Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
  • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • 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
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/624—Disulfide-stabilized antibody (dsFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• the present disclosure provides materials and methods for molecules that are capable of binding to a target (e.g., binding molecules).
• the molecule comprises an antigen-binding fragment (Fab), a single chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv comprises: a) a disulfide bond between a structurally conserved surface exposed VH position that is mutated to cysteine (Cys) and a L Cys; b) a disulfide bond between a structurally conserved surface exposed VL position that is mutated to Cys and a L Cys; or c) a first disulfide bond between a structurally conserved surface exposed VH Cys and a first L Cys and a second disulfide bond between a structurally conserved surface exposed
• Fab antigen-binding
• the molecule has improved stability, expression yields, and/or quality as compared to a comparable molecule absent a disulfide bond or two disulfide bonds, e.g., absent the first disulfide bond and the second disulfide bond.
• the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position and the L comprises a L Cys
• the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a L Cys
• the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position
• the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.
• the distance between the VH Cys and the VL Cys is from about 5 A to about 10 A or from about 7 A to about 9 A.
• the VH Cys is at H3, H5, H40, H43, H46 or H105, wherein the residue numbering is according to Chothia.
• the VL Cys is at L3, L5, L39, L42, L43, L45, L100 or L102, wherein the residue numbering is according to Chothia.
• the VH Cys is at Hl 05 and the VL Cys is at L42; the VH Cys is at H43 and the VL Cys is at L100; the VH Cys is at H3 and the VL Cys is at L3; the VH Cys is at H3 and the VL Cys is at L5; the VH Cys is at H3 and the VL Cys is at L39; the VH Cys is at H3 and the VL Cys is at L42; the VH Cys is at H3 and the VL Cys is at L45; the VH Cys is at H3 and the VL Cys is at L100; the VH Cys is at H3 and the VL Cys is at L102; the VH Cys is at H5 and the VL Cys is at L3; the VH Cys is at H5 and the VL Cys is at L5; the VH Cys is at H5 and the VL Cys is at L39; the VH Cys is at H Cys is at
• the L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region.
• Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region.
• the Ig hinge region is derived from a human Ig hinge region.
• the human Ig hinge region is an IgGl, IgG2, IgG,3 or IgG4 isotype.
• the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gin), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) or tyrosine (Tyr), and y is an integer from 1 to 3.
• X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gin), histidine (His), isoleucine
• the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer from 1 to 3.
• the L comprises the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSS
• the L comprises from about 14 to about 19 amino acids. In some embodiments, the L comprises about 14, about 15, about 16, about 17, about 18, or about 19 amino acids. In some embodiments, the L has a length of from about 14 to about 19 amino acids. In some embodiments, the L has a length of about 14, about 15, about 16, about 17, about 18, or about 19 amino acids.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 25); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, leu, Lys, Phe, Thr, Trp or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 26); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, Leu, Lys, Thr or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 27); wherein X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
• the scFv is in the VL-L-VH orientation. In some embodiments, the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H105; the VL comprises a Cys at L42; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H105; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H105; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H5; the VL comprises a Cys at L42; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H5; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H5; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H3; the VL comprises a Cys at L42; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H3; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H3; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation
• the VH comprises a Cys at H43; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H43; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H43; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H43; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H40; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H40; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H40; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H40; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H46; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H46; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H46; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H46; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the L comprises the amino acid sequence of SEQ ID NO:
• the L comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 7.
• the binding molecules comprises a heavy chain, a light chain, and a polypeptide, wherein the N-terminus of the heavy chain and the light chain form the Fab; wherein the polypeptide comprises the scFv at the N-terminus; and wherein the C- terminus of the polypeptide and the C-terminus of the heavy chain form the Fc region.
• the Fab binds to a tumor antigen and the scFv binds to a T cell antigen.
• the tumor antigen is BCMA and the T cell antigen is CD3.
• the scFv comprises the amino acid sequence of SEQ ID NO: 126 or SEQ ID NO: 128.
• the Fab comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 132, and a VL comprising the amino acid sequence of SEQ ID NO: 129; or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 137, and a VL comprising the amino acid sequence of SEQ ID NO: 135.
• the VH comprises a Cys at H105; the VL comprises a Cys at L43; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the present disclosure provides a molecule comprising an antigen- binding fragment (Fab) that binds to a first antigen, and a single chain variable fragment (scFv) that binds to a second antigen, and a fragment crystallizable region (Fc region), wherein the scFv comprises a means for stabilizing the scFv.
• Fab antigen- binding fragment
• scFv single chain variable fragment
• Fc region fragment crystallizable region
• the scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), and wherein the means for stabilizing the scFv comprises: a) a disulfide bond between a structurally conserved surface exposed VH cysteine (Cys) and a L Cys; b) a disulfide bond between a structurally conserved surface exposed VL Cys and a L Cys; or c) a first disulfide bond between a structurally conserved surface exposed VH Cys and a first L Cys and a second disulfide bond between the structurally conserved surface exposed VL Cys and a second L Cys.
• VH heavy chain variable region
• L linker
• VL light chain variable region
• the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position and the L comprises a L Cys
• the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a L Cys
• the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position
• the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position
• the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.
• the distance between the VH Cys and the VL Cys is from about 5 A to about 10 A or from about 7 A to about 9 A.
• the VH Cys is at H3, H5, H40, H43, H46 or H105, and/or wherein the VL Cys is at L3, L5, L39, L42, L43, L45, L100 or L102, and wherein the residue numbering is according to Chothia.
• the VH Cys is at Hl 05 and the VL Cys is at L42; the VH Cys is at H43 and the VL Cys is at L100; the VH Cys is at H3 and the VL Cys is at L3; the VH Cys is at H3 and the VL Cys is at L5; the VH Cys is at H3 and the VL Cys is at L39; the VH Cys is at H3 and the VL Cys is at L42; the VH Cys is at H3 and the VL Cys is at L45; the VH Cys is at H3 and the VL Cys is at L100; the VH Cys is at H3 and the VL Cys is at L102; the VH Cys is at H5 and the VL Cys is at L3; the VH Cys is at H5 and the VL Cys is at L5; the VH Cys is at H5 and the VL Cys is at L39; the VH Cys is at H Cys is at
• the L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region.
• Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region. In some embodiments, the Ig hinge region is derived from a human Ig hinge region.
• the human Ig hinge region is an IgGl, IgG2, IgG3, or IgG4 isotype.
• the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gin), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) or tyrosine (Tyr), and y is an integer from 1 to 3.
• X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gin), histidine (His), isoleucine
• the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer from 1 to 3.
• the L comprises the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47), CSS
• the L comprises from about 14 to about 19 amino acids. In some embodiments, the L comprises about 14, about 15, about 16, about 17, about 18, or about 19 amino acids. In some embodiments, the L has a length of from about 14 to about 19 amino acids. In some embodiments, the L has a length of about 14, about 15, about 16, about 17, about 18, or about 19 amino acids.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 25); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, leu, Lys, Phe, Thr, Trp or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 26); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, Leu, Lys, Thr or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 27); wherein X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
• the scFv is in the VL-L-VH orientation.
• the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H105; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H105; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H5; the VL comprises a Cys at L42; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H5; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H5; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H3; the VL comprises a Cys at L42; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H3; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H3; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H43; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H43; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H43; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H43; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H40; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H40; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H40; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H40; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H46; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H46; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H46; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the VH comprises a Cys at H46; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the L comprises the amino acid sequence of SEQ ID NO:
• the L comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 7.
• the molecules comprises a heavy chain, a light chain, and a polypeptide, wherein the N-terminus of the heavy chain and the light chain form the Fab; wherein the polypeptide comprises the scFv at the N-terminus; and wherein the C-terminus of the polypeptide and the C-terminus of the heavy chain form the Fc region.
• the Fab binds to a tumor antigen and the scFv binds to a T cell antigen.
• the tumor antigen is BCMA and the T cell antigen is CD3.
• the scFv comprises the amino acid sequence of SEQ ID NO: 126 or SEQ ID NO: 128.
• the Fab comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 132, and a VL comprising the amino acid sequence of SEQ ID NO: 129; or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 137, and a VL comprising the amino acid sequence of SEQ ID NO: 135.
• the VH comprises a Cys at H105; the VL comprises a Cys at L43; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the present disclosure provides polynucleotides encoding the molecules disclosed herein or fragments thereof, or polypeptides thereof.
• the present disclosure provides vectors comprising the polynucleotides disclosed herein.
• the present disclosure provides host cells comprising the vectors disclosed herein.
• the host cell is a prokaryotic cell.
• the host cell is an eukaryotic cell.
• the present disclosure provides methods of producing the presently disclosed molecules.
• the method comprises culturing the presently disclosed host cell in conditions so that the molecule is produced, and purifying the produced molecule.
• the method comprises introducing the presently disclosed polynucleotide into a host cell; culturing the host cell in conditions so that the molecule is produced, and purifying the produced molecule.
• compositions comprising the presently disclosed molecules.
• the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.
• the present disclosure provides means for producing the presently disclosed molecules.
• the present disclosure provides methods for directing or engaging a cell to a target cell.
• the method comprises contacting the target cell with the presently disclosed molecule.
• the Fab binds to a first antigen on the target cell and the scFv binds to a second antigen on a cell.
• the cell is an immune cell.
• the immune cell is a T cell.
• the target cell is a tumor cell.
• the method is for treating a disease or disorder in a subject.
• the disease or disorder is a tumor.
• the disease or disorder is cancer.
• the subject is a human subject.
• the present disclosure provides methods for eliminating or inhibiting a target cell.
• the method comprises contacting the target cell with the presently disclosed molecule.
• the present disclosure provides methods for treating a disease or disorder in a subject. In some embodiments, the method comprises administering to the subject the presently disclosed molecule. [00101] In one aspect, the present disclosure provides the presently disclosed molecules for use as a medicament. In one aspect, the present disclosure provides molecules for use in treating a disease or disorder.
• FIG. 1 shows an exemplary design of the stabilized bispecific BCMA/CD3 antibody.
• the CD3 scFvs of the bispecific antibody are connected by a flexible linker and the linker is stabilized to the scFv (spFv) with disulfide bonds between the staple sequence in the linker and anchor points.
• FIGS. 2A-2B show improved thermal stability of Cris7a/b domains by the stapling.
• FIG. 2A Overlay of DSC thermograms for Cris7a scFv, Cris7a spFv, Cris7b scFv and Cris7b spFv. Parameters related to protein design and enthalpy features from analysis are listed in Table 12 and Table 14.
• FIG. 2B SDS-PAGE of scFv and spFv proteins of Cris7a/Cris7b in LH orientation.
• FIGS. 3A-3D show size exclusion chromatography of Cris7b comprising bispecific molecules.
• FIGS. 4A-4D show size exclusion chromatography of CD3B219 comprising bispecific molecules.
• FIG. 5 shows thermal stability of CD3/BCMA bispecific molecules.
• FIG. 6 shows cytotoxicity of CD3/BCMA bispecific molecules.
• CD3/BCMA bispecific molecules killed BCMA + H929'GFP + cells.
• FIG. 7 shows activation of CD4 + /CD25 + effector cells by CD3/BCMA bispecific molecules.
• FIG. 8 shows activation of CD8 + /CD25 + effector cells by CD3/BCMA bispecific molecules.
• FIG. 9 shows yield of proteins by spFv bispecific molecules.
• FIG. 10 shows aggregation resistance of spFv bispecific molecules.
• FIG. 11 shows similar CD3 binding affinity of scFv bispecific molecules and spFv bispecific molecules.
• FIG. 12 shows similar CD3-mediated killing properties of scFv bispecific molecules and spFv bispecific molecules.
• FIGS. 13A-13E show stapling of scFvs.
• FIG. 13A scFv stapling to improve low stability and minimize breathing mediated aggregation.
• FIG. 13B Cartoon schematic of the “stapling” scheme, using HL configuration as an example. A similar scheme is valid for the LH construct. The dashed line indicated the flexible linker connecting the C-terminus of a leading variable region to the stapling “CPPC” motif, followed by a second dashed line connecting to the N-terminus of a trailing variable region. The segment labeled “CPPC” in the middle of the linker indicated one possible design of a “staple,” which occurs naturally in an IgGl hinge.
• FIG. 13C Graphical illustration of anchor point selection geometry consideration (HL configuration) mapped onto Fv of a germline human antibody (PDB ID 5119, GLkl).
• Anchor points for HL orientation are Chothia position 43 for VH (H43C) and position 100 for VL (L100C); for LH: Chothia position 42 in VL (L42C) and 105 in VH (H105C).
• H43C Chothia position 43 for VH
• L100C position 100 for VL
• LH Chothia position 42 in VL
• H105C 105 in VH
• FIGS. 14A-14G show structures and comparison of various scFv/spFv domains.
• FIG. 14A GLkl spFv LH.
• FIG. 14B GLkl spFv HL.
• FIG. 14C GLk2 spFv HL.
• FIG. 14D 2mFo-dFc electron density (contoured at 1.5 ⁇ ) of the staple motif CPPC and SS to anchor points for Glk2 spFv HL. Circles indicate the stapling disulfide density.
• FIG. 14E CAT2200b spFv HL.
• FIG. 14F unbound CAT2200b spFv HLL as compared to CAT2200a scFv LH bound to IL-17.
• FIG. 14G front and back views of unbound CAT2200b spFv HL as compared to CAT2200a spFv LH bound to IL- 17.
• FIG. 15 shows the staple and linker conformations in five spFv structures.
• the CPPC motif were re-labeled as Cysl, Prol, Pro2, Cys2 for clarity.
• the structures are superimposed on the mainchain of the CPPC motif.
• the dashed lines indicate C ⁇ -C ⁇ and C ⁇ -C ⁇ distances between the Cysl and Cys2 residues.
• the range of C ⁇ -C ⁇ distances observed in all copies of the linker staple Cys residues are indicated.
• N-termini are indicated with ‘Nter’
• C-termini are indicated with ‘Cter’.
• FIGS. 16A-16D show improved yields, product quality and expected disulfide formation in the stapled linker of spFv bispecific molecules.
• FIG. 16A Schematic of BCMA (Fab) ⁇ CD3 (scFv/spFv) bispecific molecular architecture. HK in Fc regions indicate the knob-in-hole (K, knob; H, hole) mutations for Fc heterodimerization. RF (H435R and Y436F) mutations in the Fab-comprising chain for purification to prevent the binding of Protein A to the RF-comprising chain monomers or homodimers.
• FIG. 16A Schematic of BCMA (Fab) ⁇ CD3 (scFv/spFv) bispecific molecular architecture. HK in Fc regions indicate the knob-in-hole (K, knob; H, hole) mutations for Fc heterodimerization.
• FIG. 16B SEC profiles of post-CHl of scFv/spFv Cris7b-comprising molecules with BCMB749 indicate the presence of oligomer species (labeled O) in the scFv proteins but absent in the spFv proteins (monomer, M).
• FIG. 16C Schematic of the expected disulfides in the stapled bispecific molecules. All Cys residues are indicated by their sequential positions/numbers in their respective polypeptide chains. Expected disulfide bonds are indicated by lines connecting them. The dotted lines represent the additional disulfide bonds in the stapled region of the single chain Fv. Inter-chain disulfide bonds are shown in solid double lines.
• TIC Total ion current
• FIGS. 17A-17D show stability and retained binding affinity to CD3 of Cris7b- comprising spFv bispecific molecules.
• FIG. 17A NanoDSF traces of Cris7b-comprising scFv/spFv bispecific molecules with BCMB749 showed ⁇ 10 C transition to higher Tm with incorporation of stapling mutations.
• FIG. 17B and 17C Cris7b spFv bispecific molecules were resistant to heat induced aggregation. SEC traces (FIG. 17B) and quantification of aggregate levels (FIG.
• FIG. 17C showed that Cris7b-comprising spFv bispecific molecules had a dramatic reduction in heat induced aggregation over 6 week time frame at either 4 °C or 40 °C.
• FIG. 17D BLI binding traces showed comparable binding features (e.g., association and dissociation) regarding their binding to recombinant CD3.
• Light gray Cris7b spFv
• Dark gray Cris7b scFv Bird
• Dashed lines Cris7b G4S.
• FIGS. 18A-18C show similar functions between spFv bispecific molecules and their non-stapled counterparts.
• FIG. 18A spFv bispecific molecules exhibited potent killing activity of BCMA + cancer cells.
• FIG. 18B and 18C scFv/spFv bispecific molecules activated CD4 + /CD25 + (FIG. 18B) and CD8 + /CD25 + (FIG. 18C) T cells with similar potency. The null control showed no killing or T cell activating activity.
• FIGS. 19A-19B illustrate anchor points selection in VL and VH sequences.
• FIG. 19A Proposed linkers between a VL (SEQ ID NO: 144) and a VH (SEQ ID NO: 144). The variable number of amino acid residues (aa) gives flexibility and allows proper linker-anchor disulfide formation but is not long enough to allow disulfide scrambling.
• FIG. 19B The VL and VH sequences are numbered according to the Chothia numbering scheme (Chothia and Lesk 1987) and indicated the above sequences. The anchor points are highlighted in pairs with a number (1 or 2) underneath a chosen position.
• Pairs 1 and 2 are for LH and HL constructs, respectively.
• Anchor points for HL orientation are Chothia position 43 for VH (H43C) and position 100 for VL (L100C); for LH: Chothia position 42 in VL (L42C) and 105 in VH (H105C).
• FIGS. 20A-20E show disulfide bond geometry (FIG. 20A) and C ⁇ -C ⁇ and C ⁇ - C ⁇ distance distributions between anchor points for stapling (FIG. 20B and FIG. 20C) and between positions of direct interchain disulfide bonds (FIG. 20D and FIG. 20E).
• FIG. 20A Cartoon to illustrate the location of C ⁇ -C ⁇ and C ⁇ -C ⁇ distances in a formed disulfide bond. Relative distances between Coc and C ⁇ residues strongly impacted the efficiency of disulfide bond formation. In evaluating the distance distributions in FIGS. 20B-20E, the two Cys residues at the anchor positions are unlikely to form disulfide bonds directly as the distances, particularly C ⁇ -C ⁇ are much longer than typical for positions that form SS bonds. For inter- VL/VH disulfide bonds (DS1 and DS2, FIG. 20D and FIG.
• FIG. 20A and FIG. 20B C ⁇ -C ⁇ and C ⁇ -C ⁇ distances, respectively, for the two anchor positions selected for LH and HL stapling.
• FIG. 20C and FIG. 20D C ⁇ -C ⁇ and C ⁇ -C ⁇ distances, respectively, for the DS1 and DS2 in known antibody structures.
• the C ⁇ and C ⁇ distances for protein disulfide bonds are from Dani et al. (2003) Protein Eng. 16(3): 187-193.
• FIGS. 21A-21C show stapling anchor to terminus geometry.
• FIG. 21A Schematic illustration of the distances. Nter: N terminus; Cter: C terminus; d: distance between and VL and VH anchor points; dl-d4: dl: leading segment from domain 1; distance from C-terminus of domain 1 (VH, in cartoon) to Cys anchor residue of domain 1 (VH, in cartoon).
• FIG. 21B and FIG. 21C Distance distributions for the same set of Fv fragments as in Figure S2 for the LH and HL configurations. Methods were the same as provided for FIG. 20.
• FIG. 22 shows H bonding between El of a trailing VL domain and backbone of the trailing linker segment of Glk2 spFv HL structure.
• FIG. 23 shows humanization and sequence alignment of BCMB749.
• Each sequence alignment comprises the parental (top), selected human acceptor germline sequence (middle) and the CDR-grafted with back mutations italicized (bottom).
• CDRs are underlined.
• Bold CDR support positions in the framework regions. Boxed: VL/VH interface residues.
• BCMB749 VL SEQ ID NO: 129
• BCMB749 VH SEQ ID NO: 132
• huKVl-12*01 SEQ ID NO: 149
• huHVl-3*01 SEQ ID NO: 150
• BCMB749h_VL SEQ ID NO: 135)
• BCMB749h_VH SEQ ID NO: 137.
• FIG. 24 shows analytical SEC traces comparing product quality of small-scale produced CD3 -comprising bispecific samples with either scFv (left) or spFv (right) arms after purification.
• Upper plots display bispecific molecules that comprise a Cris7b variant; lower plots display bispecific molecules that comprise an alternative anti-CD3 binding variant.
• Plots on the left comprise a murine anti-BCMA Fab arm; plots on the right comprise a humanized anti-BCMA Fab arm.
• FIGS. 25A-25C show mass spectrometry mapping of disulfides in Byos.
• FIG. 25A Calculated and observed mass results for all disulfide bonded di-peptide species after LysC and ProAlanase digestions.
• FIG. 25B Fc disulfide 262-322.
• FIG. 25C spFv disulfide 119-237.
• FIG. 25B and FIG. 25C (Upper left panel) MSI of the expected mass is within 2 ppm of the calculated disulfide species; (Upper right panel) Extracted ion chromatogram (XIC), depicting the retention time of the expected disulfide. The signal of the recovered peptides was in the mid-range. (Bottom panel) MS/MS coverage for both peptides of the disulfide.
• FIGS. 26A-26C show no impact on antibody binding by stapling.
• FIG. 26A ELISA titration against recombinant CD3 showed comparable binding to an antigen, independent of the presence of scFv or spFv arm, which indicates that stapling mutations do not impede antigen binding.
• FIG. 26B ELISA titration against recombinant BCMA showed comparable binding to an antigen, independent of the presence of scFv or spFv arm, which supports that stapling mutations do not impact binding of partner arm.
