816,567 and “ Antibodies : A Laboratory Manual ,” 2 nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). Vectors encodins fusion proteins
• a polynucleotide or set of polynucleotides encoding the fusion protein is generated and inserted into one or more vectors for further cloning and/or expression in a host cell.
• Polynucleotide(s) encoding the fusion protein can be produced by standard methods known in the art (see, for example, Ausubel et al, Current Protocols in Molecular Biology , John Wiley & Sons, New York, 1994 & update, and “ Antibodies : A Laboratory Manual ,” 2 nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).
• the number of polynucleotides required for expression of the fusion protein will be dependent on the format of the fusion protein, including whether or not the fusion protein comprises an antibody and the number of polypeptides within the fusion protein. For example, when a fusion protein comprises two polypeptide chains, two polynucleotides each encoding one polypeptide chain will be required. Similarly, in certain embodiments, when the fusion protein comprises a biologically functional protein in a mAb format, two polynucleotides each encoding one polypeptide chain are required. When multiple polynucleotides are required, they can be incorporated into one vector or into more than one vector.
• the polynucleotide or set of polynucleotides is incorporated into an expression vector together with one or more regulatory elements, such as transcriptional elements, which are required for efficient transcription of the polynucleotide.
• regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals.
• the expression vector can optionally further contain heterologous nucleic acid sequences that facilitate expression or purification of the expressed protein.
• the expression vector can be an extrachromosomal vector or an integrating vector.
• vectors such as expression vectors
• the polynucleotide(s) can be comprised by a single vector or by more than one vector.
• the polynucleotides are comprised by a multi cistronic vector.
• Expression vectors to be used to express polynucleotides include, but are not limited to, pTT5 and pUC15, Cells comprising vectors encoding fusion proteins.
• Suitable host cells for cloning or expression of the fusion protein polypeptides include various prokaryotic or eukaryotic cells as known in the art.
• Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells and yeast cells (such as Saccharomyces or Pichia cells).
• Prokaryotic host cells include, for example, E. coli, A. salmonicida or B. subtilis cells.
• the fusion proteins are produced in bacteria, in particular when glycosylation and Fc effector function are not needed, as described for example in U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523, and in Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003.
• Eukaryotic microbes such as filamentous fungi or yeast are suitable expression host cells in certain embodiments, in particular fungi and yeast strains whose glycosylation pathways have been “humanized” resulting in the production of an antibody with a partially or fully human glycosylation pattern (see, for example, Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al ., 2006, Nat. Biotech. 24:210-215).
• Suitable host cells for the expression of glycosylated fusion proteins are usually eukaryotic cells.
• U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 describe PLANTIBODIESTM technology for producing antibodies in transgenic plants.
• Mammalian cell lines adapted to grow in suspension are particularly useful for expression of fusion proteins. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney (HEK) line 293 or 293 cells (see, for example, Graham et al, 1977, J.
• MRC 5 cells including FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR CHO cells, see Urlaub et al, 1980, Proc Natl Acad Sci USA, 77:4216), and myeloma cell lines (such as Y0, NS0 and Sp2/0).
• CHO Chinese hamster ovary
• myeloma cell lines such as Y0, NS0 and Sp2/0.
• Exemplary mammalian host cell lines suitable for production of antibodies are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003).
• the host cell is a transient or stable higher eukaryotic cell line, such as a mammalian cell line.
• the host cell is a mammalian HEK293T, CHO, HeLa, NS0 or COS cell.
• the host cell is a stable cell line that allows for mature glycosylation of the fusion protein.
• the host cells comprising the expression vector(s) encoding the fusion protein can be cultured using routine methods to produce the fusion protein.
• host cells comprising the expression vector(s) encoding the fusion protein can be used therapeutically or prophylactically to deliver the fusion protein to a subject, or polynucleotides or expression vectors can be administered to a cell from a subject ex vivo and the cell then returned to the body of the subject.
• a host cell comprises (for example, has been transformed with) a vector comprising a polynucleotide that encodes the VL of a binding domain described herein and the VH of the binding domain.
• a host cell comprises a first vector comprising a polynucleotide that encodes the VL of a binding domain described herein and a second vector comprising a polynucleotide that encodes the corresponding VH of the binding domain.
• the host cell is eukaryotic, for example, a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g.
• the host cell is Expi293TM (Thermo Fisher, Waltham, MA). In certain embodiments, the host cell is CHO-S cells (National Research Council Canada) or HEK293 cells.
• Certain embodiments of the present disclosure relate to a method of making a fusion protein comprising culturing a host cell into which one or more polynucleotides encoding the fusion protein, or one or more expression vectors encoding the fusion protein, have been introduced, under conditions suitable for expression of the fusion protein, and optionally recovering the fusion protein from the host cell (or from host cell culture medium).
• Cell culture media that can be used include, but are not limited to, DMEM (Thermo Fisher, Waltham, MA), Opti-MEMTM (Thermo Fisher, Waltham, MA), Opti-MEMTM I Reduced Serum Medium (Thermo Fisher, Waltham, MA), RPMI-1640 medium, Expi293TM Expression Medium (Thermo Fisher, Waltham, MA), and FreeStyle CHO expression medium (Thermo Fisher Scientific, Waltham, MA).
• DMEM Thermo Fisher, Waltham, MA
• Opti-MEMTM Thermo Fisher, Waltham, MA
• Opti-MEMTM I Reduced Serum Medium Thermo Fisher, Waltham, MA
• RPMI-1640 medium Expi293TM Expression Medium
• Expi293TM Expression Medium Thermo Fisher, Waltham, MA
• FreeStyle CHO expression medium Thermo Fisher Scientific, Waltham, MA.
• the cell culture medium can be supplemented with serum, e.g., fetal bovine serum (FBS), amino acids, e.g., L-glutamine, antibiotics, e.g., penicillin, and streptomycin, and/or antimycotics, e.g., amphotericin, or any other supplements routinely used in the to support cell culture.
• serum e.g., fetal bovine serum (FBS)
• amino acids e.g., L-glutamine
• antibiotics e.g., penicillin, and streptomycin
• antimycotics e.g., amphotericin
• the fusion proteins are purified after expression.
• Proteins can be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice , 3 rd Ed., Scopes, Springer-Verlag, NY, 1994).
• Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reverse-phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC.
• Additional purification methods include electrophoretic, immunological, precipitation, dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful.
• the bacterial proteins A and G bind to the Fc region.
• the bacterial protein L binds to the Fab region of some antibodies.
• Purification can also be enabled by a particular fusion partner.
• antibodies can be purified using glutathione resin if a GST fusion is employed, Ni +2 affinity chromatography if a His-tag is employed, or immobilized anti-flag antibody if a flag-tag is used. The degree of purification necessary will vary depending on the use of the antibodies. In some instances, no purification can be necessary.
• fusion proteins are substantially pure.
• the term “substantially pure” (or “substantially purified”) when used in reference to a fusion protein described herein, means that the fusion protein is substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, such as a native cell, or a host cell in the case of recombinantly produced fusion protein.
• a fusion protein that is substantially pure is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating protein.
• Assessment of protein purification and/or homogeneity can be performed by any method known in the art, including, but not limited to, non-reducing/reducing CE-SDS, non- reducing/reducing SDS-PAGE, Ultra-high performance liquid chromatography-size exclusion chromatography (UPLC-SEC), High Performance Liquid Chromoatography (HPLC), mass spectrometry, multi angle light scattering (MALS), dynamic light scattering (DLS).
• UPLC-SEC Ultra-high performance liquid chromatography-size exclusion chromatography
• HPLC High Performance Liquid Chromoatography
• MALS multi angle light scattering
• DLS dynamic light scattering
• the fusion proteins described herein comprise one or more post-translational modifications. Such post-translational modifications can occur in vivo , or they be conducted in vitro after isolation of the fusion protein from the host cell.
• Post-translational modifications include various modifications as are known in the art (see, for example, Proteins - Structure andMolecular Properties , 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12, 1983; Seifter et al, 1990, Meth. Enzymol ., 182:626-646, and Rattan et al, 1992, Ann. N.Y. Acad. Sci ., 663:48-62).
• the fusion proteins can comprise the same type of modification at one or several sites, or it can comprise different modifications at different sites.
• post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, formylation, oxidation, reduction, proteolytic cleavage or specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease orNaBEE.