• FIG. 26A ELISA titration against recombinant CD3 showed comparable binding to an antigen, independent of the presence of scFv or spFv arm, which indicates that stapling mutations do not impede antigen binding.
• FIG. 26B ELISA titration against recombinant BCMA showed comparable binding to an antigen, independent of the presence of scFv or s
• FIG. 27 shows a comparison of biophysical properties of scFv/spFv bi- and tri- specifics.
• BsAb bispecific antibody
• TsAb trispecific antibody, where 1 and 2 indicate target 1 and 2.
• sc/sp indicate format (scFv/spFv) for the single chain moiety. All affinity values by SPR. *cell binding EC50. Values for the scFv/spFv moieties only are given.
• transitional terms “comprising,” “consisting essentially of,” and “consisting of’ are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of’ excludes any element, step, or ingredient not specified in the claim; and (iii) “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.
• Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of’ and “consisting essentially of.”
• “About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result or embodiment, “about” means within one standard deviation per the practice in the art, or a range of up to 5%, whichever is larger.
• “Alternative scaffold” refers to a single chain protein framework that contains a structured core associated with variable domains of high conformational tolerance.
• the variable domains tolerate variation to be introduced without compromising scaffold integrity, and hence the variable domains can be engineered and selected for binding to a specific antigen.
• Antibody-dependent cellular cytotoxicity refers to the mechanism of inducing cell death that depends upon the interaction of antibody-coated target cells with effector cells possessing lytic activity, such as natural killer cells (NK), monocytes, macrophages and neutrophils via Fc gamma receptors (Fc ⁇ R) expressed on effector cells.
• lytic activity such as natural killer cells (NK), monocytes, macrophages and neutrophils via Fc gamma receptors (Fc ⁇ R) expressed on effector cells.
• ADCP antibody-dependent cellular phagocytosis
• antigen refers to any molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) that is capable of mediating an immune response.
• Non-limiting exemplary immune responses include antibody production and activation of immune cells, such as T cells, B cells, or NK cells.
• antigen binding fragment refers to a portion of a protein that binds an antigen.
• Antigen binding fragments may be synthetic, enzymatically obtainable or genetically engineered polypeptides and include portions of an immunoglobulin that bind an antigen, such as a heavy chain variable region (VH), a light chain variable region (VL), a Fab, a Fab’, F(ab’)2, a Fd, and Fv fragments, domain antibodies (dAb) consisting of a VH domain or a VL domain, camelized VH domains, VHH domains, minimal recognition units consisting of amino acid residues that mimic the CDRs of an antibody, such as FR3-CDR3-FR4 portions, the HCDR1, the HCDR2 and/or the HCDR3 and the LCDR1, the LCDR2 and/or the LCDR3, alternative scaffolds that bind an antigen, and multispecific molecules comprising the antigen binding fragments.
• VH heavy chain variable region
• VL light
• Antigen binding fragments may be linked together via a linker to form various types of single antibody designs in which the VH/VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and the VL domains are expressed by separate single chains, to form a monovalent antigen binding domain, such as a single chain variable fragment (scFv) or a diabody.
• Antigen binding fragments may also be conjugated to other antibodies, proteins, antigen binding fragments or alternative scaffolds, which may be monospecific or multispecific, to engineer bispecific and multispecific molecules.
• antibodies in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen binding fragments, multispecific antibodies, such as bispecific, trispecific, tetraspecific, etc., dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity.
• “Full length antibodies” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g., IgM).
• Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CHI, hinge, CH2 and CH3).
• Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL).
• the VH and the VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR).
• CDR complementarity determining regions
• FR framework regions
• Each VH and VL are composed of three CDRs and four FR segments, arranged from amino-to-carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
• Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
• IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.
• Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (K) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
• bispecific refers to a molecule (such as an antibody) that specifically binds two distinct antigens or two distinct epitopes within the same antigen.
• a bispecific molecule may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca cynomolgus (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.
• BCMA refers to B cell maturation antigen. BCMA is also known as CD269, and TNFRSF17 (UniProt Q02223), and is a member of the tumor necrosis receptor superfamily that is expressed in differentiated plasma cells. In some embodiments, the BCMA is human BCMA. An exemplary human BCMA nucleotide sequence is provided by GenBank Accession Number BC058291. There are four major haplotypes of the BCMA gene in the human genome (Kawasaki et al., Genes Immun. 2:276-9, 2001). In accordance with the present disclosure, the term “BCMA” encompasses all four haplotypes.
• the extracellular domain of human BCMA consists of amino acids 1 to 54 of the amino acid sequence having a Uniprot Ref. No. Q02223-1.
• the term “antibody against BCMA, anti-BCMA antibody” as used herein relates to an antibody specifically binding to BCMA.
• the anti-BCMA antibody binds to human BCMA.
• the anti-BCMA antibody binds to a portion of human BCMA.
• the anti-BCMA antibody binds to the extracellular domain of human BCMA.
• chimeric antigen receptor refers to engineered T cell receptors, which graft a ligand or antigen specificity onto immune cells, e.g., T cells (including, but not limited to, naive T cells, central memory T cells, effector memory T cells, or combinations thereof). CARs are also known as artificial T- cell receptors, chimeric T-cell receptors or chimeric immunoreceptors.
• a CAR comprises an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain.
• the intracellular domain comprises a polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.
• the transmembrane domain comprises a peptide or polypeptide that is known to span the cell membrane and can function to link the extracellular domain and the intracellular domain.
• the CAR further comprises a hinge domain, which serves as a linker between the extracellular domain and the transmembrane domain.
• CD3 refers to an antigen that is expressed on T cells as part of the multimeric T cell receptor (TCR) complex.
• CD3 consists of a homodimer or heterodimer formed from the association of two or four receptor chains: CD3 epsilon, CD3 delta, CD3 zeta and CD3 gamma.
• CD3 antibodies provided herein bind to a CD3 -epsilon polypeptide, which, together with CD3 -gamma, CD3 -delta and CD3-zeta, and the T cell receptor alpha/beta and gamma/delta heterodimers, forms the T cell receptor-CD3 complex.
• CD3 includes any CD3 variant, isoform, and species homolog, which is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding proteins of interest. In certain embodiments, the CD3 is a human CD3.
• complement-dependent cytotoxicity refers to the mechanism of inducing cell death in which the Fc effector domain of a target-bound protein binds and activates complement component Clq, which in turn activates the complement cascade leading to target cell death. Activation of complement may also result in deposition of complement components on the target cell surface that facilitate CDC by binding complement receptors (e.g., CR3) on leukocytes.
• complement receptors e.g., CR3
• CDR complementarity determining regions
• VH VH
• LCDR1, LCDR2, LCDR3 VL
• CDRs may be defined using various delineations such as Kabat (Wu et al., (1970) J Exp Med 123: 211-250); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
• CDR CDR
• HCDR1 CDR1
• HCDR2 CDR3
• LCDR1 CDR2
• LCDR3 CDR3
• the term “decrease,” “lower” or “reduce,” refers generally to the ability of a test molecule to mediate a reduced response (i.e., downstream effect) when compared to the response mediated by a control or a vehicle.
• Non-limiting exemplary responses include binding of a protein to its antigen or receptor, enhanced binding to Fc ⁇ R, or enhanced Fc effector functions, such as enhanced ADCC, CDC and/or ADCP.
• Decrease may be a statistically significant difference in the measured response between the test molecule and the control (or the vehicle), or a decrease in the measured response, such as a decrease of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 30 fold or more, such as 500, 600, 700, 800, 900 or 1000 fold or more.
• the term “enhance,” “promote” or “increase,” refers generally to the ability of a test molecule to mediate a greater response (i.e., downstream effect) when compared to the response mediated by a control or a vehicle.
• Non-limiting exemplary responses include binding of a protein to its antigen or receptor, enhanced binding to Fc ⁇ R, or enhanced Fc effector functions, such as enhanced ADCC, CDC and/or ADCP.
• Enhance may be a statistically significant difference in the measured response between the test molecule and the control (or the vehicle), or an increase in the measured response, such as an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 30 fold or more, such as 500, 600, 700, 800, 900 or 1000 fold or more.
• expression vector refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.
• heterologous refers to a polypeptide or a polynucleotide that comprises two or more polypeptides or two or more polynucleotides, which are not found in the same relationship to each other in nature.
• heterologous polynucleotide refers to a polynucleotide that comprises two or more polynucleotides, which are not found in the same relationship to each other in nature.
• heterologous polypeptide refers to a polypeptide that comprises two or more polypeptides, which are not found in the same relationship to each other in nature.
• human antibody refers to an antibody that is optimized to have minimal immune response when administered to a human subject. Variable regions of human antibody are derived from human immunoglobulin sequences. If a human antibody comprises a constant region or a portion thereof, the constant region is also derived from human immunoglobulin sequences.
• a human antibody comprises heavy and light chain variable regions that are “derived from” sequences of human origin, if the variable regions of the human antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes.
• Such exemplary systems are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice or rats carrying human immunoglobulin loci.
• Human antibody typically contains amino acid differences when compared to the immunoglobulins expressed in humans due to differences between the systems used to obtain the human antibody and human immunoglobulin loci, introduction of somatic mutations or intentional introduction of substitutions into the frameworks or CDRs, or both.
• human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to an amino acid sequence encoded by human germline immunoglobulins or rearranged immunoglobulin genes.
• human antibody may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al., (2000) J Mol Biol 296:57-86, or a synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al., (2010) J Mol Biol 397:385-396, and in Int. Patent Publ. No. W02009/085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of “human antibody”.
• humanized antibody refers to an antibody in which at least one CDR is derived from non-human species and at least one framework is derived from human immunoglobulin sequences.
• a humanized antibody may include substitutions in the frameworks so that the frameworks may not be exact copies of expressed human immunoglobulin or human immunoglobulin germline gene sequences.
• isolated refers to a homogenous population of molecules (such as scFv or spFv of the present disclosure or heterologous proteins comprising the scFv or spFv of the present disclosure), which have been substantially separated and/or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step.
• molecules such as scFv or spFv of the present disclosure or heterologous proteins comprising the scFv or spFv of the present disclosure
• isolated refers to a molecule that is substantially free of other cellular material and/or chemicals and encompasses molecules that are isolated to a higher purity, such as to about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.
• modulate refers to either enhanced or decreased ability of a test molecule to mediate an enhanced or reduced response (i.e., downstream effect) when compared to the response mediated by a control or a vehicle.
• the term “monoclonal antibody” refers to an antibody obtained from a substantially homogenous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known alterations such as removal of C-terminal lysine from the antibody heavy chain or post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation.
• Monoclonal antibodies typically bind one antigenic epitope.
• a bispecific monoclonal antibody binds two distinct antigenic epitopes.
• Monoclonal antibodies may have heterogeneous glycosylation within the antibody population.
• Monoclonal antibody may be monospecific or multispecific such as bispecific, monovalent, bivalent or multivalent.
• multispecific refers to a molecule that binds two or more distinct antigens or two or more distinct epitopes within the same antigen. Multispecific molecule may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.
• homologs such as human or monkey
• Macaca fascicularis cynomolgus, cyno
• Pan troglodytes or may bind an epitope that is shared between two or more distinct antigens.
• polynucleotide refers to a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry.
• cDNA is a typical example of a polynucleotide.
• protein or “polypeptide” refers to a molecule that comprises one or more polypeptides each comprised of at least two amino acid residues linked by a peptide bond.
• a protein may be a monomer, or a protein complex of two or more subunits, the subunits being identical or distinct. Small polypeptides of less than 50 amino acids may be referred to as “peptides”.
• a protein may be a heterologous fusion protein, a glycoprotein, or a protein modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation or biotinylation.
• recombinant refers to polynucleotides, polypeptides, vectors, viruses and other macromolecules that are prepared, expressed, created or isolated by recombinant means.
• single chain variable fragment refers to a single chain protein comprising a VH, a VL and a linker between the VH and the VL.
• the scFv may have the VL and VH in either orientation, e.g., with respect to the N- to C-terminal order of the VH and the VL.
• the scFv may thus be in the orientation VL-linker-VH or VH- linker-VL.
• scFv may be engineered to comprise disulfide bonds between the VH, the VL and the linker.
• the term “specifically binds,” “specific binding,” “specifically binding” or “binds” refers to a protein (such as a scFv) binding to an antigen or an epitope within the antigen with a greater binding affinity than for other antigens.
• the protein (such as the scFv) binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (KD) of about 1 x 10 -6 M or less, about 1 x 10 -7 M or less, about 5 x 10 -8 M or less, about 1 x 10 -8 M or less, about 1 x 10 -9 M or less, about 1 x 10 -10 M or less, about 1 x 10 -11 M or less, or about 1 x 10 -12 M or less, typically with the KD that is at least one hundred fold less than its KD for binding to a non-specific antigen (e.g., BSA, casein).
• KD equilibrium dissociation constant
• staple refers to a scFv linker that comprises one or two Cys residues that are capable of forming a disulfide bond with the anchor point Cys.
• stapled single chain Fv refers to a scFv that comprises one or more disulfide bonds between the VH and the linker or between the VL and the linker.
• the spFv comprises one disulfide bond between the VH and the linker, one disulfide bond between the VL and the linker, or two disulfide bonds with one between the VH and one between the linker and the VL and the linker.
• scFv molecules that comprise disulfide bonds between the VH and the VL are excluded from the term “spFv”.
• subject includes any human or nonhuman animal.
• Nonhuman animal includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.
• subject and patient can be used interchangeably herein.
• the subject is a human subject.
• therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
• a therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual.
• treat refers to accomplishing one or more of the following: reducing the severity and/or duration of the disorder, inhibiting worsening of symptoms characteristic of the disorder being treated, limiting or preventing recurrence of the disorder in subjects that have previously had the disorder, or limiting or preventing recurrence of symptoms in subjects that were previously symptomatic for the disorder.
• trispecific refers to a molecule (such as an antibody) that specifically binds three distinct antigens or three distinct epitopes within the same antigen.
• the trispecific molecule may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca cynomolgus (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between three or more distinct antigens.
• variant refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications, for example one or more substitutions, insertions, and/or deletions.
• L351Y_F405A_Y407V refers to L351Y, F405A and Y407V mutations in one immunoglobulin constant region.
• L351Y_F405A_Y407V/T394W refers to L351Y, F405A and Y407V mutations in a first Ig constant region and T394W mutation in a second Ig constant region present in the molecule.
• VH Cysteine or “VH Cys” refers to a Cys residue that resides in a VH framework.
• VL Cysteine or “VL Cys” refers to a Cys residue that resides in a VL framework.
• thermostable refers to a scFv retaining comparable binding to an antigen when compared to a non-heated scFv sample, which is referred to as thermostable.
• the term “improved stability” refers to a spFv of the present disclosure having an elevated melting point (Tm) when compared to a parent scFv that is devoid of disulfide bonds and Cys residues introduced into the spFv.
• the elevated Tm may be an elevation of about 2°C or more, such as about 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C or 15°C.
• anchor point refers to a scFv VH or a VL framework Cys residue that can be mutagenized to Cys without an adverse effect to the overall scFv structure and is capable of forming a disulfide bond with a Cys residing in a scFv linker.
• surface exposed refers to an amino acid residue that is at least partially exposed to a surface of a protein and accessible to solvent, such as accessible to deuteriation. Algorithms are well-known in the art for predicting surface accessibility of residues based on a primary sequence or a protein. Alternatively, surface exposed residues may be identified from a crystal structure of a protein.
• LTBR refers to a polypeptide that is a cell surface receptor for lymphotoxin involved in apoptosis and cytokine release, which is a member of the tumor necrosis factor receptor superfamily.
• LTBR can also be referred to as “tumor necrosis factor receptor superfamily member 3 (TNFRSF3).”
• TNFRSF3 tumor necrosis factor receptor superfamily member 3
• LTBR is expressed on the surface of many cell types, including cells of epithelial and myeloid lineages.
• LTBR can bind the lymphotoxin membrane form (a complex of lymphotoxin-alpha and lymphotoxin-beta). Activation of LTBR can trigger apoptosis via TRAF3 and TRAF5 and can lead to release of interleukin 8.
• the LTBR is a human LTBR.
• An exemplary human LTBR comprises the amino acid sequence with a UniProt number P36941.
• Antigen binding single chain variable fragments are molecules that can be utilized as therapeutics, imaging agents, diagnostic agents, or as portions of heterologous molecules such as multispecific molecules, and the like in view of the art and the extensive teachings in the present specification.
• scFv stapled Fv
• heterologous and multispecific molecules comprising the spFv, polynucleotides encoding them, vectors, host cells and methods of making and using them.
• the present disclosure is based, at least in part, on the identification of suitable residue positions in the VH and/or the VL (herein referred to as VH anchor point or VL anchor point) and in the flexible linker (herein referred to as staple) which may be engineered to cysteine residues resulting in formation of disulfide bonds between the linker and the variable domains in the scFv.
• VH anchor point or VL anchor point
• staple flexible linker
• the “stapling” strategy described herein is widely applicable to various molecules, including, but not limited to, all VH/VL domains and pre-existing scFv molecules providing, inter alia, structural identity to scFv with improved stability.
• the spFv described herein may be conjugated into any heterologous protein, bispecific or multispecific format, including chimeric antigen receptors (CAR), T cell redirection molecules, bispecific and multispecific molecules and may be used as therapeutic, diagnostic and detection molecules.
• CAR chimeric antigen receptors
• T cell redirection molecules bispecific and multispecific molecules and may be used as therapeutic, diagnostic and detection molecules.
• the present disclosure provides various spFvs.
• the present disclose provides an isolated single chain variable fragment (scFv) comprising a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv comprises: a) a disulfide bond between a structurally conserved surface exposed VH cysteine (Cys) and a L Cys; b) a disulfide bond between a structurally conserved surface exposed VL Cys and a L Cys; or c) a first disulfide bond between a structurally conserved surface exposed VH Cys and a first L Cys and a second disulfide bond between a structurally conserved surface exposed VL Cys and a second L Cys.
• scFv isolated single chain variable fragment
• VH heavy chain variable region
• L linker
• VL light chain variable region
• the scFv comprises: a) a disulfide bond between a
• the present disclosure also provides an isolated scFv comprising a VH, a L, and a VL, wherein a) the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position and the L comprises a L Cys; b) the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a L Cys; or c) the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position, the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position, and the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.
• the disulfide bond is formed during expression of the scFv.
• a spFv consists of one disulfide bond, which is formed between a L Cys and a VH Cys or between a L Cys and a VL Cys.
• such spFvs are referred to as “half-anchored spFvs”.
• the anchor positions are the same in a spFv comprising one or two disulfide bonds.
• the linker Cys position may vary in the half- anchored spFvs as long as it satisfies distance and geometry requirements for disulfide bond formation with the anchor point.
• the half-anchored spFv restrains VL/VH relative movement similar to a VL/VH pair stabilized with two disulfide bonds. Thus, a half-anchored spFv is also stabilized.
• the VH and VL in the spFvs may be anchored in any orientation.
• the N-terminus of the VH is anchored to the C-terminus of the VL.
• the C-terminus of the VH is anchored to the N-terminus of the VL Anchor positions also depend on VL and VH orientations and not all VL and VH anchor points can be paired.
• the presently disclosed spFvs have increased stability as compared to the parent scFvs devoid of the disulfide bond(s).
• Stability includes thermal stability and mechanical stability. Thermostability may be evaluated using differential thermal calorimetry (DSC), in which DSC scans are performed using heated protein samples (such as samples heated to 100°C) followed by analyses of the resulting thermal melting profiles using 2-state or non-2-state transitions. For non-2-sate transitions, two transitions (Tml and Tm2) are recorded which correspond to the melting Tm of the VL and the VH domains, respectively.
• DSC differential thermal calorimetry
• the spFvs have increased thermal stability as compared to the parent scFv devoid of the disulfide bond(s).
• the Tm of the spFv is about 10°C higher than that of the parent scFv devoid of the disulfide bond(s) regardless of the Tm of the parent scFv.
• the presently disclosed spFvs have significantly improved yields and quality of the bispecific monomer as compared to the parent scFv devoid of the disulfide bond(s). In some embodiments, the presently disclosed spFvs have reduced aggregation upon heat stress at high concentrations as compared to the parent scFv devoid of the disulfide bond(s). In some embodiments, the presently disclosed spFv molecule is a multispecific molecule. In some embodiments, the spFv molecule is a bispecific molecule. In other embodiments, the spFv molecule is a trispecific molecule.
• the spFv molecule has improved developability as compared to the parent scFv devoid of the disulfide bond(s).
• stapling can increase the success of scFv conversion, thus allowing more scFv molecules to be available as molecular building blocks for therapeutic constructs.
• the distance between the VH Cys and the VL Cys is from about 5 A to about 10 A. In some embodiments, the distance between the VH Cys and the VL Cys is from about 7 A to about 9 A. In some embodiments, the distance between the VH Cys and the VL Cys is about 7 A. In some embodiments, the distance between the VH Cys and the VL Cys is about 8 A. In some embodiments, the distance between the VH Cys and the VL Cys is about 9 A.
• the VH Cys is at H3, H5, H40, H43, H46 or H105, wherein the residue numbering is according to Chothia.
• the VH Cys is at H3.
• the VH Cys is at H5.
• the VH Cys is at H40.
• the VH Cys is at H43.
• the VH Cys is at H46.
• the VH Cys is at Hl 05
• the VL Cys is at L3, L5, L39, L42, L43, L45, L100 or
• the VL Cys is at L3.
• the VL Cys is at L5.