• post-translational modifications include, for example, addition or removal of N-linked or O-linked carbohydrate chains, chemical modifications of N-linked or O- linked carbohydrate chains, processing of N-terminal or C-terminal ends, attachment of chemical moieties to the amino acid backbone, and addition or deletion of an N-terminal methionine residue resulting from prokaryotic host cell expression.
• Post-translational modifications can also include modification with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
• suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase and acetylcholinesterase.
• suitable prosthetic group complexes include, but are not limited to, streptavidin/biotin and avidin/biotin.
• suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin.
• luminescent material is luminol
• bioluminescent materials include luciferase, luciferin and aequorin
• radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon and fluorine.
• post-translational modifications include acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxyl ati on, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
• the fusion proteins are masked from engaging their intended target(s).
• the extent to which binding of the fusion proteins to their target(s) is decreased may be measured by standard techniques such as enzyme-linked immunosorbent assay (ELISA,), bio-layer interferometery (BLI), surface plasmon resonance (SPR), fluorescence-activated cell sorting (FACS), flow cytometry, kinetic exclusion assay (KinExA), meso scale discovey (MSD)), microfluidics, or isothermal titration calorimetry (ITC).
• the fusion protein comprises an antigen-binding domain that is masked by the ligand-receptor pair and binding of the antigen-binding domain to its cognate antigen is decreased by at least 3 -fold as compared to a corresponding unmasked anti gen -binding domain, for example, binding of the antigen-binding domain to its cognate antigen is decreased by at least 5-fold, at least 10-fold, at least 20-fold, at least 25-fold or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 70-fold or at least 80-fold, or at least 90-fold or at least 100-fold, or at least 200-fold or at least 400-fold, or at least 600-fold or at least 800-fold or at least 1000-fold or at least 2000-fold or at least 5000-fold or at least 10,000-fold.
• protease cleavage of at least one of the peptidic linkers between the ligand or receptor of the ligand-receptor pair and the biologically functional protein unmasks (activates) the fusion protein such that it can bind its intended target(s).
• the susceptibility of the peptidic linker to cleavage may be tested in vitro by standard techniques including those described in the Examples herein.
• the extent to which binding of the fusion protein to its target(s) is recovered after protease cleavage may also be tested by standard techniques such as enzyme-linked immunosorbent assay (ELISA), bio-layer interferometery (BLI), surface plasmon resonance (SPR), fluorescence-activated cell sorting (FACS), flow cytometry, kinetic exclusion assay (KinExA), meso scale discovey (MSD), microfluidics, or isothermal titration calorimetry (ITC).
• ELISA enzyme-linked immunosorbent assay
• BBI bio-layer interferometery
• SPR surface plasmon resonance
• FACS fluorescence-activated cell sorting
• flow cytometry kinetic exclusion assay
• KinExA kinetic exclusion assay
• MSD meso scale discovey
• microfluidics microfluidics
• ITC isothermal titration calorimetry
• Partial recovery of binding is defined as measurable binding of the relevant domain of the fusion protein (e.g., ligand, receptor or antigen-binding domain) to its intended target and may be, for example, between 100-fold and 2-fold less than binding of the parental domain. Partial recovery may be about 100-fold, 75-fold, 50-fold, 25-fold, 10-fold, 5-fold- or 2-fold less than the binding of the parental domain.
• the relevant domain of the fusion protein e.g., ligand, receptor or antigen-binding domain
• the present disclosure includes methods for the treatment of a disease or condition comprising administration of a fusion protein described herein to a subject in need thereof.
• the subject is a mammal. In certain embodiments, the subject is human.
• the methods disclosed herein are for the treatment of cancer.
• Cancers can include, but are not limited to, hematologic neoplasms (including leukemias, myelomas and lymphomas), carcinomas (including adenocarcinomas and squamous cell carcinomas), melanomas and sarcomas. Carcinomas and sarcomas are also frequently referred to as “solid tumors”.
• the cancer is a solid tumor.
• the cancer is leukemia.
• the cancer is lymphoma.
• the fusion protein can exert either a cytotoxic or cytostatic effect and can result in one or more of a reduction in the size of a tumor, the slowing or prevention of an increase in the size of a tumor, an increase in the disease-free survival time between the disappearance or removal of a tumor and its reappearance, prevention of an initial or subsequent occurrence of a tumor (for example, metastasis), an increase in the time to progression, reduction of one or more adverse symptom associated with a tumor, or an increase in the overall survival time of a subject having a tumor.
• a cytotoxic or cytostatic effect can result in one or more of a reduction in the size of a tumor, the slowing or prevention of an increase in the size of a tumor, an increase in the disease-free survival time between the disappearance or removal of a tumor and its reappearance, prevention of an initial or subsequent occurrence of a tumor (for example, metastasis), an increase in the time to progression, reduction of one or more adverse symptom associated with a tumor, or
• the methods disclosed herein are for the treatment of an immunodeficiency disorder or disease.
• the methods disclosed herein are for the treatment of auto- immune diseases or conditions.
• the methods described herein comprise administering a fusion protein described herein to a subject in need thereof.
• the fusion protein can be administered to a subject by an appropriate route of administration.
• the route and/or mode of administration will vary depending upon the desired results.
• immunotherapeutic antibodies are administered by systemic administration or local administration. Local administration can be at the site of a tumor or into a tumor draining lymph node.
• the fusion proteins will be administered by parenteral administration, for example, by intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous or spinal administration, such as by injection or infusion.
• Treatment is achieved by administration of a “therapeutically effective amount” of the fusion protein.
• a “therapeutically effective amount” refers to an amount that is effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
• a therapeutically effective amount can vary according to factors such as the disease state, age, sex, and weight of the subject.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the fusion protein are outweighed by the therapeutically beneficial effects.
• “Sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate an immune response to a target cell or tissue, e.g., by immunomodulatory ligand-receptor binding to an immune cell.
• a suitable dosage of the fusion protein can be determined by a skilled medical practitioner.
• the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular fusion protein employed, the route of administration, the time of administration, the rate of excretion of the polypeptide, the duration of the treatment, other drugs, compounds and/or materials used in combination with the fusion protein, e.g., anti-cancer agents, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.
• the fusion proteins described herein are administered to a subject in need thereof, for example a subject having cancer, in order to modulate the immune system of the subject.
• the fusion proteins described herein downregulate an immune response or upregulate an immune response.
• administration of a sufficient amount of fusion protein to the subject can effect one or more of the following to activate or upregulate an immune response: modulation of an immune checkpoint, modulation of T-cell receptor signaling, modulation of T-cell activation, modulation of pro-inflammatory cytokines, modulation of interferon- ⁇ production by T cells, modulation of T-cell suppression, modulation of M2 -type tumor associated macrophages (TAM) or myeloid-derived suppressor cell (MDSC) survival and/or differentiation, and/or modulation of cytotoxic or cytostatic effects on cells.
• TAM tumor associated macrophages
• MDSC myeloid-derived suppressor cell
• kits for modulating an immune response comprising inhibition of an immune checkpoint, stimulation of an immune checkpoint, immune cell activation, stimulation of T-cell receptor signaling, and stimulation of antibody- dependent cellular cytotoxicity (ADCC), T cell-dependent cytotoxicity (TDCC)), Cell-dependent cytotoxicity (CDC), or antibody-dependent cellular phagocytosis (ADCP).
• ADCC antibody-dependent cellular cytotoxicity
• TDCC T cell-dependent cytotoxicity
• CDC Cell-dependent cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• the fusion protein when activated by a protease, is capable of agonizing a target leukocyte costimulatory receptor.
• Functional effects of leukocyte costimulatory receptor agonism include activation of T effector cells, differentiation and activation of inflammatory myeloid cells and/or recruitment of B cells and/or NKT cells.
• T effector cells can result in increased production of one or more cytokines by the T cells, such as interferon gamma (IFN- ⁇ ), interleukin-2 (IL-2), interleukin- 12 (IL- 12), interleukin- 17 (IL-17), interleukin-21 (IL-21), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor- ⁇ (TNF- ⁇ ), macrophage inflammatory protein 1 ⁇ (MIP-1 ⁇ ) and/or C-X-C motif ligand 13 (CXCL13).
• IFN- ⁇ interferon gamma
• IL-2 interleukin-2
• IL-17 interleukin- 17
• IL-21 interleukin-21
• GM-CSF granulocyte-macrophage colony-stimulating factor
• TNF- ⁇ tumor necrosis factor- ⁇
• MIP-1 ⁇ macrophage inflammatory protein 1 ⁇
• CXCL13 C-X-C motif
• Increased production of IL-21 and CXCL13 by T effector cells may, for example, support the differentiation and activation of inflammatory myeloid cells in the TME, recruit anti -tumor lymphoid cells such as B and NKT cells and/or support the formation of tertiary lymphoid structures.
• the fusion protein activates T effector cells.
• the fusion protein increases production of GM-CSF, TNF- ⁇ , MIP-1 ⁇ , IL-17, IL- 12, IL-21 and/or C-X-C motif ligand 13 (CXCL13) by T effector cells.
• the fusion protein decreases CSF1 -dependent viability of monocytes and activate T effector cells.
• Certain embodiments of the present disclosure relate to methods of using the fusion proteins to modulate leukocyte costimulatory receptor agonism in vivo , for example, in order to treat cancer.
• the methods relate to inhibition or downregulation of an immune cell or immune response, e.g., for treating an autoimmune disease or disorder.
• the fusion protein is administered in a sufficient amount to modulate an immune cell.
• the downregulation of an immune response is by modulation of an immune checkpoint, modulation of T-cell receptor signaling, modulation of T cell activation, modulation of pro-inflammatory cytokines, modulation of interferon-g production by T cells, modulation of T cell suppression, modulation of M2 -type tumor associated macrophages (TAM) or myeloid-derived suppressor cell (MDSC) survival and/or differentiation, and/or modulation of cytotoxic or cytostatic effects on cells.
• TAM tumor associated macrophages
• MDSC myeloid-derived suppressor cell
• the fusion proteins described herein induce antibody dependent cell-mediated cytotoxicity (ADCC), which in turn results in increased lysis of the target cell.
• the fusion protein comprises an Fc region with increased binding affinity of the Fc for Fc ⁇ RIIIa (an activating receptor) resulting in increased antibody dependent cell-mediated cytotoxicity (ADCC) and increased lysis of the target cell.
• the Fc region is with modified CH2 domains comprising amino acid modifications that result in increased binding affinity of the Fc for Fc ⁇ RIIIa (an activating receptor) resulting in increased antibody dependent cell-mediated cytotoxicity (ADCC).
• fusion proteins described herein reduce antibody dependent cell-mediated cytotoxicity (ADCC). In certain indications, a decrease in, or elimination of, ADCC and complement-mediated cytotoxicity (CDC) is desirable.
• fusion proteins comprise and Fc region with modified CH2 domains comprising amino acid modifications that result in increased binding to Fc ⁇ RIIb or amino acid modifications that decrease or eliminate binding of the Fc region to all of the Fey receptors (“knock-out” variants) can be useful.
• the fusion protein comprises an Fc region with decreased binding to Fc ⁇ RIIb (an inhibitory receptor).
• the fusion proteins according to the present disclosure can be formulated in pharmaceutical compositions.
• These compositions can comprise, in addition to one or more of the fusion proteins, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
• a pharmaceutically acceptable excipient e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
• compositions for oral administration can be in tablet, capsule, powder or liquid form.
• a tablet can include a solid carrier such as gelatin or an adjuvant.
• Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.
• the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
• a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
• isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
• Preservatives, stabilisers, buffers, antioxidants and/or other additives can be included, as required.
• administration is preferably in a “therapeutically effective amount” that is sufficient to show benefit to the individual.
• a “prophylactically effective amount” can also be administered, when sufficient to show benefit to the individual.
• the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.
• a composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
• kits comprising one or more of the compositions described herein and instructions for use.
• kits comprising vectors for expressing a fusion protein described herein and instructions for use.
• kits comprising host cells comprising a vector for expressing a fusion protein and instructions for use.
• kits comprising a purified fusion protein and instructions for use.
• the purified fusion protein can be lyophilized or provided in a dry form, such as a powder or granules, and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized or dried component(s).
• the kit typically will comprise a container and a label and/or package insert on or associated with the container.
• the label or package insert contains instructions customarily included in commercial packages of therapeutic products, providing information or instructions about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
• the label or package insert can further include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration.
• the container holds a composition comprising the fusion protein.
• the container can have a sterile access port.
• the container can be an intravenous solution bag or a vial having a stopper that can be pierced by a hypodermic injection needle.
• the kit can comprise one or more additional containers comprising other components of the kit.
• a pharmaceutically-acceptable buffer such as bacteriostatic water for injection) (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution), other buffers or diluents.
• Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like.
• the containers can be formed from a variety of materials such as glass or plastic.
• one or more components of the kit can be lyophilized or provided in a dry form, such as a powder or granules, and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized or dried component(s).
• the kit can further include other materials desirable from a commercial or user standpoint, such as filters, needles, and syringes.
• An anti-CD3 Fab x anti-Her2 scFv Fc was appended with a mask on the anti-CD3 Fab by linking one of the ligand-receptor pair PD-1-PDL-1 to the N-terminus of the light chain of the Fab and the other to the N-terminus of the heavy chain.
• the fusion protein constructs were designed as follows.
• the fusion proteins were in a modified bispecific Fab x scFv Fc format with a half- antibody comprising the anti-CD3 heavy and light chain that forms a heterodimer with an anti- Her2 scFv fused to an Fc.
• the anti-CD3 paratope was described in US20150232557A1 (VL SEQ ID NO: 1, VH SEQ ID NO: 2).
• the anti-Her2 paratope was in an scFv format that is based on trastuzumab VL and VH (Carter, P. etal. Humanization of an anti-p185HER2 antibody for human cancer therapy.
• PD-1 and PD-L1 moieties were predicted to dimerize and sterically block epitope binding.
• either the PD-1 or the PD-L1 sequence used as one half of the mask contained mutations to increase the affinity of the PD-1:PD-L1 complex as described before (Maute, R. L. et al. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging. Proc Natl Acad Sci USA 112, E6506-6514, doi:10.1073/pnas.l519623112 (2015); SEQ ID NO: 9; Liang, Z. et al. High-affinity human PD-L1 variants attenuate the suppression of T cell activation.
• the PD-1 IgV domain attached to the heavy chain is indicated with a striped pattern in the cartoons and the PD-L1 IgV domain attached to the light chain is shown as a checkered pattern.
• Heavy chain vector inserts comprising a signal peptide (Barash et al ., 2002, Biochem and Biophys Res. Comm., 294:835-842, SEQ ID 27) and the heavy chain clone terminating at G446 (EU numbering) of CH3 were ligated into a pTT5 vector to produce heavy chain expression vectors.
• Light chain vector inserts comprising the same signal peptide and the light chain clone were ligated into a pTT5 vector to produce light chain expression vectors.
• the resulting heavy and light chain expression vectors were sequenced to confirm correct reading frame and sequence of the coding DNA.
• Opti-MEMTM I Reduced Serum Medium Prior to transfection the DNA was diluted in 1.5 mL Opti-MEMTM I Reduced Serum Medium (Thermo Fisher, Waltham, MA). In a volume of 1.42 mL Opti-MEMTM I Reduced Serum Medium, 80 ⁇ L of ExpiFectamineTM 293 reagent (Thermo Fisher, Waltham, MA) were diluted and, after incubation for five minutes, combined with the DNA transfection mix to a total volume of 3 mL. After 10 to 20 minutes the DNA-ExpiFectamineTM293 reagent mixture was added to the cell culture.
• ExpiFectamineTM 293 Enhancer 1 150 ⁇ L of ExpiFectamineTM 293 Enhancer 1 and 1.5 mL of ExpiFectamineTM 293 Enhancer 2 (Thermo Fisher, Waltham, MA) were added to each culture. Cells were incubated for five to seven days and supernatants were harvested for protein purification.
• Clarified supernatant samples were applied to lmL of slurry containing 50% mAb Select SuRe resin (GE Healthcare, Chicago, IL) in batch mode. Columns were equilibrated in PBS. After loading, columns were washed with PBS and protein eluted with 100 mM sodium citrate buffer pH 3.5. The eluted samples were pH adjusted by adding 10% (v/v) 1 M Tris pH 9 to yield a final pH of 6-7. After concentration, all of the material was injected into an AKTA Pure FPLC System (GE Life Sciences) and run on a Superdex 200 Increase 10/300 GL (GE Life Sciences) column pre-equilibrated with PBS pH 7.4.