• the VL Cys is at L39.
• the VL Cys is at L42.
• the VL Cys is at L43.
• the VL Cys is at L45.
• the VL Cys is at L100.
• the VL Cys is at L102.
• the VH Cys is at Hl 05 and the VL Cys is at L42. [00207] In some embodiments, the VH Cys is at Hl 05 and the VL Cys is at L43 [00208] In some embodiments, the VH Cys is at H43 and the VL Cys is at L100 [00209] In some embodiments, the VH Cys is at H3 and the VL Cys is at L3. [00210] In some embodiments, the VH Cys is at H3 and the VL Cys is at L5. [00211] In some embodiments, the VH Cys is at H3 and the VL Cys is at L39.
• the VH Cys is at H3 and the VL Cys is at L42. [00213] In some embodiments, the VH Cys is at H3 and the VL Cys is at L45. [00214] In some embodiments, the VH Cys is at H3 and the VL Cys is at L100. [00215] In some embodiments, the VH Cys is at H3 and the VL Cys is at L102. [00216] In some embodiments, the VH Cys is at H5 and the VL Cys is at L3. [00217] In some embodiments, the VH Cys is at H5 and the VL Cys is at L5.
• the VH Cys is at H5 and the VL Cys is at L39. [00219] In some embodiments, the VH Cys is at H5 and the VL Cys is at L42. [00220] In some embodiments, the VH Cys is at H5 and the VL Cys is at L45. [00221] In some embodiments, the VH Cys is at H5 and the VL Cys is at L100. [00222] In some embodiments, the VH Cys is at H5 and the VL Cys is at L102. [00223] In some embodiments, the VH Cys is at H40 and the VL Cys is at L3.
• the VH Cys is at H40 and the VL Cys is at L5. [00225] In some embodiments, the VH Cys is at H40 and the VL Cys is at L39. [00226] In some embodiments, the VH Cys is at H40 and the VL Cys is at L42. [00227] In some embodiments, the VH Cys is at H40 and the VL Cys is at L45.
• the VH Cys is at H40 and the VL Cys is at L100 [00229] In some embodiments, the VH Cys is at H40 and the VL Cys is at L102 [00230] In some embodiments, the VH Cys is at H43 and the VL Cys is at L3. [00231] In some embodiments, the VH Cys is at H43 and the VL Cys is at L5. [00232] In some embodiments, the VH Cys is at H43 and the VL Cys is at L39. [00233] In some embodiments, the VH Cys is at H43 and the VL Cys is at L42.
• the VH Cys is at H43 and the VL Cys is at L45. [00235] In some embodiments, the VH Cys is at H43 and the VL Cys is at L102 [00236] In some embodiments, the VH Cys is at H46 and the VL Cys is at L3. [00237] In some embodiments, the VH Cys is at H46 and the VL Cys is at L5. [00238] In some embodiments, the VH Cys is at H46 and the VL Cys is at L39. [00239] In some embodiments, the VH Cys is at H46 and the VL Cys is at L42. [00240] In some embodiments, the VH Cys is at H46 and the VL Cys is at L45.
• the VH Cys is at H46 and the VL Cys is at L100.
• the VH Cys is at H46 and the VL Cys is at L102.
• the VH Cys is at Hl 05 and the VL Cys is at L3. [00244] In some embodiments, the VH Cys is at Hl 05 and the VL Cys is at L5.
• the VH Cys is at H105 and the VL Cys is at L39.
• the VH Cys is at Hl 05 and the VL Cys is at L45.
• the VH Cys is at Hl 05 and the VL Cys is at L100
• the VH Cys is at Hl 05 and the VL Cys is at L102 [00249]
• the residue numbering of the VH and the VL regions is according to Chothia.
• Chothia numbering is well known. Other numbering systems, such as Kabat or IMGT numbering, or sequential numbering may also be used to number the VH and the VL residue positions.
• Table 1 shows the correspondence between Chothia, Kabat and sequential numbering for an exemplary VH, GLkl VH (SEQ ID NO: 60).
• Table 2 shows the correspondence between Chothia, Kabat and sequential numbering for an exemplary VL, GLkl VL (SEQ ID NO: 56).
• the L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region.
• Ig hinge region is derived from a human or a non-human Ig hinge region. Exemplary non-human Ig hinge regions are those from mouse, rat, dog, chicken and non-human primates, such as monkeys.
• the Ig hinge region is derived from a human Ig hinge region.
• the human Ig hinge region is an IgGl, IgG2, IgG3, IgG4, IgM, IgA or IgE isotype.
• the Ig hinge region includes residue 216 and terminates at residue 230 of a human IgG, wherein the residue numbering is according to the EU Index. In some instances, a lower hinge region from about residue 231 to about residue 237 may also be included in the IgG hinge region.
• the IgGl hinge region comprises the amino acid sequence of SEQ ID NO: 63, which is provided below. In some embodiments, the IgGl hinge region comprises the amino acid sequence of SEQ ID NO: 64, which is provided below.
• the hinge regions of other Ig isotypes are well known and their amino acid sequences may be obtained for example at ImMunoGeneTics web site.
• the Ig hinge region is an IgG2 hinge region. In some embodiments, the IgG2 hinge comprises the amino acid sequence of SEQ ID NO: 65, which is provided below.
• the L comprises a contiguous amino acid sequence, which is derived from an Ig hinge region.
• the L comprises at least a portion of an Ig hinge region or at least a portion of an engineered Ig hinge region.
• An engineered Ig hinge region comprises one or more mutations as compared to a wild-type Ig hinge region.
• mutations that may be introduced include substitutions of Cys residues (e.g., to reduce the number of Cys in the L to one or two), substitution of Pro residues, or any conservative modifications (such as conservative substitutions).
• Conservative modifications refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody comprising the amino acid modifications.
• Conservative modifications include amino acid substitutions, additions, and deletions.
• Conservative amino acid substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain.
• amino acids with acidic side chains e.g., aspartic acid, glutamic acid
• basic side chains e.g., lysine, arginine, histidine
• nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
• uncharged polar side chains e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan
• aromatic side chains e.g., phenylalanine, tryptophan, histidine, tyrosine
• aliphatic side chains e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine
• amide e.g., asparagine, glutamine
• any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol ScandSuppl 643:55-67; Sasaki et al., (1988) Adv Biophys 35:1-24).
• Amino acid substitutions to may be made by known methods for example by PCR mutagenesis (US Pat. No. 4,683,195).
• the resulting variant hinges may be incorporated into the spFv constructs of the disclosure and tested for their characteristics such as stability and binding to an antigen using known assays and assays described herein.
• the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gin), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) or tyrosine (Tyr), and y is an integer from 1 to 3.
• Pro may be included into the L to provide rigidity.
• Gly may be included into the L to allow maximum flexibility. Any other amino acid may also be used in the L except for Cys and Met.
• the L comprises the amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer from 1 to 3.
• the L comprises the amino acid sequence CPC, CGC, ,
• the L comprises the amino acid sequence CPC.
• the L comprises the amino acid sequence CGC.
• the L comprises the amino acid sequence CSC.
• the L comprises the amino acid sequence CPPC (SEQ ID NO: 1)
• the L comprises the amino acid sequence CGPC (SEQ ID NO: 28).
• the L comprises the amino acid sequence CPGC (SEQ ID NO: 29).
• the L comprises the amino acid sequence CGGC (SEQ ID NO: 30).
• the L comprises the amino acid sequence CSPG (SEQ ID NO: 31).
• the L comprises the amino acid sequence CPSC (SEQ ID NO: 32).
• the L comprises the amino acid sequence CSSC (SEQ ID NO: 33). [00267] In some embodiments, the L comprises the amino acid sequence CGSC (SEQ ID NO: 34).
• the L comprises the amino acid sequence CSGC (SEQ ID NO: 35).
• the L comprises the amino acid sequence CPPPC (SEQ ID NO: 36).
• the L comprises the amino acid sequence CGPPC (SEQ ID NO: 37).
• the L comprises the amino acid sequence CPGPC (SEQ ID NO: 38).
• the L comprises the amino acid sequence CPPGC (SEQ ID NO: 39).
• the L comprises the amino acid sequence CGGPC (SEQ ID NO: 40).
• the L comprises the amino acid sequence CPGGC (SEQ ID NO: 41).
• the L comprises the amino acid sequence CGGGC (SEQ ID NO: 42).
• the L comprises the amino acid sequence CSPPC (SEQ ID NO: 43).
• the L comprises the amino acid sequence CPSPC (SEQ ID NO: 44).
• the L comprises the amino acid sequence CPPSC (SEQ ID NO: 45).
• the L comprises the amino acid sequence CSSPC (SEQ ID NO: 46).
• the L comprises the amino acid sequence CPSSC (SEQ ID NO: 47).
• the L comprises the amino acid sequence CSSSC (SEQ ID NO: 48).
• the L comprises the amino acid sequence CGSPC (SEQ ID NO: 49). [00283] In some embodiments, the L comprises the amino acid sequence CPGSC (SEQ ID NO: 50).
• the L comprises the amino acid sequence CSGPC (SEQ ID NO: 51).
• the L comprises the amino acid sequence CPSGC (SEQ ID NO: 52).
• the L comprises from about 15 to about 20 amino acids. In some embodiments, the L has a length of from about 15 to about 20 amino acids.
• the L comprises from about 14 to about 19 amino acids. In some embodiments, the L has a length of from about 14 to about 19 amino acids. In some embodiments, the L comprises about 14 amino acids. In some embodiments, the L has a length of about 14 amino acids. In some embodiments, the L comprises about 15 amino acids. In some embodiments, the L has a length of about 15 amino acids. In some embodiments, the L comprises about 16 amino acids. In some embodiments, the L has a length of about 16 amino acids. In some embodiments, the L comprises about 17 amino acids. In some embodiments, the L has a length of about 17 amino acids. In some embodiments, the L comprises about 18 amino acids.
• the L has a length of about 18 amino acids. In some embodiments, the L comprises about 19 amino acids. In some embodiments, the L has a length of about 19 amino acids. In some embodiments, the L comprises about 20 amino acids. In some embodiments, the L has a length of about 20 amino acids.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 25), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, leu, Lys, Phe, Thr, Trp or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 26), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, Leu, Lys, Thr or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 27); wherein X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3, and n is an integer from 4 to 6.
• the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. [00292] In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 7.
• the L has a length of from about 5 to about 10 amino acids.
• the L comprises about 5 amino acids. In some embodiments, the L consists of about 5 amino acids. In some embodiments, the L comprises 7 amino acids. In some embodiments, the L consists of 7 amino acids. In some embodiments, the L comprises 8 amino acids. In some embodiments, the L consists of 8 amino acids. In some embodiments, the L comprises 9 amino acids. In some embodiments, the L consists of 9 amino acids. In some embodiments, the L comprises about 10 amino acids. In some embodiments, the L consists of about 10 amino acids.
• the L further comprises a trailing segment.
• the trailing segment has a length of 4 amino acids. In some embodiments, the trailing segment has a length of 5 amino acids.
• the L comprises a 9+4+5 configuration.
• the spFv is in the VL-L-VH orientation. In some embodiments, the spFv is in the VH-L-VL orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H105; the VL comprises a Cys at L42; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H105; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H105; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H5; the VL comprises a Cys at L42; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H5; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H5; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H3; the VL comprises a Cys at L42; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H3; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H3; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H43; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H43; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H43; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure also provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H43; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure also provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H40; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure also provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H40; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure also provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H40; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H40; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H46; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H46; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H46; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H46; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• the present disclosure provides a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H105; the VL comprises Cys at L43; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the L comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 7.
• the present disclosure provides molecules comprising the presently disclosed spFvs (e.g., those disclosed in Section 4.1.1).
• the molecules are multispecific molecules.
• the molecules are heterologous molecules.
• the spFv of the present disclosure may be conjugated to a second molecule.
• second molecules include half-life extending moieties, imaging agents, therapeutic agents, antibodies comprising various antibody formats and fragments thereof, antigen binding domains, Fc regions, and immunoglobulin heavy /light chains or fragments thereof.
• the molecule comprises a single chain variable fragment (scFv) comprising a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv comprises a disulfide bond between a structurally conserved surface exposed VH cysteine (Cys) and a L Cys; a disulfide bond between a structurally conserved surface exposed VL Cys and a L Cys; or a first disulfide bond between a structurally conserved surface exposed VH Cys and a first L Cys and a second disulfide bond between a structurally conserved surface exposed VL Cys and a second L Cys.
• scFv single chain variable fragment
• VH heavy chain variable region
• L linker
• VL light chain variable region
• the molecule comprises a scFv comprising a VH, a L and a VL, wherein the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position and the L comprises a L Cys; the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a L Cys; or the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position, the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.
• the distance between the VH Cys and the VL Cys is from about 5 A to about 10 A. In some embodiments, the distance between the VH Cys and the VL Cys is from about 7 A to about 9 A.
• the VH Cys is at H3, H5, H40, H43, H46 or H105, wherein the residue numbering is according to Chothia.
• the VL Cys is at L3, L5, L39, L42, L43, L45, L100 or L102, wherein the residue numbering is according to Chothia.
• the VH Cys is at Hl 05 and the VL Cys is at L42.
• the VH Cys is at Hl 05 and the VL Cys is at L43.
• the VH Cys is at H43 and the VL Cys is at L100.
• the VH Cys is at H3 and the VL Cys is at L3.
• the VH Cys is at H3 and the VL Cys is at L5.
• the VH Cys is at H3 and the VL Cys is at L39.
• the VH Cys is at H3 and the VL Cys is at L42.
• the VH Cys is at H3 and the VL Cys is at L45.
• the VH Cys is at H3 and the VL Cys is at L100.
• the VH Cys is at H3 and the VL Cys is at L102.
• the VH Cys is at H5 and the VL Cys is at L3.
• the VH Cys is at H5 and the VL Cys is at L5.
• the VH Cys is at H5 and the VL Cys is at L39.
• the VH Cys is at H5 and the VL Cys is at L42.
• the VH Cys is at H5 and the VL Cys is at L45. [00342] In some embodiments, the VH Cys is at H5 and the VL Cys is at L100.
• the VH Cys is at H5 and the VL Cys is at L102.
• the VH Cys is at H40 and the VL Cys is at L3.
• the VH Cys is at H40 and the VL Cys is at L5.
• the VH Cys is at H40 and the VL Cys is at L39.
• the VH Cys is at H40 and the VL Cys is at L42.
• the VH Cys is at H40 and the VL Cys is at L45.
• the VH Cys is at H40 and the VL Cys is at L100.
• the VH Cys is at H40 and the VL Cys is at L102.
• the VH Cys is at H43 and the VL Cys is at L3.
• the VH Cys is at H43 and the VL Cys is at L5.
• the VH Cys is at H43 and the VL Cys is at L39.
• the VH Cys is at H43 and the VL Cys is at L42.
• the VH Cys is at H43 and the VL Cys is at L45.
• the VH Cys is at H43 and the VL Cys is at L100.
• the VH Cys is at H43 and the VL Cys is at L102.
• the VH Cys is at H46 and the VL Cys is at L3.
• the VH Cys is at H46 and the VL Cys is at L5.
• the VH Cys is at H46 and the VL Cys is at L39.
• the VH Cys is at H46 and the VL Cys is at L42.
• the VH Cys is at H46 and the VL Cys is at L45.
• the VH Cys is at H46 and the VL Cys is at L100.
• the VH Cys is at H46 and the VL Cys is at L102.
• the VH Cys is at Hl 05 and the VL Cys is at L3.
• the VH Cys is at Hl 05 and the VL Cys is at L5.
• the VH Cys is at H105 and the VL Cys is at L39.
• the VH Cys is at Hl 05 and the VL Cys is at L42.
• the VH Cys is at Hl 05 and the VL Cys is at L45.
• the VH Cys is at Hl 05 and the VL Cys is at L100.
• the VH Cys is at Hl 05 and the VL Cys is at L102.
• the L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region.
• Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region.
• Non-limiting examples of non-human Ig hinge regions include those from mouse, rat, dog, chicken and non-human primates, such as monkeys.
• the Ig hinge region is derived from a human Ig hinge region.
• the human Ig hinge region is an IgGl, IgG2, IgG3, IgG4, IgM, IgA or IgE isotype.
• the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, Leu, Lys, Phe, Thr, Trp or Tyr, and y is an integer from 1 to 3.
• Pro may be included into the linker to provide rigidity.
• Gly may be included into the linker to allow maximum flexibility. Any other amino acid may also be used in the L except for Cys and Met.
• the L comprises the amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer from 1 to 3.
• the L comprises the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 47),
• the L comprises from about 15 to about 20 amino acids. In some embodiments, the L has a length of from about 15 to about 20 amino acids. In some embodiments, the L comprises from about 14 to about 19 amino acids. In some embodiments, the L has a length of from about 14 to about 19 amino acids. In some embodiments, the L comprises about 14 amino acids. In some embodiments, the L has a length of about 14 amino acids. In some embodiments, the L comprises about 15 amino acids. In some embodiments, the L has a length of about 15 amino acids. In some embodiments, the L comprises about 16 amino acids. In some embodiments, the L has a length of about 16 amino acids. In some embodiments, the L comprises about 17 amino acids.
• the L has a length of about 17 amino acids. In some embodiments, the L comprises about 18 amino acids. In some embodiments, the L has a length of about 18 amino acids. In some embodiments, the L comprises about 19 amino acids. In some embodiments, the L has a length of about 19 amino acids. In some embodiments, the L comprises about 20 amino acids. In some embodiments, the L has a length of about 20 amino acids.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 25); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, leu, Lys, Phe Thr, Trp or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 26); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, Leu, Lys, Thr or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 27); wherein X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
• the spFv is in the VL-L-VH orientation. In some embodiments, the spFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H105; the VL comprises a Cys at L42; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H105; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H105; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H5; the VL comprises a Cys at L42; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H5; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H5; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H3; the VL comprises a Cys at L42; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H3; the VL comprises a Cys at L45; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H3; the VL comprises a Cys at L39; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H43; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H43; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H43; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H43; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H40; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H40; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H40; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H40; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H46; the VL comprises a Cys at L100; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H46; the VL comprises a Cys at L102; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H46; the VL comprises a Cys at L5; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H46; the VL comprises a Cys at L3; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VH-L-VL orientation.
• a molecule comprising a scFv comprising a VH, a L and a VL, wherein the VH comprises a Cys at H105; the VL comprises a Cys at L43; the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and the scFv is in the VL-L-VH orientation.
• the L comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the L comprises the amino acid sequence of SEQ ID NO: 7.
• the scFv of the present disclosure is conjugated to a second protein, a polynucleotide, a therapeutic agent, a cytotoxic agent, or a detectable label.
• the second protein is a half-life extending moiety.
• the second protein is an antibody or a fragment thereof.
• the second protein is an antigen binding fragment.
• the second protein is a therapeutic molecule.
• the present disclosure provides, in some embodiments, molecules comprising the presently disclosed spFvs (e.g., those disclosed in Section 4.1.1) and half-life extending moieties.
• the presently disclosed spFv is conjugated to a half-life extending moiety.
• Non-limiting examples of half-life extending moieties include an immunoglobulin (Ig), a fragment of an Ig, an Ig constant region, a fragment of an Ig constant region, a Fc region, transferrin, albumin, albumin variants, an albumin binding domain, or polyethylene glycols (PEGs).
• Amino acid sequences of human Igs are well known. Human Igs include IgGl, IgG2, IgG3, IgG4, IgM, IgA, and IgE.
• the spFv of the present disclosure is conjugated to an Ig or a fragment thereof. In some embodiments, the spFv of the present disclosure is conjugated to a Fc region. In some embodiments, the spFv of the present disclosure is conjugated to transferrin. In some embodiments, the spFv of the present disclosure is conjugated to albumin. In some embodiments, the spFv of the present disclosure is conjugated to an albumin binding protein. In some embodiments, the spFv of the present disclosure is conjugated to a polyethylene glycol (PEG). Non-limiting examples of PEGs include PEG5000 and PEG20,000.
• the spFv of the present disclosure is conjugated to a fatty acid or a fatty acid ester, e.g., for desired properties.
• fatty acids and fatty acid esters include laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides), and the like.
• the half-life extending moiety may be a direct fusion with the spFv of the present disclosure and may be generated by standard cloning and expression techniques. Alternatively, well-known chemical coupling methods may be used to attach the moieties to recombinantly produced spFvs of the present disclosure.
• the present disclosure provides molecules comprising the presently disclosed spFvs (e.g., those disclosed in Section 4.1.1), which are conjugated to a therapeutic agent, a cytotoxic agent, or a detectable label.
• Such molecules may be used to direct therapeutics, mediate killing, visualize, identify, and/or purify cells that express the antigen to which the spFv binds to, in vitro or in vivo.
• Detectable label includes compositions that, when conjugated to the presently disclosed spFv, renders the latter detectable, via, for example, spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
• Non-limiting examples of detectable labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorochromes, fluorophores, fluorescent quenching agents, colored molecules, radioactive isotopes, scintillates, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni 2+ , Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors, acridinium esters, and colorimetric substrates.
• enzymes for example, as commonly used in an ELISA
• biotin digoxigenin
• haptens luminescent molecules
• chemiluminescent molecules chemiluminescent molecules
• a detectable label may emit a signal spontaneously, such as when the detectable label is a radioactive isotope. In other cases, the detectable label emits a signal as a result of being stimulated by an external field.
• radioactive isotopes include ⁇ -emitting, Auger-emitting, ⁇ -emitting, an alpha-emitting, and positron-emitting radioactive isotope.