• mAb Select SuRe resin GE Healthcare, Chicago, IL
• the protein was eluted from the column at a rate of 0.75 mL/min and collected in 0.5 mL fractions. Peak fractions were pooled and concentrated using Vivaspin 20, 30 kDa MWCO polyethersulfone concentrators (MilliporeSigma Burlington MA, USA). After sterile filtering through 0.2 ⁇ m PALL AcrodiscTM Syringe Filters with SuporTM Membrane, proteins were quantitated based on A280 nm (Nanodrop), frozen and stored at -80 °C until further use.
• Samples contained significant amounts of higher molecular weight species as determined by UPLC-SEC after protein A purification (not shown) and preparative SEC was used in order to obtain samples of high purity. Yields after preparative SEC ranged from 1.5 - 5 mg per variant. Sample purity and stability was assessed in Example 3 and Example 4. EXAMPLE 3 PURITY AND HOMOGENEITY ASSESSMENT OF MASKED ANTI-CD3 VARIANTS
• mAh samples were then denatured at 90°C for 5 mins and 35 ⁇ l of water is added to each sample well.
• the LabChip® instrument was operated using the HT Protein Express Chip (Perkin Elmer #760499) and the HT Protein Express 200 assay setting (14 kDa-200 kDa).
• UPLC-SEC was performed on an Agilent Technologies 1260 Infinity LC system using an Agilent Technologies AdvanceBio SEC 300A column at 25 °C. Before injection, samples were centrifuged at 10000 g for 5 minutes, and 5 ⁇ l was injected into the column. Samples were run for 7 min at a flow rate of 1 mL/min in PBS, pH 7.4 and elution was monitored by UV absorbance at 190-400 nm. Chromatograms were extracted at 280 nm. Peak integration was performed using the OpenLAB CDS ChemStation software.
• the masked heavy and light chains showed a significantly higher apparent molecular weight than what would be expected (110 kDa vs 63 kDa for the HC, 54 kDa vs 37 kDa for the LC). This was also reflected in the high apparent molecular weight of the non-reduced, disulfide bonded species (215 kDa vs 152 kDa). Glycosylation of both the PD1 and PD-L1 moieties in the designs is likely causing the increase in apparent molecular weight (Tan, S. et al. An unexpected N-terminal loop in PD-1 dominates binding by nivolumab.
• Thermograms of variants bearing a PD-EPD-L1 mask (30430, 30436; Figure 4) also showed two transitions at similar temperatures and with similar thermogram traces to the unmasked variant. This indicates that the fused masking domains do not affect the T m of the anti- CD3 Fab, and either unfold cooperatively with the Fab or uncooperatively but with a similar T m to Fab, scFv and CH2.
• Human Jurkat cells (Fujisaki Cell Center, Japan) were maintained in RPMI-1640 medium supplemented with 2 mM L-glutamine and 10% of heat-inactivated fetal bovine serum (FBS) with IX Penicillin/Streptomycin, in a humidified + 5% C02 incubator at 37°C.
• FBS heat-inactivated fetal bovine serum
• Samples of modified CD3 x Her2 variants from Example 5 were diluted 2X in blocking buffer, containing saturating amounts of irrelevant human Ig, followed by seven three-fold serial dilutions in blocking buffer for a total of eight concentration points. Blocking buffer alone was added to control wells to measure background signal on cells (negative/blank control).
• Binding curves of blank-subtracted OD450 versus linear or log antibody concentration were fitted with GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA). A one-site specific, four-parameter nonlinear regression curve fitting model with Hill slope was employed in order to determine Bmax and apparent Kd values for each test article.
• Antibodies were titrated in a v-bottom 96-well plate (Sarstedt AG, Niimbrecht, Germany) from 300 nM to 1.7 pM at a 1 :3 dilution in a total of 20 uL/well in FACS buffer - PBS containing 2% FBS (Thermo Fisher Scientific, Waltham, MA). Healthy donor peripheral blood pan T cells (BioIVT, Westbury, NY) were thawed and washed in medium that consisted of RPMI 1640 medium (A1049101, ATCC modification) (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA).
• the cells were counted, resuspended in FACS buffer, and added to the 96-well plate at 50,000 cells per well.
• the cells were incubated with the variants at 4°C for 1 hr and then washed 2x with FACS buffer and 1 mg/mL of secondary antibody AF647 Goat anti-human IgG Fc (Jackson ImmunoResearch, West Grove, PA).
• a 1000-fold diluted viability dye (Biolegend, San Diego, CA) was also added to the wells.
• the plate was incubated at room temperature for 30 min while shaking (200 rpm). Cells were then washed 2x in FACS buffer and resuspended in 100 uL of FACS buffer.
• Geometric mean of APC fluorescence was measured by flow cytometry on a BD LSRFortessa (BD Biosciences, San Jose, CA). Raw data was analyzed on FlowJo, LLC Software (Becton, Dickinson & Company, Ashland, OR). Graphs were generated using GraphPad Prism version 8.1.2 for Mac OS X (GraphPad Software, La Jolla, CA).
• variants containing a full PD1 :PD-L1 based mask appended to the CD3 Fab showed 40-180 fold reduced binding compared to the unmasked control (30421).
• CD3 binding of the cleavable variants 30430 and 30436 was partially restored (within 6-7 fold of the unmasked control). This partial recovery might be caused by a steric hinderance of epitope binding by the portion of the mask that is left on the mask after cleavage.
• variants containing a full PD 1 :PD-L1 based mask appended to the CD3 Fab showed > 43 fold reduced binding compared to the unmasked control (30421).
• CD3 binding of the cleavable variant 30430 was partially restored (within 29 fold of the unmasked control). This partial recovery might be caused by a steric hinderance of epitope binding by the portion of the mask that is left on the mask after cleavage.
• JIMT-1 (Leibniz Institute, Braunschweig, Germany) cultured in growth medium consisting of DMEM medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA), HCC1954 (ATCC, Manassas, VA) and HCC827 (ATCC, Manassas, VA) cultured in growth medium consisting of RPMI-1640 ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% Fetal Bovine Serum, and MCF-7 (ATCC, Manassas, VA) cultured in growth medium consisting of MEM medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% Fetal Bovine Serum and O.Olmg/mL of human recombinant Insulin (Thermo Fisher Scientific, Waltham, MA) were maintained horizontally in T-175 flasks (Corning, Corning, NY)
• the variants were titrated in triplicate at 1:3 dilution directly in 384-well cell culture treated optical bottom plates (ThermoFisher Scientific, Waltham, MA) from 5 nM to 0.08 pM.
• Tumor cells were rinsed with PBS (Thermo Fisher Scientific, Waltham, MA), harvested with TrypLE Express (Thermo Fisher Scientific, Waltham, MA), diluted in media, and counted using Vi-Cell (Beckman Coulter, Indianapolis, IN).
• a vial of primary human pan-T cells BioIVT, Westbury, NY
• Pan T cell suspension was mixed with tumor cells at 5:1 effector to target ratio, washed and resuspended at 0.55 E6 cell/ml. 20uLof the mixed cell suspension was added to the plate containing the titrated variants. The plates were incubated for 48 h in an incubator at 37 °C with 5% carbon dioxide. The samples were then subjected to a high- content cytotoxicity assessment and the supernatants were collected for IFN ⁇ analysis.
• the plate was scanned on the Celllnsight CX5 high content instrument using the SpotAnalysis.V4 Bioapplication with the following settings: Objective: lOx, Channel 1 - 386nm: Hoechst (Fixed exposure time 0.008 ms with a Gain of 2). IFN ⁇ quantification
• Her2 and PD-L1 receptor quantification were performed via flow cytometry using Quantum Simply Cellular anti-human and anti-mouse IgG kits respectively (Bangs Laboratories, Fishers, Indiana). Tumor cells were rinsed with PBS (Thermo Fisher Scientific, Waltham, MA), and harvested with TrypLE Express (Thermo Fisher Scientific, Waltham, MA). Cells were counted using Vi-Cell (Beckman Coulter, Indianapolis, IN), washed, and resuspended in FACS buffer - PBS containing 2% FBS (Thermo Fisher Scientific, Waltham, MA) at 4x10 ⁇ 6 c/mL.