• Non-limiting examples of radioactive isotopes include 3 H, 11 C, 13 C, 15 N, 18 F, 19 F, 55 Co, 57 Co, 60 Co, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 68 Ga, 72 As, 75 Br, 86 Y, 89 Zr, 90 Sr, 94m Tc, 99m Tc, 115 In, 123 1, 124 1, 125 I, 131 1, 211 At, 212 Bi, 213 Bi, 223 Ra, 226 Ra, 225 Ac and 227 Ac.
• the metal atoms are metals with an atomic number greater than 20, including, but not limited to, calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, bromine, krypton, rubidium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, antimony, tellurium, iodine, xenon, cesium, barium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, thallium, lead, bismuth, francium, radium, actinium, cerium, praseodymium, neodym
• the metal atoms are alkaline earth metals with an atomic number greater than twenty.
• the metal atoms are lanthanides.
• the metal atoms are actinides.
• the metal atoms are transition metals.
• the metal atoms are poor metals.
• the metal atoms are gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms.
• the metal atoms are metals with an atomic number of 53 (i.e., iodine) to 83 (i.e., bismuth).
• the metal atoms are atoms suitable for magnetic resonance imaging.
• the metal atoms are metal ions in the form of +1, +2, or +3 oxidation states, such as Ba 2+ , Bi 3+ , Cs + , Ca 2+ , Cr 2+ , Cr 3+ , Cr 6+ , Co 2+ , Co 3+ , Cu + , Cu 2+ , Cu 3+ , Ga 3+ , Gd 3+ , Au + , Au 3+ , Fe 2+ , Fe 3+ , F 3+ , Pb 2+ , Mn 2+ , Mn 3+ , Mn 4+ , Mn 7+ , Hg 2+ , Ni 2+ , Ni 3+ , Ag + , Sr 2 *, Sn 2+ , Sn 4+ , and Zn 2+ .
• the metal atoms may comprise a metal oxide, including, but not limited to, iron oxide, manganes
• Suitable dyes include any commercially available dyes, including, but not limited to 5(6)-carboxyfluorescein, IRDye 680RD maleimide, IRDye 800CW, ruthenium polypyridyl dyes, and the like.
• Suitable fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIR) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes.
• FITC fluorescein isothiocyanate
• fluorescein thiosemicarbazide e.g., Texas Red
• CyDyes e.g., Cy3, Cy5, Cy5.5
• Alexa Fluors e.g., Alexa488, Alexa555, Alexa594; Alexa647
• NIR near infrared
• the molecule comprising the presently disclosed scFv conjugated to a detectable label may be used as an imaging agent.
• the detectable label is also a cytotoxic agent.
• the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (c.g, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radio-conjugate).
• the cytotoxic agent is daunomycin, doxorubicin, methotrexate, vindesine, bacterial toxins such as diphtheria toxin, ricin, geldanamycin, maytansinoids or calicheamicin.
• the cytotoxic agent may elicit their cytotoxic and cytostatic effects by mechanisms including, but not limited to, tubulin binding, DNA binding, or topoisomerase inhibition.
• the cytotoxic agent is an enzymatically active toxin such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
• exotoxin A chain from Pseudomonas aeruginosa
• ricin A chain abrin A chain
• modeccin A chain alpha-sarcin
• Aleurites fordii proteins dianthin proteins
• the cytotoxic agent is a radionuclide, such as 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
• the cytotoxic agent is dolastatins or dolostatin peptidic analogs and derivatives, auristatin or monomethyl auristatin phenylalanine.
• exemplary molecules are disclosed in U.S. Pat No. 5,635,483 and 5,780,588. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division and have anticancer and antifungal activity.
• the dolastatin or auristatin drug moiety may be attached to the presently disclosed spFv through the N- terminus or the C- terminus of the peptidic drug moiety (see e.g., W002/088172), or via any cysteine engineered into a protein.
• Conjugation to a detectable label may be done using known methods.
• the detectable label is complexed with a chelating agent.
• the detectable label is conjugated to the presently disclosed spFv via a linker.
• the detectable label or the cytotoxic agent may be linked directly, or indirectly, to the spFv of the present disclosure using known methods.
• Suitable linkers are known in the art and include, but are not limited to, prosthetic groups, non-phenolic linkers (derivatives of N-succimidyl-benzoates; dodecaborate), chelating moi eties of both macrocyclics and acyclic chelators, such as derivatives of 1,4, 7, 10-tetraazacyclododecane- 1,4, 7, 10, tetraacetic acid (DOTA), derivatives of diethylenetriaminepentaacetic avid (DTP A), derivatives of S-2-(4- Isothiocyanatobenzyl)-l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA) and derivatives of l,4,8,l l-tetraazacyclodocedan-l,4,8,l l-tetraacetic acid (TETA
• the presently disclosed spFv may be conjugated to an Ig constant region or a fragment thereof.
• the present disclosure provides molecules comprising the presently disclosed spFv (e.g., one disclosed in Section 4.1.1) and an Ig constant region or a fragment thereof.
• the Ig constant region or fragment thereof can impart antibody-like properties, including Fc effector functions Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis or down regulation of cell surface receptors (e.g., B cell receptor; BCR).
• the Ig constant region or fragment thereof can also function as a half-life extending moiety as described herein.
• the presently disclosed spFv may also be engineered into full length antibodies using standard methods. The full length antibodies comprising the spFv may be further engineered as described herein.
• An immunoglobulin heavy chain constant region is comprised of subdomains CHI, hinge, CH2 and CH3.
• the CHI domain spans residues 118-215, the CH2 domain residues 231-340 and the CH3 domain residues 341-447 on the heavy chain, wherein the residue numbering is according to the EU Index.
• residue 341 is referred to as a CH2 domain residue.
• a hinge includes residue 216 and terminates at 230 of a human IgGl.
• a hinge includes a lower hinge region from about residue 231 to about residue 237 as described herein.
• An Ig Fc region comprises at least the CH2 and the CH3 domains of the Ig constant region, and therefore comprises at least a region from about 231 to 447 of an Ig heavy chain constant region.
• the Ig constant region is a heavy chain constant region.
• the Ig constant region is a light chain constant region.
• the fragment of the Ig constant region comprises a Fc region. In some embodiments, the fragment of the Ig constant region comprises a CH2 domain. In some embodiments, the fragment of the Ig constant region comprises a CH3 domain. In some embodiments, the fragment of the Ig constant region comprises a CH2 domain and a CH3 domain. In some embodiments, the fragment of the Ig constant region comprises at least portion of a hinge, a CH2 domain and a CH3 domain. A portion of the hinge refers to one or more amino acid residues of an Ig hinge. In some embodiments, the fragment of the Ig constant region comprises a hinge, a CH2 domain and a CH3 domain.
• the spFv is conjugated to the N-terminus of the Ig constant region or fragment thereof. In some embodiments, the spFv is conjugated to the C-terminus of the Ig constant region or fragment thereof.
• the molecule comprising the presently disclosed spFv and the Ig constant region or fragment thereof may be assessed for their functionality using several known assays. Binding to a target antigen may be assessed using methods described herein. Altered properties imparted by the Ig constant domain or fragment thereof (e.g., a Fc region) may be assayed in Fc receptor binding assays using soluble forms of the receptors, such as Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII or FcRn, or using cell-based assays measuring for example ADCC, CDC or ADCP.
• ADCC may be assessed using an in vitro assay using cells that express the antigen to which the spFv of the present disclosure binds to as target cells and NK cells as effector cells. Cytolysis may be detected by the release of a label (e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells.
• a label e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins
• target cells are used with a ratio of 1 target cell to 4 effector cells.
• Target cells are pre- labeled with BATDA and combined with effector cells and the test antibody. The samples are incubated for 2 hours and cell lysis is measured by measuring released BATDA into the supernatant. Data are normalized to maximal cytotoxicity with 0.67% Triton X-100 (Sigma Aldrich) and minimal control determined by spontaneous release of BATDA from target cells in the absence of any antibody.
• ADCP may be evaluated by using monocyte-derived macrophages as effector cells and any cells that express the antigen to which the presently disclosed spFv binds to as target cells which are engineered to express GFP or other labeled molecule.
• effectortarget cell ratio may be for example 4: 1. Effector cells may be incubated with target cells for 4 hours with or without the antibody of the invention. After incubation, cells may be detached using accutase. Macrophages may be identified with anti-CDl lb and anti- CD14 antibodies coupled to a fluorescent label, and percent phagocytosis may be determined based on % GFP fluorescence in the CD11 + CD14 + macrophages using standard methods.
• CDC of cells may be measured for example by plating Daudi cells at 1 x 10 5 cells/well (50 pL/well) in RPMI-B (RPMI supplemented with 1% BSA), adding 50 pL of a test protein to the wells at a final concentration of between 0 and 100 pg/mL, incubating the reaction for 15 min at room temperature, adding 11 pL of pooled human serum to the wells, and incubating the reaction for 45 min at 37° C. Percentage (%) lysed cells may be detected as % propidium iodide stained cells in FACS assay using standard methods.
• the molecule comprises an antigen-binding fragment (Fab), a single chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv comprises: a disulfide bond between a structurally conserved surface exposed VH cysteine (Cys) and a L Cys; a disulfide bond between a structurally conserved surface exposed VL Cys and a L Cys; or a first disulfide bond between a structurally conserved surface exposed VH Cys and a first L Cys and a second disulfide bond between a structurally conserved surface exposed VL Cys and a second L Cys, wherein the molecule has improved stability, expression yields, and/or quality as compared to a molecule absent a disulfide bond, e.g.,
• the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position and the L comprises a L Cys
• the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a L Cys
• the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position
• the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.
• the distance between the VH Cys and the VL Cys is from about 5 A to about 10 A. In some embodiments, the distance between the VH Cys and the VL Cys is from about 7 A to about 9 A.
• the VH Cys is at H3, H5, H40, H43, H46 or H105, wherein the residue numbering is according to Chothia. In some embodiments, the VL Cys is at L3, L5, L39, L42, L43, L45, L100 or L102, wherein the residue numbering is according to Chothia.
• the VH Cys is at Hl 05 and the VL Cys is at L42; the VH Cys is at H43 and the VL Cys is at L100; the VH Cys is at H3 and the VL Cys is at L3; the VH Cys is at H3 and the VL Cys is at L5; the VH Cys is at H3 and the VL Cys is at L39; the VH Cys is at H3 and the VL Cys is at L42; the VH Cys is at H3 and the VL Cys is at L45; the VH Cys is at H3 and the VL Cys is at L100; the VH Cys is at H3 and the VL Cys is at L102; the VH Cys is at H5 and the VL Cys is at L3; the VH Cys is at H5 and the VL Cys is at L5; the VH Cys is at H5 and the VL Cys is at L39; the VH
• the molecule comprises a scFv (or spFv) that binds CD3 and a Fab that binds BCMA.
• the scFv comprises the amino acid sequence of SEQ ID NO: 125.
• the scFv comprises the amino acid sequence of SEQ ID NO: 126.
• the scFv comprises the amino acid sequence of SEQ ID NO: 127.
• the scFv comprises the amino acid sequence of SEQ ID NO: 128.
• the scFv comprises a VH, a L and a VL. The L links the VH and the VL.
• the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
• the L comprises the amino acid sequence of SEQ ID NO: 2.
• the linker comprises SEQ ID NO: 3.
• the L comprises the amino acid sequence of SEQ ID NO: 4.
• the L comprises the amino acid sequence of SEQ ID NO: 5.
• the L comprises the amino acid sequence of SEQ ID NO: 6.
• the L comprises the amino acid sequence of SEQ ID NO: 7.
• the VH comprises a Cys at Hl 05.
• the VL comprises a Cys at L43.
• the VH comprises a Cys at Hl 05, and the VL comprises a Cys at L43.
• the scFv is in the VL-L-VH orientation.
• the scFv (or spFv) that binds to CD3 is conjugated to an Ig constant region.
• the Ig constant region comprises the amino acid sequence of SEQ ID NO: 133.
• the Ig constant region comprises the amino acid sequence of SEQ ID NO: 139.
• the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 140.
• the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 141.
• the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 142.
• the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 143.
• the Fab that binds BCMA comprises a VH and a VL.
• the VH of the Fab comprises the amino acid sequence of SEQ ID NO: 132.
• the VH of the Fab comprises the amino acid sequence of SEQ ID NO: 137.
• the VL of the Fab comprises the amino acid sequence of SEQ ID NO: 129.
• the VL of the Fab comprises the amino acid sequence of SEQ ID NO: 135.
• the BCMA VH/VL comprises SEQ ID NO: 132 and SEQ ID NO: 129.
• the VH of the Fab comprises the amino acid sequence of SEQ ID NO: 132 and the VL of the Fab comprises the amino acid sequence of SEQ ID NO: 135. In other embodiments, the VH of the Fab comprises SEQ ID NO: 137 and SEQ ID NO: 129. In some embodiments, the BCMA VH/VL comprises the amino acid sequence of SEQ ID NO: 137 and the VL of the Fab comprises the amino acid sequence of SEQ ID NO: 135. In a further embodiment, the BCMA VH is conjugated to an Ig constant region. In some embodiments, the Ig constant region comprises the amino acid sequence of SEQ ID NO: 133.
• the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 134. In some embodiments, the molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 138. [00457] In some embodiments, the molecule is a bispecific molecule.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131 or SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134 or SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO: 143.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 140.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 141.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 142. In some embodiments, the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 143.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 140.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 141.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 142. In some embodiments, the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 143.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 140.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 141.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 142.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 143.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 140.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 141.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 142.
• the bispecific molecule comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 136, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 138, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 143.
• the present disclosure provides chimeric antigen receptors (CARs) comprising the presently disclosed spFvs (e.g., those disclosed in Section 4.1.1).
• the CAR comprising the spFv of the disclosure may be monospecific or multispecific, comprising, as its extracellular domain, one or more scFvs of the present disclosure.
• Chimeric antigen receptors are genetically engineered receptors. These engineered receptors can be readily inserted into and expressed by immune cells, including T cells in accordance with techniques known in the art. With a CAR, a single receptor can be programmed to both recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen. When these antigens exist on target cells, an immune cell that expresses the CAR can target and kill the target cell.
• a CAR comprises an extracellular domain that binds the antigen ad an optional linker, a transmembrane domain, and an intracellular domain comprising a signaling domain.
• the extracellular domain of the CAR may comprise any polypeptide that binds a desired antigen.
• the extracellular domain of the CAR comprises the scFv (or spFv) disclosed herein.
• CARs may also be engineered to bind two or more desired antigens that may be arranged in tandem and separated by linker sequences.
• one or more scFvs (or spFvs) of the present disclosure, domain antibodies, llama VHH antibodies or other VH only antibody fragments may be organized in tandem via a linker to generate bispecific or multispecific CARs.
• the transmembrane domain of CAR may be derived from the transmembrane domain of CD8, an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), 4- 1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD 19, IL2R beta, IL2R gamma, IL7Ra, ITGA1 , VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, I
• the intracellular domain of the CAR further comprises a co-stimulatory domain.
• the co-stimulatory domain may be derived from the intracellular domains of one or more co-stimulatory molecules.
• Co-stimulatory molecules are well-known cell surface molecules other than antigen receptors or Fc receptors and provide a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen.
• Non-limiting examples of co-stimulatory molecules include 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.
• the intracellular domain of CAR may be derived from the signaling domains of for example CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD66d or CD39.
• An intracellular domain of a CAR refers to a part of a CAR polypeptide that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited following antigen binding to the extracellular CAR domain.
• a linker is positioned between the extracellular domain and the transmembrane domain.
• the linker is a polypeptide of about 2 to 100 amino acids in length.
• the linker may include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non-cleavable. An exemplary cleavable linker includes 2A. [00472] An exemplary CAR comprises an extracellular domain comprising the scFv (or spFv) of the present disclosure, a transmembrane domain comprising a transmembrane domain of CD8, and an intracellular domain comprising a signaling domain of CD3 ⁇ .
• An exemplary CAR comprises an extracellular domain comprising the scFv (or spFv) of the present disclosure, a transmembrane domain comprising a transmembrane domain of CD8 or a transmembrane domain of CD28, and an intracellular domain comprising a signaling domain of CD3 ⁇ and a co-stimulatory domain comprising an intracellular domain of CD28, an intracellular domain of 4-1BB, or an intracellular domain of OX40.
• CARs are generated by standard molecular biology techniques.
• the molecule is monospecific.
• the molecule is multispecific.
• the molecule is bispecific.
• the molecule is trispecific.
• the molecule is tetraspecific.
• a scFv (e.g., spFv) structure defined by the atomic coordinates provided in Table 18. In other embodiments, provided herein is a scFv (e.g., spFv) structure defined by one or more subsets of the atomic coordinates provided in Table 18. In some embodiments, provided herein is a scFv (e.g., spFv) structure defined by the atomic coordinates provided in Table 19. In other embodiments, provided herein is a scFv (e.g., spFv) structure defined by one or more subsets of the atomic coordinates provided in Table 19.
• a scFv (e.g., spFv) structure defined by the atomic coordinates provided in Table 20. In other embodiments, provided herein is a scFv (e.g., spFv) structure defined by one or more subsets of the atomic coordinates provided in Table 20. In some embodiments, provided herein is a scFv (e.g., spFv) structure defined by the atomic coordinates provided in Table 21. In other embodiments, provided herein is a scFv (e.g., spFv) structure defined by one or more subsets of the atomic coordinates provided in Table 21.
• a scFv (e.g., spFv) structure defined by the atomic coordinates provided in Table 22. In other embodiments, provided herein is a scFv (e.g., spFv) structure defined by one or more subsets of the atomic coordinates provided in Table 22. In some embodiments, provided herein is a scFv (e.g., spFv) structure defined by the atomic coordinates provided in Table 23. In other embodiments, provided herein is a scFv (e.g., spFv) structure defined by one or more subsets of the atomic coordinates provided in Table 23.
• the presently disclosed spFv may be engineered into molecules of any known format using known recombinant technologies, expression and purification protocols.
• the presently disclosed spFv may be engineered into full length multispecific antibodies having one or more mutations in the CH3 domain which promoter stability of the two half molecules. These multispecific antibodies may be generated in vitro using Fab arm exchange or by co-expression of the various chains.
• Fab arm exchange two monospecific bivalent antibodies are engineered to have the one or more substitutions in the CH3 domain, the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the multispecific antibody by Fab arm exchange.
• Non-limiting examples of reducing agents that may be used include 2- mercaptoethylamine (2-MEA), dithiothreitol (DTT), di thioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta- mercaptoethanol.
• a reducing agent is selected from the group consisting of 2- mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine.
• incubation for at least 90 min at a temperature of at least 20 °C in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.
• CH3 mutations that may be used include technologies such as Knob-in-Hole mutations (Genentech), electrostatically-matched mutations (Chugai, Amgen, NovoNordisk, Oncomed), the Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Duobody® mutations (Genmab), and other asymmetric mutations (e.g., Zymeworks).
• technologies such as Knob-in-Hole mutations (Genentech), electrostatically-matched mutations (Chugai, Amgen, NovoNordisk, Oncomed), the Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Duobody® mutations (Genmab), and other asymmetric mutations (e.g., Zymeworks).
• Knob-in-hole mutations are disclosed for example in WO 1996/027011 and include mutations on the interface of CH3 region in which an amino acid with a small side chain (hole) is introduced into the first CH3 region and an amino acid with a large side chain (knob) is introduced into the second CH3 region, resulting in preferential interaction between the first CH3 region and the second CH3 region.
• Non-limiting examples of CH3 region mutations forming a knob and a hole include T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366 S_L368 A_Y407 V.
• Heavy chain heterodimer formation may be promoted by using electrostatic interactions by substituting positively charged residues on the first CH3 region and negatively charged residues on the second CH3 region as described in US2010/0015133, US2009/0182127, US2010/028637 or US2011/0123532.
• asymmetric mutations that can be used to promote heavy chain heterodimerization include, but are not limited to, L351Y F405A Y407V/T394W, T366I K392M T394W/F405 A_Y407 V, T366L_K392M_T394W/F405 A_Y407 V, L351Y_Y407A/T366A_K409F, L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, or T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W, as described in US2012/0149876 or US2013/0195849 (Zymeworks).
• SEEDbody mutations involve substituting select IgG residues with IgA residues to promote heavy chai heterodimerization as
• Duobody® mutations are disclosed for example in US2014/0303356 and include mutations F405L/K409R, wild-type/F405L_R409K, T350I K370T F405L/K409R, K370W/K409R, D399AFGHILMNRSTVWY/K409R, T366 ADEFGHILMQ VY/K409R, L368 ADEGHNRSTVQ/K409 AGRH, D399FHKRQ/K409AGRH, F405IKLSTVW/K409AGRH and Y407LWQ/K409AGRH.
• DVD Dual Variable Domain Immunoglobulins
• VH1 -linker- VH2-CH the heavy chain having a structure VH1 -linker- VH2-CH and the light chain having the structure VLl-linker- VL2-CL; linker being optional
• structures that include various dimerization domains to connect the two antibody arms with different specificity such as leucine zipper or collagen dimerization domains
• ScFv-, diabody -based, and domain antibodies include but are not limited to, Bispecific T Cell Engager (BiTE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain antibodies.
• BiTE Bispecific T Cell Engager
• Tiandab Tandem Diabody
• DART Dual Affinity Retargeting Technology
• AIT TCR-like Antibodies
• AIT ReceptorLogics
• Human Serum Albumin ScFv Fusion Merrimack
• COMBODY Epigen Biotech
• the scFv (or spFv) of the present disclosure may also be engineered into multispecific molecules comprising three antigen binding domains.
• at least one antigen binding domain is in the form of a scFv (or spFv) of the present disclosure.
• Exemplary designs include (in which “1” indicates the first antigen binding domain, “2” indicates the second antigen binding domain, and “3” indicates the third antigen binding domain:
• Design 1 Chain A) scFvl- CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1- hinge-CH2-CH3
• Design 2 Chain A) scFvl - hinge- CH2-CH3; Chain B) VL2-CL; Chain C) VH2- CHl-hinge-CH2-CH3
• Design 3 Chain A) scFvl- CHl-hinge- CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CHl-hinge-CH2-CH3
• Design 4 Chain A) CH2-CH3 -scFvl; Chain B) VL2-CL; Chain C) VH2-CH1- hinge-CH2-CH3
• CH3 engineering may be incorporated to the Designs 1-4, including, but not limited to, mutations L351Y_F405A_Y407V/T394W,
• the Ig constant region or fragment thereof, such as a Fc region present in the presently disclosed molecules may be of any allotype or isotype.
• the Ig constant region or fragment thereof is an IgGl isotype. In some embodiments, the Ig constant region or fragment thereof is an IgG2 isotype. In some embodiments, the Ig constant region or fragment thereof is an IgG3 isotype. In some embodiments, the Ig constant region or fragment thereof is an IgG4 isotype. [00498] The Ig constant region or fragment thereof may be of any allotype. In some embodiments, the allotype has no influence on properties of the Ig constant region, such as binding or Fc-mediated effector functions.
• Immunogenicity of therapeutic proteins comprising Ig constant regions or fragments thereof is associated with increased risk of infusion reactions and decreased duration of therapeutic response (Baert et al., (2003) N Engl J Med 348:602-608).
• the extent to which therapeutic proteins comprising Ig constant regions or fragments thereof induce an immune response in the host may be determined in part by the allotype of the Ig constant region (Stickler et al., (2011) Genes and Immunity 12:213-221).
• Ig constant region allotype is related to amino acid sequence variations at specific locations in the constant region sequences of the antibody. Table 3 shows select IgGl, IgG2 and IgG4 allotypes.
• C-terminal lysine may be removed from the Ig constant region by endogenous circulating carboxypeptidases in the blood stream (Cai et al., (2011) Biotechnol Bioeng 108:404-412). During manufacturing, CTL removal may be controlled to less than the maximum level by control of concentration of extracellular Zn 2+ , EDTA or EDTA - Fe 3+ as described in U.S. Patent Publ. No. US2014/0273092. CTL content of proteins may be measured using known methods.
• the spFv conjugated to the Ig constant region has a C- terminal lysine content of from about 10% to about 90%. In some embodiments, the C- terminal lysine content is from about 20% to about 80%. In some embodiments, the C- terminal lysine content is from about 40% to about 70%. In some embodiments, the C- terminal lysine content is from about 50% to about 80%. In some embodiments, the C- terminal lysine content is from about 60% to about 80%. In some embodiments, the C- terminal lysine content is from about 50% to about 70%. In some embodiments, the C- terminal lysine content is from about 60% to about 70%. In some embodiments, the C- terminal lysine content is from about 55% to about 70%. In some embodiments, the C- terminal lysine content is about 60%.
• Fc region mutations may be made to the presently disclosed molecules comprising the Ig constant region or fragment thereof to modulate their effector functions such as ADCC, ADCP and/or ADCP and/or pharmacokinetic properties. This may be achieved by introducing mutation(s) into the Fc that modulate binding of the mutated Fc to activating Fc ⁇ Rs (Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIII), inhibitory Fc ⁇ RIIb and/or to FcRn.
• the presently disclosed molecule comprises at least one mutation in the Ig constant region or fragment thereof. In some embodiments, the at least one mutation is in the Fc region.
• the presently disclosed molecule comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen mutations in the Fc region.
• the presently disclosed molecule comprises at least one mutation in the Fc region that modulates binding of the molecule to FcRn.
• Fc positions that may be mutated to modulate half-life include, but are not limited to, positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434 and 435.
• Non-limiting examples of mutations that may be made singularly or in combination include mutations T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R.
• Non-limiting examples of singular or combination mutations that may be made to increase the half-life include mutations M428L/N434S, M252Y/S254T/T256E, T250Q/M428L, N434A and T307A/E380A/N434A.
• Non-limiting examples of singular or combination mutations that may be made to reduce the half-life include mutations H435A, P257I/N434H, D376V/N434H, M252Y/S254T/T256E/H433K/N434F, T308P/N434A and H435R.
• the presently disclosed molecule comprises M252Y/S254T/T256E mutation in the Fc region.
• the presently disclosed molecule comprises at least one mutation in the Fc region that reduces binding of the molecule to an activating Fey receptor (Fc ⁇ R) and/or reduces Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP).
• Fc ⁇ R activating Fey receptor
• Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP).
• Fc positions that may be mutated to reduce binding of the presently disclosed molecule to the activating Fc ⁇ R and subsequently to reduce effector function include, but are not limited to, positions 214, 233, 234, 235, 236, 237, 238, 265, 267, 268, 270, 295, 297, 309, 327, 328, 329, 330, 331 and 365.
• Non-limiting examples of mutations that may be made singularly or in combination include mutations K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, D265S, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331S in IgGl, IgG2, IgG3 or IgG4.
• Non-limiting examples of combination mutations that result in reduced ADCC include mutations L234A/L235A on IgGl, L234A/L235A/D265S on IgGl, V234A/G237A/ P238S/H268A/V309L/A330S/P331S on IgG2, F234A/L235A on IgG4, S228P/F234A/ L235A on IgG4, N297A on all Ig isotypes, V234A/G237A on IgG2, K214T/E233P/ L234V/L235A/G236- deleted/A327G/P331A/D365E/L358M on IgGl, H268Q/V309L/A330S/P331S on IgG2, S267E/L328F on IgGl, L234F/L235E/D265A on IgGl, L234A/
• An exemplary mutation that results in reduced CDC is a K322A mutation.
• a S228P mutation may be made in IgG4 to enhance IgG4 stability.
• the presently disclosed molecule comprises at least one mutation in the Fc region, wherein the at least one mutation is selected from the group consisting of K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331S.
• the at least one mutation comprises L234A, L235A, D265S.
• the at least one mutation comprises L234A or L235A.
• the presently disclosed molecule comprises at least one mutation in the Fc region that enhances binding of the molecule to Fc ⁇ R and/or enhances Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody- dependent cell-mediated cytotoxicity (ADCC) and/or phagocytosis (ADCP).
• CDC complement dependent cytotoxicity
• ADCC antibody- dependent cell-mediated cytotoxicity
• ADCP phagocytosis
• Fc positions that may be mutated to increase binding of the molecule to the activating Fc ⁇ R and/or enhance Fc effector functions include, but are not limited to, positions 236, 239, 243, 256,290,292, 298, 300, 305, 312, 326, 330, 332, 333, 334, 345, 360, 339, 378, 396 or 430 (the residue numbering is according to the EU index).
• Non-limiting examples of mutations that may be made singularly or in combination include G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305L, K326A, A330K, I332E, E333A, K334A, A339T and P396L.
• Non-limiting examples of combination mutations that result in increased ADCC or ADCP include a S239D/I332E, S298A/E333A/K334A, F243L/R292P/Y300L, F243L/R292P/Y300L/P396L, F243L/R292P/Y300L/V305I/P396L and G236A/S239D/I332E.
• Fc positions that may be mutated to enhance CDC include, but are not limited to, positions 267, 268, 324, 326, 333, 345 and 430.
• Non-limiting examples of mutations that may be made singularly or in combination include S267E, F1268F, S324T, K326A, K326W, E333A, E345K, E345Q, E345R, E345Y, E430S, E430F and E430T.
• Non-limiting examples of combination mutations that result in increased CDC include K326A/E333A, K326W/E333A, H268F/S324T, S267E/H268F, S267E/S324T and S267E/H268F/S324T.
• the mutations are present in a wild-type IgGl, a wild-type IgG2, or a wild-type IgG4.
• the wild-type IgGl comprises the amino acid sequences of SEQ ID NO: 66, which is provided below.
• the wild- type IgG2 comprises the amino acid sequences of SEQ ID NO: 67, which is provided below.
• the wild-type IgG4 comprises the amino acid sequences of SEQ ID NO: 68, which is provided below.
• Glycoengineering [00516] The ability of the presently disclosed molecule comprising an Ig constant region or a fragment thereof to mediate ADCC can be enhanced by engineering the oligosaccharide component of the Ig constant region or fragment thereof.
• Human IgG1 or IgG3 are N- glycosylated at Asn297 with the majority of the glycans in the well-known biantennary G0, G0F, G1, G1F, G2 or G2F forms.
• Ig constant region containing proteins may be produced by non-engineered CHO cells typically have a glycan fucose content of about at least about 85%.
• Such molecules can be achieved using different methods reported to lead to the successful expression of relatively high defucosylated immunoglobulins bearing the biantennary complex-type of Fc oligosaccharides such as control of culture osmolality (Konno et al., (2012) Cytotechnology 64:249-265), application of a variant CHO line Lec13 as the host cell line (Shields et al.,(2002) J Biol Chem 277:26733-26740), application of a variant CHO line EB66 as the host cell line (Olivier et al., (2010) MAbs;2: 405-415), application of a rat hybridoma cell line YB2/0 as the host cell line (Shinkawa et al., (2003) J Biol Chem 278:3466-3473), introduction of small interfering RNA specifically against the a 1,6- fucosyltrasferase (FUT8) gene (Mori et al., (2004) Biotechnol Bio
• the presently disclosed molecule comprising the Ig constant region or fragment thereof has a biantennary glycan structure with fucose content of about between about 1% to about 15%, for example, about 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.
• the presently disclosed molecule comprising the Ig constant region or fragment thereof has a glycan structure with fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, or 20%.
• “Fucose content” refers to the amount of the fucose monosaccharide within the sugar chain at Asn297.
• the relative amount of fucose is the percentage of fucose-containing structures related to all glycostructures. These may be characterized and quantified by multiple methods, for example: 1) using MALDI-TOF of N-glycosidase F treated sample (e.g., complex, hybrid and oligo- and high-mannose structures) as described in Int Pat. Publ. No.
• the oligosaccharides thus released can be labeled with a fluorophore, separated and identified by various complementary techniques which allow: fine characterization of the glycan structures by matrix-assisted laser desorption ionization (MALDI) mass spectrometry by comparison of the experimental masses with the theoretical masses, determination of the degree of sialylation by ion exchange HPLC (GlycoSep C), separation and quantification of the oligosaccharide forms according to hydrophilicity criteria by normal-phase HPLC (GlycoSep N), and separation and quantification of the oligosaccharides by high performance capillary electrophoresis-laser induced fluorescence (HPCE-LIF).
• MALDI matrix-assisted laser desorption ionization
• Low fucose or “low fucose content” refers to the presently disclosed molecule comprising the Ig constant region or fragment thereof with fucose content of about between about 1% and about 15%.
• Normal fucose or “normal fucose content” refers to the presently disclosed molecule comprising the Ig constant region or fragment thereof with fucose content of great than about 50%, e.g., greater than about 80% or greater than about 85%.
• Anti-idiotypic antibodies are antibodies that specifically bind to the presently disclosed spFv.
• the present disclose also provides anti -idiotypic antibodies that specifically binds to the presently disclosed spFv.
• the anti -idiotypic antibody binds to the disulfide bond in the presently disclosed spFv.
• the anti -idiotypic antibody binds to the antigen binding domain of the presently disclosed spFv.
• the present disclosure also provides polynucleotides encoding the presently disclosed spFv. Further provided are vectors comprising such polynucleotides.
• the vector is an expression vector.
• Expression vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, vectors for prokaryotic expression, vectors for eukaryotic expression, transposon based vectors or any other vector suitable for introduction of the presently disclosed polynucleotide into a given cell or organism.
• the polynucleotide encoding the presently disclosed spFv may be operably linked to control sequences in the expression vector that facilitate the expression of the spFv.
• regulatory elements may include, but are not limited to, a transcriptional promoter, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation.
• Expression vectors may also include one or more nontranscribed elements such as an origin of replication, other 5’ or 3’ flanking nontranscribed sequences, 5’ or 3’ nontranslated sequences (such as necessary ribosome binding sites), splice donor and acceptor sites, or selection markers.
• the polynucleotide may be a cDNA.
• the promoter driving spFv expression may be strong, weak, tissue-specific, inducible or developmental-specific promoter.
• Non-limiting examples of promoters include hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, and others.
• viral promoters function constitutively in eukaryotic cells and are suitable for use with the described embodiments.
• Such viral promoters include, but are not limited to, Cytomegalovirus (CMV) immediate early promoter, the early and late promoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, the long terminal repeats (LTRs) of Maloney leukemia virus, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), and other retroviruses, and the thymidine kinase promoter of Herpes Simplex Virus.
• CMV Cytomegalovirus
• MMTV Mouse Mammary Tumor Virus
• LTRs long terminal repeats
• HCV Human Immunodeficiency Virus
• EBV Epstein Barr Virus
• RSV Rous Sarcoma Virus
• thymidine kinase promoter Herpes Simplex Virus.
• Inducible promoters such as the metallothionein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter, promoters that contain one or more interferon-stimulated response elements (ISRE) such as protein kinase R 2’,5’- oligoadenylate synthetases, Mx genes and AD ARI.
• Vectors of the disclosure may also contain one or more Internal Ribosome Entry Site(s) (IRES). Inclusion of an IRES sequence into fusion vectors may be beneficial for enhancing expression of some proteins.
• Vectors of the present disclosure may be circular or linear. They may be prepared to comprise a replication system functional in a prokaryotic or eukaryotic host cell.
• Replication systems can be derived, e.g., from ColE1, SV40, 2 ⁇ plasmid, ⁇ , bovine papilloma virus, and the like.
• the expression vectors can be designed for either transient expression, for stable expression, or for both.
• the expression vectors can be made for constitutive expression or for inducible expression.
• Exemplary vectors that may be used are Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden).
• Eukaryotic pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia), pEE6.4 (Lonza) and pEE12.4 (Lonza).
• Additional vectors include the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.).
• Bacteriophage vectors such as ⁇ GT10, ⁇ GT11, ⁇ EMBL4, and ⁇ NM1149, ⁇ ZapII (Stratagene) can be used.
• Exemplary plant expression vectors include pBI01, pBI01.2, pBI121, pBI101.3, and pBIN19 (Clontech).
• Exemplary animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech).
• the expression vector may be a viral vector, e.g., a retroviral vector, e.g., a gamma retroviral vector.
• the present disclosure also provides host cells comprising the presently disclosed vectors.
• the host cell is a prokaryotic cell. In some embodiments, the host cell is an eukaryotic cell.
• “Host cell” refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but to the progeny of such a cell, and also to a stable cell line generated from the particular subject cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells or archeal cells.
• Escherichia coli, bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species are examples of prokaryotic host cells.
• Other microbes, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells.
• Exemplary eukaryotic cells may be of mammalian, insect, avian or other animal origins.
• Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines such as SP2/0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines.
• An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196).
• Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologies, Walkersville, MD), CHO-K1 (ATCC CRL-61) or DG44.
• the present disclosure also provides methods of producing the presently disclosed spFv.
• the method comprises culturing a presently disclosed host cell in conditions so that the spFv is produced, and recovering the spFv produced by the host cell. Methods of making scFvs and purifying them are known.
• the spFv may be purified according to standard procedures, including, but not limited to, ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer- Verlag, N.Y., (1982)).
• the scFv may be substantially pure, e.g., at least from about 80% to 85% pure, at least from about 85% to 90% pure, at least from about 90% to 95% pure, or at least from about 98% to 99%, or more, pure, e.g., free from contaminants such as cell debris, macromolecules, etc. other than the subject protein
• polynucleotides encoding the spFv of the disclosure may be incorporated into vectors using standard molecular biology methods. Host cell transformation, culture, antibody expression and purification are done using well known methods.
• compositions comprising the spFvs or the molecules disclosed herein.
• the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
• Carrier refers to a diluent, adjuvant, excipient, or vehicle with which the spFv or molecule is administered.
• vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
• 0.4% saline and 0.3% glycine may be used.
• These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques e.g., filtration).
• the compositions may comprise pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc.
• the concentration of the spFv or molecule in the composition may vary, from less than about 0.5%, usually to at least about 1% to as much as 15 or 20% by weight and may be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the mode of administration selected.
• Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.
• the mode of administration of the spFv, molecule, or composition disclosed herein may be any suitable route, including, but not limited to, parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, transmucosal (oral, intranasal, intravaginal, rectal) or other means appreciated by a skilled artisan.
• parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous
• transmucosal oral, intranasal, intravaginal, rectal
• the present disclosure further provides processes for preparing the presently disclosed spFvs (e.g., those disclosed in Section 4.1.1).
• the process comprises: providing a heavy chain variable region (VH) and a light chain variable region (VL) that form an antigen binding site; providing a linker (L) that comprises or is engineered to comprise a first L Cys; engineering the VH to comprise a VH Cys at a structurally conserved surface exposed VH framework residue position; and forming a disulfide bond between the VH Cys and the first L Cys to prepare a stabilized scFv.
• VH heavy chain variable region
• VL light chain variable region
• L linker
• the process comprises: providing a VH and a VL that form an antigen binding site; providing a L that comprises or is engineered to comprise a second L Cys; engineering the VL to comprise a VL Cys at a structurally conserved surface exposed VL framework residue position; and forming a disulfide bond between the VL Cys and the second L Cys to prepare a stabilized scFv.
• the process comprises: providing a heavy chain variable region (VH) and a light chain variable region (VL) that form an antigen binding site; providing a linker (L) that comprises or is engineered to comprise a first L Cys and a second L Cys; engineering the VH to comprise a VH Cys at a structurally conserved surface exposed VH framework residue position; engineering the VL to comprise a VL Cys at a structurally conserved surface exposed VL framework residue position; and forming a disulfide bond between the VH Cys and the first L Cys and a disulfide bond between the VL Cys and the second L Cys to prepare a stabilized scFv.
• VH heavy chain variable region
• VL light chain variable region
• the disulfide bond is formed during expression of the scFv.
• Any known VH/VL pair of scFv that forms an antigen binding domain may be engineered into the stabilized scFvs.
• antigen binding VH/VL pairs of interest may be identified de novo using known methods and the resulting VH/VL pairs may be engineered into spFv format.
• the hybridoma method of Kohler and Milstein may be used to identify VH/VL pairs that bind an antigen of interest and the resulting VH/VL pairs may be engineered as spFvs.
• transgenic animals such as mice, rat or chicken carrying human immunoglobulin (Ig) loci in their genome may be used to generate antigen binding fragments, and are described in for example U.S. Patent No. 6,150,584, Int. Patent Publ. No. WO1999/45962, Int. Patent Publ. Nos. W02002/066630, WO2002/43478, W02002/043478 and W01990/04036.
• the endogenous immunoglobulin loci in such animal may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the genome of the animal using homologous or non-homologous recombination, using transchromosomes, or using minigenes. Companies such as Regeneron (http://_www_regeneron_com), Harbour Antibodies (http://_www_harbourantibodies_com), Open Monoclonal Technology, Inc.
• Phage display may also be used to generate antigen binding fragments which can be engineered as spFvs.
• the spFv is humanized. In some embodiments, the spFv is human. In some embodiments, the spFv is non-human.
• the process comprises expressing a presently disclosed polynucleotide (e.g., one disclosed in Section 4.6) in a host cell to produce a stabilized scFv.
• a presently disclosed polynucleotide e.g., one disclosed in Section 4.6
• a host cell e.g., one disclosed in Section 4.6
• stabilized scFv e.g., one disclosed in Section 4.6
• spFvs exemplary spFvs, Molecules, and Linkers
• the presently disclosed spFvs or molecules comprise one or more the amino acid sequences set forth in Table 4.
• the “Cris7a VL-VH scFv” comprises the amino acid sequence of SEQ ID NO: 125.
• the “Cris7a VL-VH spFv” comprises the amino acid sequence of SEQ ID NO: 126.
• the “Cris7b VL-VH scFv” the amino acid sequence of SEQ ID NO: 127.
• the “Cris7b VL-VH spFv” comprises the amino acid sequence of SEQ ID NO: 128.
• the “BCMB749 VL” comprises the amino acid sequence of SEQ ID NO: 129.
• the “Human CL” comprises the amino acid sequence of SEQ ID NO: 130.
• the “BCMB749 LC” comprises the amino acid sequence of SEQ ID NO: 131.
• the “BCMB749 VH” of comprises the amino acid sequence of SEQ ID NO: 132.
• the “Human HC ConstantDomains 1” comprises the amino acid sequence of SEQ ID NO: 133
• the “BCMB749 HC1” comprises the amino acid sequence of SEQ ID NO: 134.
• the “BCMB749h_VL” comprises the amino acid sequence of SEQ ID NO: 135.
• the “BCMB749h LC” comprises the amino acid sequence of SEQ ID NO: 136.
• the “BCMB749h_VH” comprises the amino acid sequence of SEQ ID NO: 137.
• the “BCMB749h HC1” comprises the amino acid sequence of SEQ ID NO: 138.
• the “Human HC ConstantDomains 2” comprises the amino acid sequence of SEQ ID NO: 139.
• the “Cris7b VL-VH scFv HC2” comprises the amino acid sequence of SEQ ID NO: 140.