• the potency of an unmasked control (30421) was determined to be between 0.03 pM (HCC1954: high Her2, high PD-L1) and 3 pM (MCF-7: medium Her2, low PD-L1) for cytotoxicity of the different cell lines.
• the potency of this unmasked control as determined by IFN ⁇ release was between 8.4 pM (HCC1954: high Her2, high PD-L1) and 50 pM (HCC829: low Her2, medium PD-L1).
• a cell line with very low PD-L1 expression showed no significant differentiation in the cytotoxicity readout between unmasked control (30421) and those variants capable of engaging PD-L1 (31929, 30421 + 120 nM atezolizumab, 30430 +uPa).
• these variants with an anti-PD-L1 moiety did show a higher potency in IFN ⁇ release for all tested cell lines compared to the unmasked control (30421).
• An irrelevant anti-RSV antibody (22277) showed no activity in the TDCC for either of the cell lines.
• binding of the modified variants to CHO cells expressing PD-L1 and PD-1 was determined as follows.
• CHO-S cells (National Research Council Canada) were cultured in FreeStyle CHO expression medium (Thermo Fisher Scientific, Waltham, MA) with 1% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA). Neon Transfection system (Thermo Fisher Scientific, Waltham, MA) was used to perform transfection. CHO-S cells were counted and washed 2x with PBS and once in Resuspension buffer R (Thermo Fisher Scientific, Waltham, MA) before being resuspended at 100 E6 cells/mL. PD-1, PDL-1 or GFP plasmid DNA (GenScript, Piscataway, NJ) was added at 1 ug/1 E6 cells.
• Neon tube was filled with 3mL Electrolytic buffer E2 (Thermo Fisher Scientific, Waltham, MA). Using a 100 ⁇ L Neon tip (Thermo Fisher Scientific, Waltham, MA), transfection for each plasmid was carried out at the following settings: Voltage - 1620, Width - 10, Pulse - 3. Transfected cells were transferred to a pre-warmed flask at a concentration of 1 E6 cells/mL for each condition.
• Variants purified in Example 2 and uPa treated in Example 5 were titrated directly in a v-bottom 96-well plate (VWR, Radnor, PA, USA) from 200 nM at a 1:3 dilution.
• CHO-PD1, CHO-PDL-1, and CHO-GFP cells were thawed and washed in RPMI 1640 medium (A1049101, ATCC modification) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA, USA) and resuspended in FACS buffer (PBS + 2% FBS).
• Each of the CHO-PD1 and CHO-PDL-1 cells were combined 2:1 with CHO-GFP cells and 20 uL of cell suspension was added to the plate with the titrated variants. The cells were incubated with the variants at 4 °C for 1 h. Following incubation, the cells were washed 2x with FACS buffer and 1 ug/mL of secondary antibody AF647 Goat anti-human IgGFc (Jackson ImmunoRe search, West Grove, PA, USA) along with 1000-fold diluted viability dye (Biolegend, San Diego, CA, USA) was added to the wells. Plate was incubated at room temperature for 30min. Cells were washed 2x in FACS buffer and resuspended in 50 uL of FACS buffer.
• Geometric mean of APC fluorescence was measured by flow cytometry on a BD LSRFortessa (BD Life Sciences, Gurugram, India). Non-specific binding was determined by measuring APC fluorescence Geometric mean of GFP positive cells. Graphs were generated using GraphPad Prism version 8.1.2 for Mac OS X (GraphPad Software, La Jolla, CA, USA).
• the uncleavable variants did not bind to PD-L1 or PD-1 when treated with protease (+uPa) whereas partial binding was recovered for the uPa-treated samples that contain a uPa cleavage sequence between the Fab and PD-1:PD-L1 mask. Specifically, binding to PD-L1 was partially recovered for 30430, within 53 fold of the relevant one-sided mask control 31929 (A). Binding to PD-1 was partially recovered for 30436, within 12 fold of the one sided mask control 31931 (B).
• JIMT-1 (Leibniz Institute, Braunschweig, Germany) cultured in growth medium consisting of DMEM medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA), HCC1954 (ATCC, Manassas, VA) and HCC827 (ATCC, Manassas, VA) cultured in growth medium consisting of RPMI-1640 ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% Fetal Bovine Serum, MCF-7 (ATCC, Manassas, VA) cultured in growth medium consisting of MEM medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% Fetal Bovine Serum and O.Olmg/mL of human recombinant Insulin (Thermo Fisher Scientific, Waltham, MA) and Jurkat T cells stably expressing human PD-1 and NF AT -induced lucifer
• the variants were titrated in triplicate at 1:3 dilution directly into 384- well Low Flange White Flat Bottom Polystyrene TC-treated Microplates, (Coming Cat# 3570, Coming, NY) from 150 nM to 0.85 pM in 20 uL total volume per well.
• Tumor cells were dissociated using cell dissociation buffer and mixed with Jurkat cells at a 1:1 ratio in RPMI 1640 supplemented with 1% Fetal Bovine serum. 20 uL of the mixed cell suspension was added to the plate containing the titrated variants. The plates were incubated for 16 h at 37°C with 5% carbon dioxide.
• a cleavable masked variant (30430) When treated with uPa (+uPa), a cleavable masked variant (30430) showed activity in the RGA that was higher than that of the unmasked control (30421) at variant concentrations above 100 pM, pointing to an unmasking of the CD3 paratope as well as a blocking of the PD-1:PD-L1 checkpoint engagement by the functional PD-1 moiety of the mask left on the variant after cleavage.
• a control with just the PD-1 domain attached to the heavy chain of the CD3 Fab showed a similar profile and increased activity in the RGA at variant concentrations higher than 100 pM when compared to the unmasked control (30421).
• An irrelevant anti-RSV antibody (22277) showed no activity in the RGA.
• a cell line with very low PD- L1 expression (MCF-7) showed no differentiation of unmasked control (30421) and those capable of engaging PD-L1 (31929, 30421 + 120 nM atezolizumab, 30430 +uPa).
• An irrelevant anti-RSV antibody (22277) showed no activity in the RGA for either of the cell lines.
• variable domains of mAbs targeted against several different epitopes were appended with a masking domain comprising a PD-1:PD-L1 complex.
• the fusion protein constructs were designed as follows.
• FIG. 1 A schematic of the construct design for the masked Fab as well as the intended mechanism of action (MoA) is shown in Figure 1.
• FIG. 10 A schematic of the final design, a bivalent, fully masked mAb with two identical heavy and light chains, is shown in Figure 10. The used sequences of the final variants are listed in Table B. Table B: Sequences of paratopes investigated for compatibility with mask
• the apparent molecular weight of this species is significantly higher than what would be expected (> 250 kE)a vs 200 kE)a).
• Reducing CE-SDS of representative variants targeted against c-Met and CDH3 showed only bands corresponding to the intact heavy and light chains. These show the same high apparent molecular weight as described in Example 3.
• Glycosylation of both the PD1 and PD-L1 moieties in the designs is likely causing the increase in apparent molecular weight (Tan, S. et al. An unexpected N-terminal loop in PD-1 dominates binding by nivolumab. Nat Commun 8, 14369, doi:10.1038/ncommsl4369 (2017), Li, C. W. et al. Glycosylation and stabilization of programmed death ligand- 1 suppresses T-cell activity. Nat Commun 7, 12632, doi:10.1038/ncommsl2632 (2016)).
• EXAMPLE 13 UP A CLEAVAGE OF MASKED ANTI-EGFR, ANTI-MESOTHELIN, ANTI-TF, ANTI-CD 19, ANTI-CMET AND ANTI-CDH3 VARIANTS [00391]
• select samples produced in Example 11 were treated with uPa in vitro. Reactions were monitored by reducing SDS-PAGE as follows.
• Preparative cleavage assays of the modified variants targeting different epitopes were set up as described in Example 5 and analyzed by non-reducing SDS-PAGE.
• the SDS-PAGE was set up as described in Example 12 with the exception of the usage of reducing Laemmli buffer for the denaturation of the sample.
• the reducing buffer was obtained by supplementing 4X Laemmli buffer with 10 % b-ME.
• Target binding of the different paratope/epitope pairs was assessed by SPR and flow cytometry on the samples produced in Example 11 and treated by uPa in Example 13 as follows.
• Modified variants targeted against the different epitopes were diluted 2X in complete medium, followed by three-fold serial dilutions in cold complete medium for a total of eight to ten concentration points starting at 300 nM or 150 nM.