• the “Cris7b VL-VH spFv HC2” comprises the amino acid sequence of SEQ ID NO: 141.
• the “CD3B219a99v scFv HC2” comprises the amino acid sequence of SEQ ID NO: 142.
• the “CD3B219a99v spFv HC2” comprises the amino acid sequence of SEQ ID NO: 143.
• the scFv linker of the disclosure comprises the amino acid sequences set forth in Table 5.
• the “GLkl scFv VL-VH” linker comprises the amino acid sequence of SEQ ID NO:2
• the “GLkl spFv VL-VH” linker comprises the amino acid sequence of SEQ ID NO:3. [00563] In some embodiments, the “GLkl scFv VH-VL” linker comprises the amino acid sequence of SEQ ID NO:2.
• the “GLkl spFv VH-VL” linker comprises the amino acid sequence of SEQ ID NO:3.
• the “GLk2 scFv VL-VH” linker comprises the amino acid sequence of SEQ ID NO:2.
• the “GLk2 spFv VL-VH” linker comprises the amino acid sequence of SEQ ID NO:3.
• the “GLk2 scFv VH-VL” linker comprises the amino acid sequence of SEQ ID NO:2.
• the “GLk2 spFv VH-VL” linker comprises the amino acid sequence of SEQ ID NO:4.
• the “CAT2200a scFv VL-VH” linker of the disclosure comprises the amino acid sequence of SEQ ID NO:2.
• the “CAT2200a spFv VL-VH” linker of the disclosure comprises the amino acid sequence of SEQ ID NO:5.
• the “CAT2200b scFv VL-VH” linker of the disclosure comprises the amino acid sequence of SEQ ID NO:2.
• the “CAT2200a spFv VL-VH” linker of the disclosure comprises the amino acid sequence of SEQ ID NO:6.
• the “CAT2200a scFv VH-VL” linker of the disclosure comprises the amino acid sequence of SEQ ID NO:2.
• the “CAT2200b spFv VH-VL” linker of the disclosure comprises the amino acid sequence of SEQ ID NO:7.
• a molecule comprising an antigen-binding fragment (Fab), a single chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv comprises: a) a disulfide bond between a structurally conserved surface exposed VH position which is mutated to cysteine (Cys) and a L Cys; b) a disulfide bond between a structurally conserved surface exposed VL position which is mutated to Cys and a L Cys; or c) a first disulfide bond between a structurally conserved surface exposed VH Cys and a first L Cys and a second disulfide bond between a structurally conserved surface exposed VL Cys and a second L Cys, optionally wherein the molecule has improved stability, expression yields and/or quality
• VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position and the L comprises a L Cys
• VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a L Cys
• the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position
• the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position
• the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.
• A3 The molecule of embodiment Al or A2, wherein the distance between the VH Cys and the VL Cys is from about 5 A to about 10 A or from about 7 A to about 9 A.
• A4 The molecule of any one of embodiments A1-A3, wherein the VH Cys is at H3, H5, H40, H43, H46 or H105, wherein the residue numbering is according to Chothia.
• A5. The molecule of any one of embodiments A1-A4, wherein the VL Cys is at L3, L5, L39, L42, L43, L45, L100 or L102, wherein the residue numbering is according to Chothia.
• A6 The molecule of any one of embodiments A1-A5, wherein a) the VH Cys is at Hl 05 and the VL Cys is at L42; b) the VH Cys is at H43 and the VL Cys is at L100; c) the VH Cys is at H3 and the VL Cys is at L3; d) the VH Cys is at H3 and the VL Cys is at L5; e) the VH Cys is at H3 and the VL Cys is at L39; f) the VH Cys is at H3 and the VL Cys is at L42; g) the VH Cys is at H3 and the VL Cys is at L45; h) the VH Cys is at H3 and the VL Cys is at L100; i) the VH Cys is at H3 and the VL Cys is at L102; j) the VH Cys is at H5 and the VL Cys is at L3
• the VH Cys is at H105 and the VL Cys is at L5; mm) the VH Cys is at H105 and the VL Cys is at L39; nn) the VH Cys is at H105 and the VL Cys is at L45; oo) the VH Cys is at H105 and the VL Cys is at L100; pp) the VH Cys is at H105 and the VL Cys is at L102, or qq) the VH Cys is at H105 and the VL Cys is at L43, wherein the residue numbering is according to Chothia.
• A7 The molecule of any one of embodiments A1-A6, wherein the L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region.
• Ig immunoglobulin
• A8 The molecule of any one of embodiments A1-A7, wherein the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region.
• A9 The molecule of any one of embodiments A1-A8, wherein the Ig hinge region is derived from a human Ig hinge region.
• Ig hinge region is an IgGl, IgG2, IgG3, or IgG4 isotype.
• Al 1 The molecule of any one of embodiments A1-A10, wherein the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gin), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) or tyrosine (Tyr), and y is an integer from 1 to 3.
• X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu
• A12 The molecule of embodiment Al l, wherein the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer from 1 to 3.
• A13 The molecule of any one of embodiments A1-A12, wherein the L comprises the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO:
• A14 The molecule of any one of embodiments A1-A13, wherein the L comprises from about 14 to about 19 amino acids, such as about 14, about 15, about 16, about 17, about 18 or about 19 amino acids; and/or the L has a length of from about 14 to about 19 amino acids, such as about 14, about 15, about 16, about 17, about 18 or about 19 amino acids.
• A15 The molecule of any one of embodiments A1-A14 wherein the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 25); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, leu, Lys, Phe, Thr, Trp or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• Al 6 The molecule of embodiment Al 5, wherein the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 26); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, Leu, Lys, Thr or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, Leu, Lys, Thr or Tyr
• m is an integer from 6 to 9
• y is an integer from 1 to 3
• n is an integer from 4 to 6.
• Al 7 The molecule of embodiment Al 6, wherein the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 27); wherein X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• A18 The molecule of any one of embodiments Al -Al 7, wherein the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
• Al 9 The molecule of any one of embodiments Al -Al 8, wherein the scFv is in the VL-L-VH orientation.
• A20 The molecule of any one of embodiments Al -Al 8, wherein the scFv is in the VH-L-VL orientation.
• A21 The molecule of any one of embodiments A1-A19, wherein a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L42; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• A22 The molecule of any one of embodiments Al -Al 9, wherein a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• A23 The molecule of any one of embodiments Al -Al 9, wherein a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• A24 The molecule of any one of embodiments Al -Al 9, wherein a) the VH comprises a Cys at H5; b) the VL comprises a Cys at L42; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• A25 The molecule of any one of embodiments Al -Al 9, wherein a) the VH comprises a Cys at H5; b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• A26 The molecule of any one of embodiments Al -Al 9, wherein a) the VH comprises a Cys at H5; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• A27 The molecule of any one of embodiments Al -Al 9, wherein a) the VH comprises a Cys at H3; b) the VL comprises a Cys at L42; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• A28 The molecule of any one of embodiments A1-A19, wherein a) the VH comprises a Cys at H3; b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• A29 The molecule of any one of embodiments Al -Al 9, wherein a) the VH comprises a Cys at H3; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• A30 The molecule of any one of embodiments A1-A18 and A20, wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A31 The molecule of any one of embodiments Al -Al 8 and A20, wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A32 The molecule of any one of embodiments Al -Al 8 and A20, wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A33 The molecule of any one of embodiments A1-A18 and A20, wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A34 The molecule of any one of embodiments Al -Al 8 and A20, wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A35 The molecule of any one of embodiments Al -Al 8 and A20, wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A36 The molecule of any one of embodiments A1-A18 and A20, wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A37 The molecule of any one of embodiments A1-A18 and A20, wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A38 The molecule of any one of embodiments A1-A18 and A20, wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A39 The molecule of any one of embodiments A1-A18 and A20, wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A40 The molecule of any one of embodiments Al -Al 8 and A20, wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A41 The molecule of any one of embodiments Al -Al 8 and A20, wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• A42 The molecule of any one of embodiments A21-A41, wherein the L comprises the amino acid sequence of SEQ ID NO: 3.
• A43 The molecule of any one of embodiments A21-A41, wherein the L comprises the amino acid sequence of SEQ ID NO: 6.
• A44 The molecule of any one of embodiments A21-A41, wherein the L comprises the amino acid sequence of SEQ ID NO: 7.
• A45 The molecule of any one of embodiments A1-A44, wherein the binding molecules comprises a heavy chain, a light chain and a polypeptide, wherein the N-terminus of the heavy chain and the light chain form the Fab; wherein the polypeptide comprises the scFv at the N-terminus; and wherein the C-terminus of the polypeptide and the C-terminus of the heavy chain form the Fc region.
• A46 The molecule of any one of embodiments Al to A45, wherein the Fab binds to a tumor antigen and the scFv binds to a T cell antigen; and wherein optionally the tumor antigen is BCMA and the T cell antigen is CD3.
• A47 The molecule of embodiment A46, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 126 or SEQ ID NO: 128.
• Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 132, and a VL comprising the amino acid sequence of SEQ ID NO: 129; or (ii) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 137, and a VL comprising the amino acid sequence of SEQ ID NO: 135.
• A49 The molecule of embodiment A47 or embodiment A48, wherein a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L43; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• A50 A polynucleotide encoding the molecule of any one of embodiments
• A1-A49 or a fragment or a polypeptide thereof or a fragment or a polypeptide thereof.
• A51 A vector comprising the polynucleotide of embodiment A50.
• A52 A host cell comprising the vector of embodiment A51.
• A53 A method of producing a binding molecule, comprising culturing the host cell of embodiment A52 in conditions so that the molecule is produced, and purifying the binding molecule.
• A54 The method of embodiment A53, wherein the host cell is a prokaryotic cell.
• A55 The method of embodiment A53, wherein the host cell is an eukaryotic cell.
• compositions comprising a molecule of any one of embodiments Al- A49, optionally wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient, optionally wherein the molecule comprises an antigen-binding fragment (Fab), a single chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv comprises a heavy chain variable region (VH), a linker (L) and a light chain variable region (VL), wherein the scFv comprises: a) a disulfide bond between a structurally conserved surface exposed VH cysteine (Cys) and a L Cys; b) a disulfide bond between a structurally conserved surface exposed VL Cys and a L Cys; or c) a first disulfide bond between a structurally conserved surface exposed VH Cys and a first L Cys and a second disulfide bond between a structurally conserved surface exposed
• composition of embodiment Bl wherein a) the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position and the L comprises a L Cys; b) the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a L Cys; or c) the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position, the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.
• Cys is at H3, H5, H40, H43, H46 or H105, wherein the residue numbering is according to Chothia.
• Cys is at L3, L5, L39, L42, L43, L45, L100 or L102, wherein the residue numbering is according to Chothia.
• B6 The composition of any one of embodiments B1-B5, wherein a) the VH Cys is at Hl 05 and the VL Cys is at L42; b) the VH Cys is at H43 and the VL Cys is at L100; c) the VH Cys is at H3 and the VL Cys is at L3; d) the VH Cys is at H3 and the VL Cys is at L5; e) the VH Cys is at H3 and the VL Cys is at L39; f) the VH Cys is at H3 and the VL Cys is at L42; g) the VH Cys is at H3 and the VL Cys is at L45; h) the VH Cys is at H3 and the VL Cys is at L100; i) the VH Cys is at H3 and the VL Cys is at L102; j) the VH Cys is at H5 and the VL Cys is at L3;
• the VH Cys is at H105 and the VL Cys is at L5; mm) the VH Cys is at H105 and the VL Cys is at L39; nn) the VH Cys is at H105 and the VL Cys is at L45; oo) the VH Cys is at H105 and the VL Cys is at L100; pp) the VH Cys is at H105 and the VL Cys is at L102, qq) the VH Cys is at H105 and the VL Cys is at L43, wherein the residue numbering is according to Chothia.
• Ig immunoglobulin
• composition any one of embodiments B1-B7, wherein the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region.
• B10 The composition of any one of embodiments B1-B9, wherein the human Ig hinge region is an IgGl, IgG2, IgG3, or IgG4 isotype.
• B 11 The composition of any one of embodiments B 1 -B 10, wherein the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gin), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) or tyrosine (Tyr), and y is an integer from 1 to 3.
• X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu),
• composition of embodiment Bl 1, wherein the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer from 1 to 3.
• B14 The composition of any one of embodiments B1-B13, wherein the L comprises from about 14 to about 19 amino acids, such as about 14, about 15, about 16, about 17, about 18 or about 19 amino acids.
• Bl 5 The composition of any one of embodiments Bl -Bl 4 wherein the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 25); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, leu, Lys, Phe, Thr, Trp or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, leu, Lys, Phe, Thr, Trp or Tyr
• m is an integer from 6 to 9
• y is an integer from 1 to 3
• n is an integer from 4 to 6.
• Bl 6 The composition of embodiment Bl 5, wherein the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 26); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, Leu, Lys, Thr or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, Leu, Lys, Thr or Tyr
• m is an integer from 6 to 9
• y is an integer from 1 to 3
• n is an integer from 4 to 6.
• Bl 7 The composition of embodiment Bl 6, wherein the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 27); wherein X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• Bl 8 The composition of any one of embodiments Bl -Bl 7, wherein the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
• Bl 9 The composition of any one of embodiments Bl -Bl 8, wherein the scFv is in the VL-L-VH orientation.
• B20 The composition of any one of embodiments Bl -Bl 8, wherein the scFv is in the VH-L-VL orientation.
• B23 The composition of any one of embodiments Bl -Bl 9, wherein a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• B26 The composition of any one of embodiments Bl -Bl 9, wherein a) the VH comprises a Cys at H5; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• B27 The composition of any one of embodiments Bl -Bl 9, wherein a) the VH comprises a Cys at H3; b) the VL comprises a Cys at L42; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00660] B28.
• B29 The composition of any one of embodiments Bl -Bl 9, wherein a) the VH comprises a Cys at H3; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• B30 The composition of any one of embodiments B1-B18 and B20, wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• B31 The composition of any one of embodiments B 1 -B 18 and B20, wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• B32 The composition of any one of embodiments Bl -Bl 8 and B20, wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00665] B33.
• composition of any one of embodiments Bl -Bl 8 and B20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• B34 The composition of any one of embodiments Bl -Bl 8 and B20, wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• B35 The composition of any one of embodiments Bl -Bl 8 and B20, wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• B36 The composition of any one of embodiments B1-B18 and B20, wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• composition of embodiment Bl wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• composition of any one of embodiments B1-B18 and B20 wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• B39 The composition of any one of embodiments B1-B18 and B20, wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• B40 The composition of any one of embodiments Bl -Bl 8 and B20, wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• B41 The composition of any one of embodiments Bl -Bl 8 and B20, wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation.
• B42 The composition of any one of embodiments B21-B41, wherein the L comprises the amino acid sequence of SEQ ID NO: 3.
• B43 The composition of any one of embodiments B21-B41, wherein the L comprises the amino acid sequence of SEQ ID NO: 6.
• B44 The composition of any one of embodiments B21-B41, wherein the L comprises the amino acid sequence of SEQ ID NO: 7.
• B45 The composition of any one of embodiments B1-B43, wherein the binding molecules comprises a heavy chain, a light chain and a polypeptide, wherein the N-terminus of the heavy chain and the light chain form the Fab; wherein the polypeptide comprises the scFv at the N-terminus; and wherein the C-terminus of the polypeptide and the C-terminus of the heavy chain form the Fc region.
• Fab binds to a tumor antigen and the scFv binds to a T cell antigen; and wherein optionally the tumor antigen is BCMA and the T cell antigen is CD3.
• composition of embodiment B46, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 126 or SEQ ID NO: 128.
• B48 The composition of embodiment B46 or embodiment B47, wherein (i) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 132, and a VL comprising the amino acid sequence of SEQ ID NO: 129; or (ii) the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 137, and a VL comprising the amino acid sequence of SEQ ID NO: 135.
• CL A method of producing a binding molecule, comprising introducing a polynucleotide encoding the molecule or a fragment thereof into a host cell; culturing the host cell in conditions so that the molecule is produced, and purifying the binding molecule, wherein the molecule comprises an antigen-binding fragment (Fab), a single chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv comprises a heavy chain variable region (VH), a linker (L) and a light chain variable region (VL), wherein the scFv comprises: a) a disulfide bond between a structurally conserved surface exposed VH cysteine (Cys) and a L Cys; b) a disulfide bond between a structurally conserved surface exposed VL Cys and a L Cys; or c) a first disulfide bond between the structurally conserved surface exposed VH Cys and a first L Cy
• VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position and the L comprises a L Cys
• VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a L Cys
• the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position
• the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a first L Cys and a second L Cys
• the VH Cys and the first L Cys are capable of forming a disulfide bond
• the VL Cys and the second L Cys are capable of forming a disulfide bond.
• C5. The method of any one of embodiments C1-C4, wherein the VL Cys is at L3, L5, L39, L42, L43, L45, L100, or L102, wherein the residue numbering is according to Chothia.
• C6 The method of any one of embodiments C1-C5, wherein a) the VH Cys is at Hl 05 and the VL Cys is at L42; b) the VH Cys is at H43 and the VL Cys is at L100; c) the VH Cys is at H3 and the VL Cys is at L3; d) the VH Cys is at H3 and the VL Cys is at L5; e) the VH Cys is at H3 and the VL Cys is at L39; f) the VH Cys is at H3 and the VL Cys is at L42; g) the VH Cys is at H3 and the VL Cys is at L45; h) the VH Cys is at H3 and the VL Cys is at L100; i) the VH Cys is at H3 and the VL Cys is at L102; j) the VH Cys is at H5 and the VL Cys is at L3;
• the VH Cys is at H105 and the VL Cys is at L5; mm) the VH Cys is at H105 and the VL Cys is at L39; nn) the VH Cys is at H105 and the VL Cys is at L45; oo) the VH Cys is at H105 and the VL Cys is at L100; pp) the VH Cys is at H105 and the VL Cys is at L102, qq) the VH Cys is at H105 and the VL Cys is at L43, wherein the residue numbering is according to Chothia.
• C7 The method of any one of embodiments C1-C6, wherein the L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region.
• Ig immunoglobulin
• C8 The method of any one of embodiments C1-C7, wherein the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region.
• C11 The method of any one of embodiments Cl -CIO, wherein the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gin), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) or tyrosine (Tyr), and y is an integer from 1 to 3.
• X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu),
• C12 The method of embodiment Cl 1, wherein the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer from 1 to 3.
• C13 The method of any one of embodiments Cl -Cl 2, wherein the L comprises the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO:
• C14 The method of any one of embodiments Cl -Cl 3, wherein the L comprises from about 14 to about 19 amino acids, such as about 14, about 15, about 16, about 17, about 18 or about 19 amino acids; and/or the L has a length of from about 14 to about 19 amino acids, such as about 14, about 15, about 16, about 17, about 18 or about 19 amino acids.
• C15 The method of any one of embodiments Cl -Cl 3, wherein the L comprises from about 14 to about 19 amino acids, such as about 14, about 15, about 16, about 17, about 18 or about 19 amino acids; and/or the L has a length of from about 14 to about 19 amino acids, such as about 14, about 15, about 16, about 17, about 18 or about 19 amino acids.
• the L comprises the amino acid sequence (X)mC(X)yC(X)n (SEQ ID NO: 25); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, leu, Lys, Phe, Thr, Trp or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6. [00698] C16.
• the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 26); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Leu, Lys, Thr or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6. [00699] C17.
• C20 The method of any one of embodiments C1-C18, wherein the scFv is in the VH-L-VL orientation.
• C21 The method of any one of embodiments C1-C19, wherein a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L42; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00704] C22.
• the VH comprises a Cys at H105; b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00705] C23.
• the VH comprises a Cys at H105; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00706] C24.
• the VH comprises a Cys at H5; b) the VL comprises a Cys at L42; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00707] C25.
• the VH comprises a Cys at H5; b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00708] C26.
• the VH comprises a Cys at H5; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00709] C27.
• the VH comprises a Cys at H3; b) the VL comprises a Cys at L42; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00710] C28.
• the VH comprises a Cys at H3; b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00711] C29.
• the VH comprises a Cys at H3; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00712] C30.
• any one of embodiments C1-C18 and C20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00713] C31.
• any one of embodiments C1-C18 and C20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00714] C32.
• any one of embodiments C1-C18 and C20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00715] C33.
• any one of embodiments C1-C18 and C20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00716] C34.
• any one of embodiments C1-C18 and C20 wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00717] C35.
• any one of embodiments C1-C18 and C20 wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00718] C36.
• any one of embodiments C1-C18 and C20 wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00719] C37.
• any one of embodiments C1-C18 and C20 wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00720] C38.
• any one of embodiments C1-C18 and C20 wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00721] C39.
• any one of embodiments C1-C18 and C20 wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00722] C40.
• any one of embodiments C1-C18 and C20 wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00723] C41.
• a method for directing or engaging a cell to a target cell comprising contacting the target cell with a binding molecule, wherein the molecule comprises an antigen-binding fragment (Fab), a single chain variable fragment (scFv), and a fragment crystallizable region (Fc region), wherein the scFv comprises a heavy chain variable region (VH), a linker (L), and a light chain variable region (VL), wherein the scFv comprises: a) a disulfide bond between a structurally conserved surface exposed VH cysteine (Cys) and a L Cys; b) a disulfide bond between a structurally conserved surface exposed VL Cys and a L Cys; or c) a first disulfide bond between a structurally conserved surface exposed VH Cys and a first L Cys and a second disulfide bond between a structurally conserved surface
• Fab antigen-binding fragment
• scFv single chain variable fragment
• D2 The method of embodiment D1, wherein a) the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position and the L comprises a L Cys; b) the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a L Cys; or c) the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position, the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a first L Cys and a second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.