• Detection of bound variants was achieved by an additional incubation with a fluorescently labeled, Fc-specific secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for an hour. Cells were washed twice by centrifugation and cell pellets were resuspended in complete medium with Propidium Iodide (Invitrogen, Carlsbad, CA, USA), filtered using a 0.60 pm size-pore 96 well filter plate (MilliporeSigma, Burlington, MA, USA) and analyzed by flow cytometry using the HTS automated sampler unit (installed on BD-LSRII or BD- LSRFortessa). Two thousands alive/single-cell events were acquired per sample.
• SPR surface plasmon resonance binding assays for determining kinetics and affinities of a subset of the different antigens (EGFR, TF, Mesothelin) to the modified mAb variants were carried out on BiacoreTM T200 instrument (GE Healthcare, Mississauga, ON, Canada) with PBS- T (PBS + 0.05% (v/v) Tween 20, pH 7.4) running buffer at a temperature of 25 °C.
• CM5 Series S sensor chip, Biacore amine coupling kit (NHS, EDC and 1 M ethanolamine) and 10 mM sodium acetate buffers were all purchased from GE Healthcare.
• PBS running buffer with 0.05% (v/v) Tween20 was purchased from Teknova Inc. (Hollister, CA). Goat polyclonal anti-human Fc antibody was purchased from Jackson Immuno Research Laboratories Inc. (West Grove, PA).
• Recombinant protein of the extracellular domain of human EGFR Genscript, Cat# Z03194-50
• mature human mesothelin R&D systems, Cat# 3265-MS-050
• the screening of mAb variants for binding to the different antigens occurred in two steps: an indirect capture of mAb variants onto the anti-human Fc-specific polyclonal antibody surface, followed by injection of five concentrations of SEC-purified antigen.
• the anti-human Fc surface was prepared on a CM5 Series S sensor chip by standard amine coupling methods as described by the manufacturer (GE Healthcare). Briefly, immediately after EDC/NHS activation, a 25 ⁇ g/mL solution of anti-human Fc in 10 mM NaOAc, pH 4.5 was injected at a flow rate of 10 ⁇ L/min for 7 min until approximately 4500 resonance units (RUs) were immobilized on all four flow cells.
• RUs resonance units
• the remaining active groups were quenched by an injection of 1 M ethanolamine at 10 uL/min for 7 min.
• MAbs for analysis were indirectly captured onto the anti-Fc surfaces (flow cells 2 - 4) by injecting 2-20 ⁇ g/mL solutions at a flow rate of 10 ⁇ L/min for 60 s, resulting in mAh capture levels ranging from 130 - 470 RUs depending on the mAh variant.
• mAh capture levels ranging from 130 - 470 RUs depending on the mAh variant.
• five concentrations of a two-fold dilution series of the antigens were sequentially injected at 40 ⁇ L/min over all flow cells, including reference flow cell 1, and a buffer blank injection over all flow cells served as control. For details on concentration ranges and contact and dissociation times of analytes, see Table 53.
• the anti -human Fc surfaces were regenerated to prepare for the next injection cycle by one pulse of 10 mM glycine/HCl, pH 1.5, for 120 s at 30 ⁇ L/min. Double reference-subtracted sensograms were analyzed using BiacoreTM T200 Evaluation Software v3.0 and then fit to the 1:1 Langmuir binding model.
• Figure 13 shows that antigen binding for all uncleavable variants (variants 31722, 31728, 31736, 31732, 28647, 28664 for EGFR, MSLN, TF, CD19, cMet, CDH3, respectively) was reduced 30-190 fold when compared to the respective unmasked controls (variants 32474, 16417, 6323, 4372, 17606, 17214 for EGFR, MSLN, TF, CD19, cMet, CDH3, respectively) as determined by on-cell binding studies. Where cleavable variants were included, samples were tested without (-uPa) and with uPa treatment (+uPa).
• uncleavable variants 31722, 31728, 31736, 31732 for EGFR, MSLN, TF, CD19, respectively
• cleavable samples 31723, 31729, 31737, 31733 for EGFR, MSLN, TF, CD 19, respectively
• binding levels were similar to uncleavable variants before being subjected to the protease while upon uPa cleavage, binding was recovered within 1.3-85 fold of the unmasked control.
• SPR binding results show the same trends of masking and recovery of binding after cleavage.
• NCI-H292 cells were routinely grown in 75cm 2 (T75) flasks at 37°C +5% CO 2 and passaged twice a week in FBS culture medium without the addition of antibiotic. Cells were seeded the day before addition of antibodies at 300, 1000 and 125 cells / 25 ⁇ L / well in 384-well plates (Corning 3570) in their culture media with the addition of 1000 units of penicillin, 1000 ⁇ g of streptomycin, and 2.5 ⁇ g of Amphotericin B per mL.
• RLU Relative Luminescence Unit
• % survival RLU Ab / RLU non treated X 100.
• an anti-EGFR antibody based on Cetuximab inhibited growth of NCI-H292 cells with an IC50 of 0.11 nM. Treatment with uPa only minimally affected this function.
• PD-1:PD-L1 masked variants (31722, 31723) were less potent (40-80 fold increased IC50) without treatment with uPa.
• the uncleavable variant 31722 was still significantly inhibited for function (100 fold)
• the cleavable 31723 showed recovery of function within 2.5 fold of the unmasked v32474.
• An irrelevant antibody (22277) showed no function in the growth inhibition assay
• a masked CTLA4:CD80 CD3 Fab was designed to be equivalent to the PD1 :PD-L1 masked variants in Example 1. Briefly, sequences of the IgV domains of human CD80 and CTLA4 (West, S. M. & Deng, X. A. Considering B7-CD28 as a family through sequence and structure. Exp Biol Med (Maywood), 1535370219855970, doi:10.1177/1535370219855970 (2019); SEQ ID 25, 26) were appended to the N-termini of heavy and light chains of the CD3 Fab, respectively, using one of the linker combinations described in Example 1 and Example 10.
• CTLA4 IgV domain was fused to the LC with a uPa-cleavable sequence while the CD80 moiety could not be removed by the protease.
• a schematic of the architecture of the investigated variant is shown in Figure 15. Additionally, to reduce homo-dimerization via CD80 that was described previously (C. C. Stamper et al. , Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses. Nature 410, 608-611 (2001)), mutations were introduced in the CD80 moiety in some variants. Sequences of the individual chains of the variant are listed in Table F. Antibodies were produced, their sample purity and cleavage by uPa assessed and binding to CD3- bearing Jurkat cells assessed as in Example 2, Example 3, Example 5 and Example 6, respectively.
• the CD80 IgV domain attached to the heavy chain is indicated with a striped pattern in the cartoons and the CTLA-4 IgV domain attached to the light chain is shown as a checkered pattern.
• the immunomodulatory pairs (e.g. PD-1:PD-L1 (Table G), CD80:CTLA-4) are used as untargeted, conditionally activated molecules in this example.
• the immunomodulatory pairs do not serve a masking function with regard to a particular paratope but are directly fused to an Fc as follows.
• the constructs investigated here are based on IgV domains of immunomodulator pairs such as PD-1:PD-L1 that are N-terminally fused to the hinge of a heterodimeric IgG Fc.
• the Fc portion of these constructs contains mutations in the CH3 domain that drive heterodimeric pairing of the two chains as described previously (for example: Von Kreudenstein, T. S. et al. Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design.
• MAbs 5, 646-654, doi: 10.4161/mabs.25632 (2013); SEQ ID 4,5; other heterodimeric Fc forming mutations are also available in the literature).
• mutations are also introduced in both CH2 domains to abrogate binding to the Fc gamma receptors (SEQ ID 6,). While one immunomodulator IgV domain (e.g. a high-affinity version of PD-1, Maute, R. L. et al. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging.
• immunomodulator IgV domain e.g. a high-affinity version of PD-1, Maute, R. L. et al. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging.
• This design results in a conditionally active, monovalent, PD-L1 targeting molecule that is directly fused to an IgG Fc via a protease cleavable peptidic linker( Figure 19).
• the high affinity PD-1:PD-L1 dimer is formed intramolecularly and undesired systemic binding to PD-L1 is prevented.
• TME tumor microenvironment
• PD-L1 is released and the PD-1 moiety can bind to PD-L1 expressed on tumor cells.
• checkpoint activity is thereby selectively blocked and the susceptibility of tumor cells to cytotoxic T-cells is enhanced.
• CD80:CTLA-4 or SIRPa:CD47 are also used as masks.
• CD80:CTLA-4 only in the presence of the right TME-associated protease, CTLA-4 is released and the remaining CD80 can bind to CD28 or CTLA-4 on T-cells and in turn exert its immunomodulatory function.
• SIRPa:CD47 mask the CD47 moiety is released by proteolytic cleavage in the TME, leaving SIRP ⁇ free to bind to CD47 on macrophages, thereby inhibiting the checkpoint activity and increasing phagocytosis and tumor cell killing.
• Variants with or without treatment with uPa are tested for binding to PD-L1 by flow cytometry as described in Example 8.
• the same samples are tested in a reporter gene assay (RGA) sensitive to PD-1:PD-L1 checkpoint inhibition (Promega, Madison, WI, USA).
• RGA reporter gene assay
• the RGA is performed similar to the RGA in Example 9, with the exception that PD-L1 expressing and TCR directed CHO cells are used together with the modified Jurkat T-cells as per the manufacturers protocol.
• the PD-1 IgV is indicated with a striped pattern in the cartoons and the PD-L1 IgV domain is shown as a checkered pattern.
• ZW Fcl does not bind to PD-L1 in a flow cytometry assay. This is due to the tight intramolecular interaction of the high affinity version of the PD-1 IgV domain with PD-L1 in the Fc assembly.
• ZW Fcl binds PD-L1 tightly in SPR and flow cytometry assays. This is expected as after cleavage of the uPa-specific sequence in the linker, the PD-L1 moiety is released and the PD-1 domain remaining on the Fc is free to bind PD-L1 in the assays.
• PD-1:PD-L1 masked versions of full sized antibodies containing a previously described anti-CD40 paratope (R. H. Vonderheide etal. , Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody. J Clin Oncol 25, 876-883 (2007)) were constructed as described in Example 10. The resulting constructs and their sequences are summarized in Table H.
• Table H Sequences of anti-CD40 variants
• Heavy and light chain sequences of the described variants were ported into expression vectors, expressed in Expi293FTM cells and purified using the 2-step purification process described in Example 11. Purified samples were then assessed for purity and sample homogeneity by UPLC- SEC and non-reducing gel electrophoresis as described in Example 3. After purification, samples were treated with uPa and their processing assessed by non-reducing CE-SDS as described in Example 5. Both uPa-untreated and uPa-treated samples were then assessed by flow cytometry for target binding to Raji cells as described in Example 14.
• HEK Blue CD40L cells (Invivogen, San Diego, CA, USA, hkb-cd40 Lot 38-01-hkbcd40) cells were detached with PBS then resuspended at 2.78 x 10 5 cells/mL in pre-warmed test media (GibcoTM DMEM (Thermo Fisher Scientific, Waltham, MS, USA, 1195-040) plus 10 % heat inactivated GibcoTM FBS (Thermo Fisher Scientific, Waltham, MS, USA, 12483-020 Lot 1996160) (56 °C, 30 min) and 100 U/mL GibcoTM Pen-Strep (Thermo Fisher Scientific, Waltham, MS, USA, 15070-063 Lot 1989510)).
• WT-CHOK1 (ATCC, Manassas, VA, USA, ATCC CCL-61, Lot 70014310) and FcgR2B-CHOKl cells (BPS Bioscience, San Diego, CA, USA, 79511, Lot 191104-41) were detached with trypsin and resuspended at 5.56 x 10 5 cells/mL with test media.
• 25,000 HEK Blue CD40 cells (90 ⁇ L) were then added to 20 ⁇ L of variants serially diluted in test media (10 ⁇ g/mL - 0.000001 ⁇ g/mL), followed by addition of 50,000 WT-CHOK1, FcYR2B-CHOKl cells (90 ⁇ L) or 90 ⁇ L of test media.
• Test articles included uPa-untreated and uPa-treated CD40- targeted variants as well as an irrelevant control antibody targeted against RSV and CD40L (Invivogen, San Diego, CA, USA) as negative and positive controls, respectively.
• the antiCD40 variants showed one predominant species in UPLC-SEC at a purity of 92 % - 100 % with low amounts of higher molecular weight species (7-8 %) present for the masked variants v32478 and v32479.
• Non- reducing CE-SDS analysis ( Figure 20 D) also showed a single predominant species for all variants.
• the apparent molecular weight of both heavy and light was also higher than expected ( ⁇ 100 kDa vs 63 kDa for the HC, ⁇ 50 kDa vs 37 kDa for the LC), likely due to glycosylation of both PD-1 and PD-L1 and as seen in Example 3 and Example 12.
• EXAMPLE 19 SIRPa:CD47 IMMUNOMODULATORY PAIRS AS MASKS [00426] To determine whether immunomodulatory pairs outside the B7:CD28 family can be utilized to mask a Fab efficiently, a CD47:SIRP ⁇ -masked version of the anti-EGFR antibodies described in Example 10 was produced and assessed for EGFR binding as follows.
• a CD47 : SIRP ⁇ -masked anti-EGFR antibody was designed to be equivalent to thePD1 :PD-L1 masked variants described in Example 10. Briefly, sequences of the IgV domains of human CD47 and a modified, affinity increased variant of human SIRP ⁇ (K. Weiskopf el al ., Engineered SIRPalpha variants as immunotherapeutic adjuvants to anticancer antibodies. Science 341, 88-91 (2013)) were appended to the N-termini of heavy and light chains of the anti-EGFR Fab, respectively, using uPa cleavable linkers described in Example 1 and Example 10. A schematic of the architecture of the investigated variant is shown in Figure 27.
• the SIRPa IgV domain attached to the heavy chain is indicated with a striped pattern in the cartoons and the CD47 IgV domain attached to the light chain is shown as a checkered pattern.
• NCI-H292 cell line expressing EGFR was maintained in RPMI-1640, supplemented with L-glutamine and 10% FBS (complete medium) in a humidified + 5% C02 incubator at 37°C. On the day before the assay, exponentially growing cells were harvested using 0.05% trypsin (Gibco®), resuspended in complete medium at a cell density of 1.2xl0 5 cells/mL.
• Detection of bound variants was achieved by an additional incubation with a fluorescent labeling mix containing AF488-labeled, human Fc-specific secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA), Deep Red CellMask (Molecular Probes, Eugene, Oregon, USA) and Hoechst33342 (Molecular probes, Eugene, Oregon, USA) in the presence of FBS (Wisent Bioproduct, St-Bruno, Quebec, Canada) for an hour. Cells were washed twice (3-cycle, 150 ⁇ L/well washes each time) in the BioTek EL405 select (BioTek, Winooski, VT, USA) plate washer.
• Baseline values were calculated with the average normalized green background fluorescence signal of the control wells (these are wells that were incubated only with the fluorescent labeling mix of secondary antibody). Baseline values in each plate were subtracted from all data before applying the non-linear fit model. Specific Total Intensity per cell area (baseline corrected) versus log antibody concentration were fitted with a “One-Site-Specific binding with Hill slope” nonlinear regression curve fit model for each test article.
• the band for the CD47-modified light chain shows a significantly higher than expected apparent molecular weight in the reducing CE-SDS profile, overlapping with the modified heavy chain. Similar to the PD-1:PD-L1 based modifications in Example 3, this is likely caused by extensive glycosylation of CD47 (W. J. Mawby, C. H. Holmes, D. J. Anstee, F. A. Spring, M. J. Tanner, Isolation and characterization of CD47 glycoprotein: a multispanning membrane protein which is the same as integrin-associated protein (IAP) and the ovarian tumour marker OA3. Biochem J 304 ( Pt 2), 525-530 (1994)).
• both the CD47 as well as the SIRP ⁇ moiety were effectively removed from the light chain as seen in Figure 29.
• the bands corresponding to the modified heavy and light chains disappeared upon cleavage and bands corresponding to the molecular weight of the unmasked heavy and light chain appeared.
• the released CD47 and SIRP ⁇ components could not unambiguously identified after cleavage, likely due to their small size and heterogeneity caused by glycosylation.
• EXAMPLE 20 CO-ENGAGEMENT AND BRIDGING OF TARGETS BY ANTI-CD3 TRISPECIFIC VARIANTS
• Her2 and CD3 can be engaged simultaneously by the anti-CD3 variants described in Examples 1-9, Her2- PD-L1 co-engagement as well as T-cell bridging studies were performed as follows.