• D3 The method of embodiment DI or D2, wherein the distance between the VH Cys and the VL Cys is from about 7 A to about 9 A or from about 7 A to about 9 A.
• D4 The method of any one of embodiments DI -D3, wherein the VH Cys is at H3, H5, H40, H43, H46 or H105, wherein the residue numbering is according to Chothia.
• D5. The method of any one of embodiments D1-D4, wherein the VL Cys is at L3, L5, L39, L42, L43, L45, L100 or L102, wherein the residue numbering is according to Chothia.
• D6 The method of any one of embodiments D1-D5, wherein a) the VH Cys is at Hl 05 and the VL Cys is at L42; b) the VH Cys is at H43 and the VL Cys is at L100; c) the VH Cys is at H3 and the VL Cys is at L3; d) the VH Cys is at H3 and the VL Cys is at L5; e) the VH Cys is at H3 and the VL Cys is at L39; f) the VH Cys is at H3 and the VL Cys is at L42; g) the VH Cys is at H3 and the VL Cys is at L45; h) the VH Cys is at H3 and the VL Cys is at L100; i) the VH Cys is at H3 and the VL Cys is at L102; j) the VH Cys is at H5 and the VL Cys is at L3;
• the VH Cys is at H105 and the VL Cys is at L5; mm) the VH Cys is at H105 and the VL Cys is at L39; nn) the VH Cys is at H105 and the VL Cys is at L45; oo) the VH Cys is at H105 and the VL Cys is at L100; pp) the VH Cys is at H105 and the VL Cys is at L102, qq) the VH Cys is at H105 and the VL Cys is at L43, wherein residue numbering is according to Chothia.
• D7 The method of any one of embodiments D1-D6, wherein the L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region.
• Ig immunoglobulin
• D8 The method of any one of embodiments D1-D7, wherein the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region. [00743] D9. The method of any one of embodiments D1-D8, wherein the Ig hinge region is derived from a human Ig hinge region. 5 [00744] D10. The method of any one of embodiments D1-D9, wherein the human Ig hinge region is an IgG1, IgG2, IgG3, or IgG4 isotype. [00745] D11.
• the L comprises an amino acid sequence C(X)yC (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid 10 (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) or tyrosine (Tyr), and y is an integer from 1 to 3.
• X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid 10 (Asp), glutamic acid (Glu), glutamine (Gln), histidine (His), iso
• D12 The method of embodiment D11, wherein the L comprises an amino acid sequence C(X)yC (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer 15 from 1 to 3. [00747] D13.
• the L comprises the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID 20 NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO: 46), CPSSC (SEQ ID NO: 1), CGPC (SEQ ID NO
• D14 The method of any one of embodiments D1-D13, wherein the L comprises from about 14 to about 19 amino acids, such as about 14, about 15, about 16, about 17, about 18 or about 19 amino acids; and/or the L has a length of from about 14 to about 19 amino acids, such as about 14, about 15, about 16, about 17, about 18 or about 19 amino 30 acids.
• D15 The method of any one of embodiments D1-D14 wherein the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 25); wherein X is Gly, - 117 - 14620-710-228 / 362327-228710 / JBI6602WOPCT1 NAI-1534348088
• the L comprises the amino acid sequence (X)mC(X)yC(X)n (SEQ ID NO: 26); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, 5 Asp, Glu, Gln, His, Ile, Leu, Lys, Thr or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6. [00751] D17.
• the L comprises the amino acid sequence (X)mC(X)yC(X)n (SEQ ID NO: 27); wherein X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6. 10 [00752] D18.
• D20 The method of any one of embodiments D1-D18, wherein the scFv is in the VH-L-VL orientation.
• D21 The method of any one of embodiments D1-D19, wherein a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L42; 20 c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00756] D22.
• the VH comprises a Cys at H105; 25 b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00757] D23.
• the VH comprises a Cys at H105; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and - 118 - 14620-710-228 / 362327-228710 / JBI6602WOPCT1 NAI-1534348088
• the scFv is in the VL-L-VH orientation.
• D24 The method of any one of embodiments D1-D19, wherein a) the VH comprises a Cys at H5; b) the VL comprises a Cys at L42; 5 c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00759] D25.
• the VH comprises a Cys at H5; 10 b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00760] D26.
• any one of embodiments D1-D19 wherein a) the VH comprises a Cys at H3; b) the VL comprises a Cys at L42; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and 25 d) the scFv is in the VL-L-VH orientation. [00762] D28.
• any one of embodiments D1-D19 wherein a) the VH comprises a Cys at H3; b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID 30 NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00763] D29.
• VH comprises a Cys at H3; - 119 - 14620-710-228 / 362327-228710 / JBI6602WOPCT1 NAI-1534348088
• the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. 5 [00764] D30.
• any one of embodiments D1-D18 and D20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and 10 d) the scFv is in the VH-L-VL orientation. [00765] D31.
• any one of embodiments D1-D18 and D20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID 15 NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00766] D32.
• any one of embodiments D1-D18 and D20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L5; 20 c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00767] D33.
• any one of embodiments D1-D18 and D20 wherein a) the VH comprises a Cys at H43; 25 b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00768] D34.
• the VH comprises a Cys at H40; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and - 120 - 14620-710-228 / 362327-228710 / JBI6602WOPCT1 NAI-1534348088
• D35 The method of any one of embodiments D1-D18 and D20, wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L102; 5 c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00770] D36.
• any one of embodiments D1-D18 and D20 wherein a) the VH comprises a Cys at H40; 10 b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00771] D37.
• any one of embodiments D1-D18 and D20 wherein 15 a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. 20 [00772] D38.
• any one of embodiments D1-D18 and D20 wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and 25 d) the scFv is in the VH-L-VL orientation. [00773] D39.
• any one of embodiments D1-D18 and D20 wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID 30 NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00774] D40.
• VH comprises a Cys at H46; - 121 - 14620-710-228 / 362327-228710 / JBI6602WOPCT1 NAI-1534348088
• the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. 5 [00775] D41.
• any one of embodiments D1-D18 and D20 wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and 10 d) the scFv is in the VH-L-VL orientation. [00776] D42. The method of any one of embodiments D21-D41, wherein the L comprises the amino acid sequence of SEQ ID NO: 3. [00777] D43.
• the VH comprises a Cys at H105; b) the VL comprises a Cys at L43; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and 5 d) the scFv is in the VL-L-VH orientation.
• D50 The method of any one of embodiments D1 to D49, wherein the target cell is a tumor cell, thereby eliminating the tumor cell.
• D51 The method of any one of embodiments D1 to D50, wherein the method is for treating a disease or disorder in a subject. 10 [00786] D52.
• a molecule comprising an antigen-binding fragment (Fab) that binds 20 to a first antigen, and a single chain variable fragment (scFv) that binds to a second antigen, and a fragment crystallizable region (Fc region), wherein the scFv comprises a means for stabilizing the scFv.
• Fab antigen-binding fragment
• scFv single chain variable fragment
• Fc region fragment crystallizable region
• the scFv comprises a heavy chain variable region (VH), a linker (L) and a light chain variable region (VL), and wherein 25 the means for stabilizing the scFv comprises: a) a disulfide bond between a structurally conserved surface exposed VH cysteine (Cys) and a L Cys; b) a disulfide bond between a structurally conserved surface exposed VL Cys and a L Cys; or 30 c) a first disulfide bond between a structurally conserved surface exposed VH Cys and a first L Cys and a second disulfide bond between a structurally conserved surface exposed VL Cys and a second L Cys.
• VH heavy chain variable region
• L linker
• VL light chain variable region
• E3 The molecule of embodiment E2, wherein - 123 - 14620-710-228 / 362327-228710 / JBI6602WOPCT1 NAI-1534348088 a) the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position and the L comprises a L Cys; b) the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a L Cys; or c) the VH comprises a VH Cys at a structurally conserved surface exposed VH framework residue position, the VL comprises a VL Cys at a structurally conserved surface exposed VL framework residue position and the L comprises a first L Cys and the second L Cys, wherein the VH Cys and the first L Cys are capable of forming a disulfide bond, and the VL Cys and the second L Cys are capable of forming a disulfide bond.
• E4 The molecule of embodiment E2 or E3, wherein the distance between the VH Cys and the VL Cys is from about 5 A to about 10 A or from about 7 A to about 9 A.
• E5. The molecule of any one of embodiments E1-E3, wherein the VH Cys is at H3, H5, H40, H43, H46 or H105, and/or wherein the VL Cys is at L3, L5, L39, L42, L43, L45, L100 or L102, and wherein the residue numbering is according to Chothia.
• E6 The molecule of any one of embodiments E1-E5, wherein a) the VH Cys is at Hl 05 and the VL Cys is at L42; b) the VH Cys is at H43 and the VL Cys is at L100; c) the VH Cys is at H3 and the VL Cys is at L3; d) the VH Cys is at H3 and the VL Cys is at L5; e) the VH Cys is at H3 and the VL Cys is at L39; f) the VH Cys is at H3 and the VL Cys is at L42; g) the VH Cys is at H3 and the VL Cys is at L45; h) the VH Cys is at H3 and the VL Cys is at L100; i) the VH Cys is at H3 and the VL Cys is at L102; j) the VH Cys is at H5 and the VL Cys is at L3
• the VH Cys is at H105 and the VL Cys is at L5; mm) the VH Cys is at H105 and the VL Cys is at L39; nn) the VH Cys is at H105 and the VL Cys is at L45; oo) the VH Cys is at H105 and the VL Cys is at L100; pp) the VH Cys is at H105 and the VL Cys is at L102, qq) the VH Cys is at H105 and the VL Cys is at L43, wherein the residue numbering is according to Chothia.
• E7 The molecule of any one of embodiments E1-E6, wherein the L comprises a contiguous amino acid sequence derived from an immunoglobulin (Ig) hinge region.
• Ig immunoglobulin
• E8 The molecule of any one of embodiments E1-E7, wherein the Ig hinge region is derived from a human Ig hinge region or a non-human Ig hinge region. [00799] E9. The molecule of any one of embodiments E1-E8, wherein the Ig hinge region is derived from a human Ig hinge region.
• Ig hinge region is an IgGl, IgG2, IgG3, or IgG4 isotype.
• El 1 The molecule of any one of embodiments E1-E10, wherein the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 23), wherein X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gin), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) or tyrosine (Tyr), and y is an integer from 1 to 3.
• X is glycine (Gly), serine (Ser), proline (Pro), alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), glutamic acid (Glu
• E12 The molecule of embodiment El l, wherein the L comprises an amino acid sequence C(X) y C (SEQ ID NO: 24), wherein X is Gly, Ser or Pro, and y is an integer from 1 to 3.
• E13 The molecule of any one of embodiments E1-E12, wherein the L comprises the amino acid sequence CPC, CGC, CSC, CPPC (SEQ ID NO: 1), CGPC (SEQ ID NO: 28), CPGC (SEQ ID NO: 29), CGGC (SEQ ID NO: 30), CSPG (SEQ ID NO: 31), CPSC (SEQ ID NO: 32), CSSC (SEQ ID NO: 33), CGSC (SEQ ID NO: 34), CSGC (SEQ ID NO: 35), CPPPC (SEQ ID NO: 36), CGPPC (SEQ ID NO: 37), CPGPC (SEQ ID NO: 38), CPPGC (SEQ ID NO: 39), CGGPC (SEQ ID NO: 40), CPGGC (SEQ ID NO: 41), CGGGC (SEQ ID NO: 42), CSPPC (SEQ ID NO: 43), CPSPC (SEQ ID NO: 44), CPPSC (SEQ ID NO: 45), CSSPC (SEQ ID NO:
• E14 The molecule of any one of embodiments E1-E13, wherein the L comprises from about 14 to about 19 amino acids, such as about 14, about 15, about 16, about 17, about 18 or about 19 amino acids.
• E15 The molecule of any one of embodiments E1-E14 wherein the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 25); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, leu, Lys, Phe, Thr, Trp or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6.
• X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gin, His, Ile, leu, Lys, Phe, Thr, Trp or Tyr
• m is an integer from 6 to 9
• y is an integer from 1 to 3
• n is an integer from 4 to 6.
• E16 The molecule of embodiment E15, wherein the L comprises the amino acid sequence (X) m C(X) y C(X) n (SEQ ID NO: 26); wherein X is Gly, Ser, Pro, Ala, Arg, Asn, Asp, Glu, Gln, His, Ile, Leu, Lys, Thr or Tyr, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6. [00807] E17.
• the molecule of embodiment E16, wherein the L comprises the amino acid sequence (X)mC(X)yC(X)n (SEQ ID NO: 27); wherein X is Gly or Pro, m is an integer from 6 to 9, y is an integer from 1 to 3 and n is an integer from 4 to 6. [00808] E18.
• E19 The molecule of any one of embodiments E1-E18, wherein the scFv is in the VL-L-VH orientation.
• E20 The molecule of any one of embodiments E1-E18, wherein the scFv is in the VH-L-VL orientation.
• E21 The molecule of any one of embodiments E1-E19, wherein a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L42; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00812] E22.
• the VH comprises a Cys at H105; b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• E23 comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• the molecule of any one of embodiments E1-E18 wherein a) the VH comprises a Cys at H105; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00814] E24.
• the VH comprises a Cys at H5; b) the VL comprises a Cys at L42; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H5; b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• the molecule of any one of embodiments E1-E18 wherein a) the VH comprises a Cys at H5; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00817] E27.
• the VH comprises a Cys at H3; b) the VL comprises a Cys at L42; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• the VH comprises a Cys at H3; b) the VL comprises a Cys at L45; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation.
• the molecule of any one of embodiments E1-E18 wherein a) the VH comprises a Cys at H3; b) the VL comprises a Cys at L39; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VL-L-VH orientation. [00820] E30.
• the molecule of any one of embodiments E1-E18 and E20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00821] E31.
• the molecule of any one of embodiments E1-E18 and E20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00822] E32.
• the molecule of any one of embodiments E1-E18 and E20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00823] E33.
• the molecule of any one of embodiments E1-E18 and E20 wherein a) the VH comprises a Cys at H43; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00824] E34.
• the molecule of any one of embodiments E1-E18 and E20 wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00825] E35.
• the molecule of any one of embodiments E1-E18 and E20 wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00826] E36.
• the molecule of any one of embodiments E1-E18 and E20 wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00827] E37.
• the molecule of any one of embodiments E1-E18 and E20 wherein a) the VH comprises a Cys at H40; b) the VL comprises a Cys at L3; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00828] E38.
• the molecule of any one of embodiments E1-E18 and E20 wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L100; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00829] E39.
• the molecule of any one of embodiments E1-E18 and E20 wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L102; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00830] E40.
• the molecule of any one of embodiments E1-E18 and E20 wherein a) the VH comprises a Cys at H46; b) the VL comprises a Cys at L5; c) the L comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; and d) the scFv is in the VH-L-VL orientation. [00831] E41.
• E21-E41 The molecule of any one of embodiments E21-E41, wherein the L comprises the amino acid sequence of SEQ ID NO: 6.
• E44 The molecule of any one of embodiments E21-E41, wherein the L comprises the amino acid sequence of SEQ ID NO: 7.
• E45 The molecule of any one of embodiments E1-E44, wherein the binding molecules comprises a heavy chain, a light chain and a polypeptide, wherein the N-terminus of the heavy chain and the light chain form the Fab; wherein the polypeptide comprises the scFv at the N-terminus; and wherein the C-terminus of the polypeptide and the C-terminus of the heavy chain form the Fc region.
• E46 E46.
• E52 A method for eliminating or inhibiting a target cell comprising contacting the target cell with the molecule of any one of embodiments E1-E49.
• E53 A method for treating a disease or disorder in a subject comprising administering to the subject the molecule of any one of embodiments E1-E49.
• F1 The molecule of any one of embodiments A1-A55 for use in a medicament.
• F2 The molecule of any one of embodiments A1-A55 for use in treating a disease or disorder.
• Particular embodiments of this invention are described herein.
• the column was prepared with a gradient of 0-100% Elution Buffer (Wash Buffer: 50 mM Tris, pH 7.5, 500 mM NaCl, 20 mM Imidazole; Elution Buffer: 50 mM Tris, pH 7.5, 500 mM NaCl, 500 mM Imidazole) to remove loosely bound nickel and then re-equilibration in DPBS.
• the cleared supernatant was first adjusted to 50 mM Tris, pH 7.5 and 20 mM imidazole and then loaded over 1 mL HisTRAP HP column @ 4 °C 0.8 mL/min. The column was then washed with PBS until stable baseline was obtained.
• scFv/spFv Stability by Differential Scanning Calorimetry Conformational stability of the Cris7a or Cris7b scFvs and their stapled spFvs were measured by differential scanning calorimetry (DSC) using a Microcal Capillary DSC instrument (Malvern Instruments) with an autosampler. Samples with the matching buffer were scanned at a rate of 60 °C/hr in the range of 25 ⁇ 100 °C with no feedback option. Six buffer-buffer only scans were performed before protein samples to establish thermal history and stable baseline. Raw DSC data were subjected to buffer blank subtraction, normalized by their protein concentration and baseline subtraction.
• Goat anti-human Fc ⁇ protein (Jackson ImmunoResearch 109-005-098), was directly immobilized on a CM4 chip (Series S CM4 Sensor chip, Cat# BR100534) using standard amine coupling. Final ⁇ 4000 Rus were immobilized on each channel.
• Samples with Cris7b scFv or spFv containing bi-specifics were captured by the anti- human Fc ⁇ surface with levels ranging 100 ⁇ 250 Rus, followed by the binding of a series of 5 antigen concentrations of Human CD3E-CD3D Heterodimer Protein (Acro Cat# CDDH52W1) starting at 300 nM in 3 fold dilution (300 nM ⁇ 3.7 nM) using single cycle kinetics method. Association and dissociation times were 150 s and 600 s, respectively. The surface was regenerated using 0.85% phosphoric acid with three short pulses, 20 s each at 50 ⁇ l/min flow rate to remove the captured/bound antibody/antigen complexes before the next round of interaction.
• Running buffer was 0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% v/v Surfactant P20.
• Raw binding data were processed by double referencing via subtracting 1) signals from the antigen binding to the empty chip surface (FC1 on each channel) and 2) signals from the proceeding buffer blank injection. Processed data were then subject to a 1:1 simple Langmuir binding model analysis to derive the kinetic (ka, kd) and affinity (KD) parameters using the Biacore Insight Evaluation software version 2 (Cytiva).
• Cris7a and Cris7b were derived from anti-CD3 variants of CRIS7 that had potential for T cell redirecting.
• the Tm of their scFv moieties had less than ideal thermal stability (FIG.2A) with Tm at 59.7 and 57.1 °C, respectively. See Table 6.
• Table 6 Tm of Cris7a and Cris7b scFvs and spFvs [00852] Stapling was applied to the scFv constructs in FIG.2A in the VL-linker-VH format.
• BCMB749 The anti-BCMA arm, BCMB749, was a mouse monoclonal antibody (provided by Xie-fan Lin- Schmidt).
• BCMB749h was a humanized variant in which BCMB749 CDRs (AbM definition) were grafted onto a human VH and VL with several back mutations (1 VH and VL acceptors were selected).
• BCMB749 and BCMB749h Fabs were then paired with two different anti- CD3 (Cris7b and CD3B219) scFv and spFv to generate BCMA targeting bi-specific molecules.
• Table 7 The various constructs are given in Table 7.
• Table 7 The CD3/BCMA bi-specific molecules generated for this study.
• the column was equilibrated and washed in 1 ⁇ DPBS; final samples were eluted in 0.1 M sodium acetate pH 3.5. Once the protein was eluted, samples were immediately neutralized with 2.5M Tris-HCl pH 7.5, and then loaded over 0.5 mL CaptureSelect CH1-XL Affinity Matrix (ThermoFisher). CH1 column was equilibrated and washed in 1X DPBS, and final samples were eluted in 0.1 M sodium acetate pH 3.5.
• the unfolding was measured by loading each sample into 24 well capillary (NanoTemper, Cat# PR-AC002) from a 384 well sample plate (ThermoNunc, Cat# 264573), with a heating ramp of 1 °C/minute between 20 ⁇ 95 °C using the Prometheus NT.48 instrument (NanoTemper Technologies GmbH). Each sample was measured at 1 mg/ml in phosphate buffer saline (PBS) in duplicates. The intrinsic fluorescence of each sample at 330 and 350 nm was used to monitor unfolding during temperature ramp and recorded as changes in fluorescence intensity over time. Thermal melting data were processed using the PR.STABILITYANALYSIS v1.0.2 software.
• the processed data contains integrated data and first derivation analysis for 330nm, 350nm, Ratio 330/350, and scatter data for all the samples.
• Final analysis results with the annotated transition data for each sample were exported in excel table format.
• the spFv containing molecules B1052 and B1050 with murine and humanized Fab arm, respectively both had 99% or higher bi-specific monomer, whereas their scFv containing counterparts, B1056 and B1055, had 60-80% bi- specific monomer and 20-40% dimer based on SEC results (FIGS.3A-3D).
• Detection of killing and T cell activation status was assessed 72 h later by flow cytometry. Endogenous GFP expressed in H929 was used to separate T cells from target cells. Cytotoxicity was measured using Near-IR Live/Dead stain (ThermoFisher, CatL34976), while activation in CD4 and CD8 T cells was assessed with anti-human CD25-BV650 (BD Biosciences), anti-human CD4-BV510 (Biolegend) and anti-human CD8-PE/Cy7 (Biolegend). The cytotoxicity and CD25 MFI data were exported and analyzed with PRISM (GRAPHPAD). The data were log-transformed, and 4 parameter logistically fit to generate regression curves for reporting of EC50.