• JIMT-1 (Leibniz Institute, Braunschweig, Germany) cultured in growth medium consisting of DMEM medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA) were maintained horizontally in T- 175 flasks (Coming, Corning, NY) in an incubator at 37 °C with 5% carbon dioxide.
• Antibodies were titrated in a 96-well v-bottom plate (Thermo Fisher Scientific, Waltham, MA) from 100 nM to 1.7 pM at a 1:3 dilution in a total of 20 uL/well in FACS buffer - PBS containing 2% FBS (Thermo Fisher Scientific, Waltham, MA). Tumor cells were rinsed with PBS (Thermo Fisher Scientific, Waltham, MA), harvested with TrypLE Express (Thermo Fisher Scientific, Waltham, MA), diluted in media, and counted using Countess automated cell counter (Thermo Fisher Scientific, Waltham, MA).
• the tumor cells were washed and resuspended in FACS buffer, and added to the 96-well plate at 50,000 cells per well.
• the cells were incubated with the variants at 4°C for 1 hr. Following incubation, the cells were washed 2x with FACS buffer and 1 mg/mL of secondary antibody AF647 Goat anti -human IgGFc (Jackson ImmunoResearch, West Grove, PA) along with 1000-fold diluted viability dye (Thermo Fisher Scientific, Waltham, MA) was added to the wells. Plate was incubated at room temperature for 30 min. Cells were washed 2x in FACS buffer and resuspended in 100 uL of FACS buffer.
• Geometric mean of APC fluorescence was measured by flow cytometry on a BD Celesta (BD Biosciences, San Jose, CA). Raw data was analyzed on FlowJo, LLC Software (Becton, Dickinson & Company, Ashland, OR). Graphs were generated using GraphPad Prism version 8.1.2 for Mac OS X (GraphPad Software, La Jolla, CA).
• JIMT-1 (Leibniz Institute, Braunschweig, Germany) cultured in growth medium consisting of DMEM medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific, Waltham, MA) were maintained horizontally in T- 175 flasks (Coming, Corning, NY) in an incubator at 37 °C with 5% carbon dioxide. Tumor cells were rinsed with PBS (Thermo Fisher Scientific, Waltham, MA), harvested with TrypLE Express (Thermo Fisher Scientific, Waltham, MA), diluted in PBS, and washed twice in PBS.
• DMEM medium Thermo Fisher Scientific, Waltham, MA
• Fetal Bovine Serum Fetal Bovine Serum
• a vial of primary human Pan-T cells (BioIVT, Westbury, NY), was thawed in a 37 °C water bath, washed in growth medium consisting of RPMI-1640 ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% Fetal Bovine Serum, subsequently washed in PBS, and resuspended in PBS. T cells and tumor cells were counted using Countess automated cell counter (Thermo Fisher Scientific, Waltham, MA) and resuspended at 5 M/mL in PBS. Cell Proliferation dye-eF670 (Thermo Fisher Scientific, Waltham, MA) was added to tumor cells at 1.25uM.
• T cells were added to T cells at 2 uM.
• T cells and Tumor cells were incubated at 37 °C for 20 min in the dark and washed twice in FACS buffer - PBS containing 2% FBS (Thermo Fisher Scientific, Waltham, MA).
• Antibodies were titrated down a v-bottom 96-well plate (Thermo Fisher Scientific, Waltham, MA) from 10 nM to 0.2 pM at a 1 :6 dilution in a total of 50 uL/well in FACS buffer.
• Pan T cells were mixed with tumor cells at 5:1 effector to target ratio at 1.44 E6 cell/mL.
• Binding to an endogenous Her2+/PD-L1+ cancer cell line was measured by flow cytometry (Figure 30A).
• EXAMPLE 21 IN VIVO FUNCTIONAL EVALUATION OF ANTI-CD3 X ANTI-HER2 T CELL-ENGAGER FUSION PROTEINS
• mice are implanted subcutaneously with 5x106 cells from a human Her2+ tumor line (JIMT-1) and simultaneously engrafted intravenously with 1x107 PBMCs from healthy human donors. After establishment and initial growth of the tumor to approximately 150-200 mm3, the mice are dosed intravenously with antibody variants described and produced in examples 1-9. Mice are monitored for both body weight and tumor growth (measured by caliper) twice per week for duration of the study.
• JIMT-1 Her2+ tumor line
• a variant with a functional PD-1 domain (31929) also shows additional tumor growth inhibition when compared to the equivalent construct with a non-functional PD-1 domain (32497).
• variants with complete PD-1:PD-L1 based masks are evaluated, a construct with uncleavable linkers on both appended domains (30423) shows rapid tumor growth.
• a construct with a cleavable linker between Fab and PD-L1 (30430) shows high anti-tumor activity, similar to an unmasked, trispecific control (31929) when a tumor cell line with high expression of the relevant protease is used in the model.
• the same cleavable variant (30430) shows rapid tumor growth, similar to an uncleavable construct (30430).
• EXAMPLE 22 CD80-CTLA-4, CD80-CD28, AND CD80-PD-L1 LIGAND-RECEPTOR PAIRS AS MASKS
• CD80 affinities for CTLA-4, CD28, and PD-L1 are 0.2 uM, 4 uM, and 1.7 uM, respectively (Butte, M. J. et al , Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity , 27, 111-122, doi:10.1016/j.immuni.2007.05.016 (2007)).
• mutations are introduced into CD80 IgV domain that are known to selectively increase affinity for CD28 (patent: US20210155668A1).
• CD80 mask format multiple constructs were designed to evaluate what geometry optimally potentiates T cell activation. Briefly, the IgV domain of human CD80 with mutations to prevent CD80 homodimerization (as described above) and/or CD80 with mutations predicted to increase affinity for CD28 are appended to the N-termini of heavy or light chain of anti-CD3 Fab using an (EAAAK)2 linker and paired in a heterodimeric Fc format with anti-TAA scFv x Fc.
• EAAAK EAAAK2 linker
• CD80 IgV domain is appended to the N- termini of heavy or light chain of anti-TAA Fab using an (EAAAK)2 linker and paired in a heterodimeric Fc format with an anti-CD3 scFv x Fc.
• EAAAK EAAAK2 linker
• CD80 can bind CTLA-4, CD28, and PD-L1, all three are used as dimeric mask partners (CD80:CTLA-4, CD80:CD28, CD80:PD-L1).
• the resulting masked constructs were designed using a CD80 IgV domain with mutations to prevent CD80 homodimerization and known to increase affinity for CD28.
• the CTLA-4, CD28, or PD-L1 IgV domains are fused to the heavy or light chain with a protease-cleavable sequence while the CD80 moiety is fused to the light or heavy chain with an alpha helical peptide linker sequence designed to not be removed by an endogenous protease.
• a high affinity version of the CD80 IgV domain and a wildtype, human CTLA-4 IgV domain are appended to the N- termini of heavy and light chains of the anti-CD3 Fab, respectively, using peptide linkers and paired with an anti-TAA scFv Fc.
• a high affinity version of the CD80 IgV domain and a wildtype, human CD28 IgV domain are appended to the N-termini of heavy and light chains of the anti-CD3 Fab, respectively, using peptide linkers and paired with an anti-TAA scFv Fc.
• CD80:PD-L1 a CD80 IgV domain with mutations to prevent CD80 homodimerization and predicted to increase affinity for CD28 and PD-L1 (patent: US20210155668A1) and wildtype, human PD-L1 IgV domain are appended to the N-termini of heavy and light chains of the anti-CD3 Fab, respectively, using peptide linkers and paired with an anti-TAA scFv Fc.
• molecules are designed wherein the CD80-containing mask (CD80:CTLA-4, CD80:CD28 or CD80:PD-L1) is used to block an anti-TAA Fab paratope and the chain is paired with an anti-CD3 scFv.
• Table J Schematics of CD80 one-sided mask variants and fully masked CD80 variants. * The a-CD3 arms are shaded in dark grey and the a-TAA arms are shaded light grey. The CD80 IgV domain is indicated with a striped pattern in the cartoons and the CTLA-4, CD28, or PD-L1 IgV domains are shown with a checkered pattern. The thunderbolt indicates a protease-cleavable linker sequence.

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