• PRISM PRISM
• Table 10 EC50 values of CD3/BCMA bispecific molecule cytotoxicity assay.
• TD01B46, TD01B48 and TD01B49 were further expressed at a larger scale expression at (500 ml or 1 L) as described as for small volumes. These proteins were purified to high homogeneity using a slightly different process as follows. Half a liter of clarified cell culture supernatants of select samples were loaded onto pre-equilibrated 5 mL prepacked HITRAP MABSELECT PRISMA columns (Cytiva) with 1xdPBS (pH 7.2) on AKTA PURE System (GE Healthcare). Columns were washed with 5 column volumes (CVs) of 1 ⁇ dPBS (pH 7.2).
• the bound antibodies were then eluted off of the columns using 5 CVs of 100 mM sodium acetate (pH 3.5) and the enriched fractions were collected. The collected material was neutralized to final volume of 15% (v/v) 2.5 M Tris-HCl (pH 7.5) and syringe-filtered with 0.2- ⁇ m filters. Samples were diluted 8-10-fold with 20 mM MES (pH 5.5) and loaded onto 180 mL CAPTO S IMPACT column (Cytiva) using AKTA PURE System (GE Healthcare) which was equilibrated with 20 mM MES (pH 5.5).
• the column was washed with 1 CV of 20 mM MES (pH 5.5) and 3 CVs of 20 mM MES (pH 6.5) to remove loosely bound impurities.
• the proteins were eluted with 15 CVs of buffer B (20 mM MES, 1M sodium chloride, pH 6.5) over a linear gradient (0–30 % buffer B). Fractions were pooled and passed through 0.2- ⁇ m filters. All samples were loaded onto a pre-equilibrated with 1xdPBS (pH 7.2) 120 mL Superdex 200 pg SEC column (Cytiva) using AKTA AVANT System (GE Healthcare).
• Proteins were eluted off the column with 1.5 CVs 1 ⁇ dPBS (pH 7.2). The concentration of purified protein was determined by absorbance at 280 nm with 0.1 and 0.7 ⁇ mm pathlengths on a DROPSENSE spectrophotometer.
• the quality of the purified protein was assessed by SDS-PAGE (3.5 ⁇ g of sample, Invitrogen, NuPage 4-12% Bis-Tris) and analytical size exclusion HPLC (20 ⁇ g sample; column: TSKgel BioAssist G3SW ⁇ l, 7.8 mm ID ⁇ 30cm H, 5 ⁇ m, TOSOH; guard column: TSKgel BioAssist SWxl guard column, 6 mm ID ⁇ 4cm H, 7 ⁇ m, TOSOH; Agilent HPLC system) at 1 mL/min for 20 min using 200 mM sodium phosphate (pH 6.8) as the running buffer.
• the endotoxin level was measured using a turbidometric LAL assay (PYROTELL®-T, Associates of Cape Cod; Falmouth, MA).
• a turbidometric LAL assay PYROTELL®-T, Associates of Cape Cod; Falmouth, MA.
• One set of constructs was chosen to move into larger scale expression and purification to generate large batches of highly purified recombinant antibodies that could be used in a panel of analyses to further test the stapling technology.
• three Cris7b containing molecules were chosen – TD01B46 (BCMB749 x Cris7b spFv), TD01B48 (BCMB749 x Cris7b scFv G4S) and TD01B49 (BCMB749 x Cris7b scFv Bird).
• the spFv bispecific TD01B46 Over a 6 week incubation period at 4 °C, the spFv bispecific TD01B46 (left panel) remained a monomer, while at 40 °C it showed a modest increase to about 5% aggregate species at 6 weeks. By contrast, over the same period at either 4 °C or 40 °C, the scFv bispecific TD01B49 (right panel) showed a large increase to about 18% and 32% aggregate species, respectively.
• Bispecific samples were loaded to antigen coated SA sensors at 7 antibody concentrations starting at 100 nM in 2-fold dilution (100 nM ⁇ 1.5 nM), diluted in 1 ⁇ DPBS with 0.05% tween-20 to prevent non-specific interactions. Association and dissociation times were 900 s, respectively.
• anti-human IgG Fc (AHC) capture biosensors (Sartorius) were loaded with 3 ug/mL bispecific sample of interest in PBS. After loading sensor tips were washed in 1 ⁇ DPBS + 0.02% Tween 20 + 1 mg/mL Bovine Serum Albumin (BSA) for blocking.
• AHC anti-human IgG Fc
• Recombinant CD3 antigen were loaded to antibody coated sensors at 7 concentrations, starting at 100 nM in 2-fold dilutions (100 nM ⁇ 1.5 nM), diluted in 1X DPBS with 0.02% tween 20 and 1 mg/mL BSA to prevent non-specific interactions. Association was monitored for 1800 s and dissociation for 900 s, respectively. All measurements were performed at 30 °C with agitation at 1,200 rpm. Sensorgrams were referenced for buffer effects and then analyzed using the FORTEBIO Data Analysis HT Software (V.12.0.1.55).
• Binding measurements showed that the scFv and spFv bi-specifics bind CD3 similarly, indicating incorporation of ‘stapling’ to the scFv did not alter CD3 binding (FIG.11, BLI sensorgrams).
• the sensograms from BLI were well fit with a heterogeneous binding mode, but poorly with a simple 1:1 binding mode.
• the kinetic values (KD1/KD2, ka1/ka2, kd1/kd2) showed the stapled anti-CD3 molecule had comparable if not slightly enhanced affinity to either scFv molecule (Table 11).
• Table 11 CD3 binding of scFv and spFv bispecific molecules by BLI, using 2:1 heterogenous ligand fitting model. 6.2.7 Cytotoxicity assay [00868] To determine the effect of BCMA targeting test molecules upon T cell activation and killing potential of tumor cells, the Intellicyt iQue3 and ForeCyt software (Sartorius) was utilized. H929-Fluc-GFP cells served as target cells for two human donor Pan T cells (Hemacare). The assay was set up in a 96-well plate at a T cell to target ratio of 3:1. Test molecules were added at a starting concentration of 10 nM and serially diluted at 1:4 in complete media. All molecules were tested in duplicate at minimum.
• Detection of killing and T cell activation status was assessed 72 hrs later by flow cytometry. Endogenous GFP expressed in H929 was used to separate T cells from target cells. Cytotoxicity was measured using Near-IR Live/Dead stain (ThermoFisher), while activation in CD4 and CD8 T cells was assessed with anti-human CD25-BV650 (BD Biosciences), anti-human CD4-BV510 (Biolegend) and anti-human CD8-PE/Cy7 (Biolegend). Using Prism software (Graphpad), cytotoxicity and CD25 MFI data was exported, log-transformed, and 4 parameter logistically fit to generate regression curves for reporting of EC50.
• H929-Fluc-GFP cells served as target cells for two human donor pan-T cells. Detection of killing and T cell activation status was assessed 72 h later by flow cytometry. All bispecific proteins potently killed BCMA+ H929-GFP+ cells in a cytotoxicity assay with very similar EC50 (data not shown), whereas a negative control bispecific with a Cris7b scFv/ non-targeting Fab (CD8B24) showed no killing activity.
• spFv containing bispecifics also displayed minimal aggregation upon heat stress at high concentrations, whereas the corresponding scFv molecules displayed significant aggregation. This was also true for a number of other spFv containing bi- and tri-specifics proteins.
• spFv can lead to equally potent biotherapeutics such as bi-, multispecifics with significantly improved developability.
• stapling can also increase the success of scFv conversion, thus allowing more scFv molecules to be available as molecular building blocks for therapeutic constructs.
• the column was prepared with a gradient of 0-100% Elution Buffer (Wash Buffer: 50 mM Tris, pH 7.5, 500 mM NaCl, 20 mM Imidazole; Elution Buffer: 50 mM Tris, pH 7.5, 500 mM NaCl, 500 mM Imidazole) to remove loosely bound nickel and then re-equilibration in DPBS.
• the cleared supernatant was first adjusted to 50 mM Tris, pH 7.5 and 20 mM imidazole and then loaded over 1mL HisTrap HP column at 4 °C 0.8 mL/min. The column was then washed with PBS until stable baseline was obtained.
• CAT2200a scFv LH was purchased from Sino Biological, which was produced in HEK293. Concentration was 0.77 mg/mL in DPBS, pH 7.2.
• a mutant of IL-17 (12-132 with K70Q A132Q C106S mutations, IL-17 hereafter for simplicity) was purchased from Accelagen (CA). The protein was refolded from E. coli inclusion body following their proprietary refolding protocol and provided at 1.50 mg/mL in 20 mM NaCl, 20 mM MES, pH 6.0.
• the column was prepared with a gradient of 0- 100% Elution Buffer (Wash Buffer: 50 mM Tris, pH 7.5, 500 mM NaCl, 20 mM Imidazole; Elution Buffer: 50 mM Tris, pH 7.5, 500 mM NaCl, 500 mM Imidazole) to remove loosely bound nickel and then re-equilibration in DPBS.
• the cleared supernatant was first adjusted to 50 mM Tris, pH 7.5 and 20 mM imidazole and then loaded over 1 mL HisTrap HP column at 4 °C 0.8 mL/min. The column was then washed with PBS until stable baseline was obtained.
• FIG.23 shows humanization and sequence alignment of BCMB749. Each sequence alignment contained the parental (top), selected human acceptor germline sequence (middle) and the CDR-grafted with back mutations italicized (bottom). CDRs are underlined. Bold: CDR support positions in the framework regions. Boxed: VL/VH interface residues.
• the mouse parental sequences of the VH and VL domains were annotated as shown in the sequence alignment in FIG.23.
• CDRs were defined according to the AbM convention and were underlined. Framework positions were classified into CDR support (in bold font) and VL/VH interface (boxed).
• the parental sequence was aligned against human germline sequences and one VH and one VL were selected according to sequence identity.
• human IGKV1-12*01 was chosen and IGHV1-3*01 for the VH.
• the selected J segment was highlighted in gray based on sequence identity.
• the first sequence was mouse parental, middle one was the chosen human germline acceptor and the bottom one was the humanized variant.
• CDRs as defined above were grafted into the corresponding regions in the acceptor human GLs. Positions that were classified as CDR support and/or VL/VH interfaces were then back mutated to their parental amino acids if they were different between the parental and human acceptors. There were three and five back mutations in the humanized VL and VH domains, respectively. 6.3.1.3 SDS-PAGE Analysis of protein samples [00875] Cris7a and Cris7b scFv and spFv domain proteins were prepared at 0.3 mg/mL in 1 ⁇ dPBS with the addition of 1 ⁇ NuPAGE LDS Sample Buffer (Invitrogen).
• the column was equilibrated and washed in 1 ⁇ DPBS; final samples were eluted in 0.1 M sodium acetate pH 3.5. Once the protein was eluted, samples were immediately neutralized with 2.5M Tris-HCl pH 7.5, and then loaded over 0.5 mL CAPTURESELECT CH1-XL Affinity Matrix (ThermoFisher). CH1 column was equilibrated and washed in 1 ⁇ DPBS, and final samples were eluted in 0.1 M sodium acetate pH 3.5.
• the collected material was pooled together of each protein separately, and syringe-filtered with 0.2- ⁇ m filter.
• Samples were loaded onto pre-equilibrated 5 mL CaptureSelect CH1-XL prepacked affinity columns (Thermo Scientific) on AKTA XPRESS systems (Amersham Biosciences Corp.) and washed with 4 CVs of 1 ⁇ dPBS (pH 7.2).
• the bound antibodies were then eluted off of the columns using 5 CVs of 100 mM sodium acetate (pH 3.5) and the enriched fractions were collected.
• the collected material was neutralized to final volume of 15% (v/v) 2.5 M Tris-HCl (pH 7.5) and syringe-filtered with 0.2- ⁇ m filters.
• the quality of the purified protein was assessed by SDS-PAGE (3.5 ug of sample, Invitrogen, NuPage 4-12% Bis-Tris) and analytical size exclusion HPLC (20 ug sample; column: TSKgel BIOASSIST G3SWxl, 7.8 mm ID x 30 cm H, 5 ⁇ m, TOSOH; guard column: TSKgel BIOASSIST SW ⁇ l guard column, 6mm ID x 4cm H, 7um, TOSOH; Agilent HPLC system) at 1 mL/min for 20 min using 200 mM sodium phosphate (pH 6.8) as the running buffer.
• the endotoxin level was measured using a turbidometric LAL assay (PYROTELL-T, Associates of Cape Cod; Falmouth, MA). 6.3.1.6 Differential Scanning Calorimetry (DSC) [00878] The scFv and spFv proteins were dialyzed overnight against 1 ⁇ DPBS (Gibco) for GLk1 and CAT2200a/CAT2200b or MES (25 mM MES, pH 6.0, 100 mM NaCl) for GLk2. Dialysis buffer was then 0.22 micron filtered and used as the reference solution and for buffer-buffer blanks in the DSC experiment.
• DPBS Gibco
• MES 25 mM MES, pH 6.0, 100 mM NaCl
• Proteins were diluted to ⁇ 0.5 mg/mL in the filtered buffer and 400 ⁇ L of each protein or buffer sample was loaded into a 96-well plate (MicroLiter Analytical Supplies, 07-2100) and kept at 4 °C in the autosampler drawer over the course of the experiment.
• a MICROCAL Capillary DSC with Autosampler (Malvern) was used to perform the DSC experiments. DSC scans were performed from 25-95 °C at a 60 °C/h scan rate with no sample rescans. No feedback was selected and the filtering period was set at 15 s. After each sample, cells were cleaned with a 10% Contrad-70 solution and a buffer-buffer blank was run.
• Thermal melt profiles were collected using a linear heating ramp of 0.3 °C/minute between 20 ⁇ 85 °C, with a 60 s incubation period and 30 s plate hold period. Data was collected and analyzed concurrently with Uncle software and Tm and Tagg (Tonset) values were directly exported.
• Crystallization was set up for each protein in sitting drop format in Corning 3550 crystal trays using a MOSQUITO robot. Each well contained 100 nl of protein and 100 nl of reservoir solution and incubated against 70 ul of reservoir at 20 ⁇ C.
• the reservoir solutions were IH1 and IH2 custom conditions as well as PEG Ion Screen HT (Hampton Research). Some initial conditions were refined by varying reservoir components in optimization attempts. Diffraction quality crystals were obtained for some of scFv and spFv proteins. Crystals were soaked for a few seconds in the mother liquor supplemented with 20% glycerol and flash frozen in liquid N2. X-ray data were collected at IMCA-CAT Beamline 17ID at Argonne National Lab.
• IL-17/ CAT2200a scFv VL-VH complex was generated by mixing 333 ⁇ L of IL17 (1.5 mg/ml) with 1.74 ml of Cat2200a scFv (0.69 mg/mL) and incubating for 3 hours at 4 °C. The mixture was concentrated with 10 kDa cutoff AMICON Ultra concentrator to about 400 ⁇ L and loaded onto a SUPERDEX75 column equilibrated in 250 mM NaCl, 20 mM HEPES, pH 7.5.
• the fractions corresponding to the complex were pooled and concentrated to a volume of 150 ⁇ L.
• the sample was diluted and concentrated 4 times: addition of 350 ⁇ L 50 mM NaCl, 20 mM HEPES, pH 7.5 and concentration to just under 150 ⁇ L.
• the volume was brought to ⁇ 105 ⁇ L and concentration determined to be 2.69 mg/mL.
• Crystallization was set up in a sitting drop format using a MOSQUITO crystallization robot with 150 nL protein + 150 nL reservoir in Corning3550 plates against 80 ⁇ L reservoir, which was a set of buffer and precipitant conditions pre-formulated in-house. The plates were incubated at 20 °C.
• X-ray diffraction data were collected at IMCA-CAT ID17 at Argonne National Laboratory.
• the IL-17/ CAT2200a spFv VL-VH complex were generated by mixing 167 ⁇ l of IL-17 (250 ⁇ g) with 154 ⁇ l MSCW274 (467 ⁇ g in 250 mM NaCl, 20 mM MES, pH 6.5) and incubating at 4 ⁇ C overnight.
• the mixture was concentrated in a 10 kDa MWCO Amicon Ultra 0.5 mL concentrator to ⁇ 100 uL, then repeatedly diluted and concentrated 5 times: concentrate to ⁇ 150 ⁇ L and added 350 ⁇ L 50 mM NaCl, 20 mM HEPES, pH 7.5. The final volume was 100 ⁇ L and the concentration of the complex was determined to be 6.0 mg/ml. Crystallization was set up similarly as for scFv/IL-17 complex in sitting drops using the MOSQUITO robot. The sitting drop were composed of 150 nL protein + 120 nL reservoir + 30 nL seeds (scFv/IL-17 above). The reservoir solution were a set of conditions varying PEG 3350 concentration and salts.
• the crystallization plates were incubated at 20 °C. Small crystals were obtained from 15.5 % PEG 3350, 0.4 M NaH2PO4. Crystals were transferred into 16% PEG 3350, 0.2 M NaH2PO4, 20% Glycerol, and flash frozen LN2.
• X-ray diffraction data were collected at IMCA-CAT ID17 at Argonne National Laboratory. [00884] All X-ray diffraction data were processed with XDS (Kabsch et al. Acta crystallographica. Section D, Biological crystallography 66, 125-132 (2010)) and CCP4 (Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta crystallographica.
• Section D Biological crystallography 53, 240-255 (1994)). All crystal structures were solved by molecular replacement (MR) using Phaser (Read et al. Acta crystallographica. Section D, Biological crystallography 57, 1373-1382 (2001).) with homology models generated in MOE (Montreal, Canada) except for scFv CAT2200a scFv VL-VH/IL-17 complex, for which the structure of pdb id 2vxs (Gerhardt et al. Journal of molecular biology 394, 905-921 (2009)) was used as search models. The structural models were refined in PHENIX (Adams et al.
• Atomic coordinates for CATscFv_LH/IL-17 are shown in Table 22. Atomic coordinates for CATspFv_LH/IL-17 (mscw374) are shown in Table 23.
• Table 13 X-ray data collection and refinement statistics Table 13, cont. Table 13, cont. 6.3.1.10 Disulfide Mapping Analysis: Sample Preparation, Instrument Parameters and Data Analysis [00885] Non-reduced digestion of the bispecific molecule was performed using an enzyme combination of recombinant LysC (rLysC) and PROALANASE (Promega, Cat. #VA2161).
• the digestion protocol used was a modified version of the Promega ACCUMAP Low pH Protein Digestion protocol for nonreduced disulfide mapping protocol.
• the bispecific antibody (40 ⁇ g) was denatured using 8M Guanidine HCl and alkylated using 0.2 M N- ethylmaleimide (NEM, Promega, Cat#VB102A) at 37 o C for 30 min.40 ⁇ g of denatured and alkylated protein was directly mixed with rLysC.
• the denatured and alkylated antibody (43 ⁇ l) was mixed with 25 ⁇ L of rLysC (Promega low pH resistant rLysC, Promega, Cat.
• Thermo Scientific Single-Use RED plate with inserts in 0.1% TFA (aq) (pH 1.8).
• the sample was transferred to the sample holding well ( ⁇ 67 ⁇ L) while in the dialysate well 325 ⁇ L of 0.1% TFA was added, and the dialysis plate was placed on a shaker at 37 o C for 40 min. This step was performed 3 times and the sample was recovered at the same concentration ( ⁇ 40 ⁇ g).
• the enzyme was added to an enzyme-to-protein ratio of 1:40 (w/w) and the digestion was incubated at 37 o C overnight.
• LC/MS peptide mapping data were acquired on a ThermoFisher Scientific (San Jose, CA) VANQUISH Dual UHPLC connected to an ORBITRAP Eclipse Mass Spectrometer.
• the HPLC column used was a Waters Acquity Premier CSH C182.1 x 150 mm heated at a temperature of 60 o C.
• Peptides ( ⁇ 15 ⁇ g of the sample) from the two-enzyme digestion were separated by a 120-minute gradient at a flow rate of 0.4 ml/min.
• the LC gradient consisted of an initial setting of 2% B (0.1 % Formic acid/ Acetonitrile) to 42% B in 114 minutes.
• the ORBITRAP Eclipse instrument was operated in ESI Positive mode using an Ion Max NG H-ESI source. Source settings were spray voltage of 3500 V, sheath gas (arb units) 35, aux gas (arb units) 7, sweep gas (arb units) 1. Ion transfer tube temperature and vaporizer temperature were 275 o C. Instrument was operated in Data Dependent Acquisition (DDA) mode. MS1 parameters were orbitrap resolution of 120,000, scan range from 350 – 2000 m/z with RF lens of 30%.
• DDA Data Dependent Acquisition
• Dependent scan 1 was ddMS2 OT HCD (Higher-energy C- trap dissociation) with isolation window 1.6 m/z, collision energy fixed at 29%, orbitrap detection, resolution was 15,000 with a maximum injection time of 50 msec.
• Dependent scan 2 was ddMS2 OT ETD (Electron-transfer dissociation) with isolation window 1.6 m/z, ETD reaction time 110 ms, ETD reagent target 2.0e5, Max ETD reagent injection time was 200 ms.
• DDMS2 detection was set to orbitrap with resolution of 15,000 and a max injection time of 50 msec.
• the unfolding was measured by loading each sample into 24 well capillary (NanoTemper, Cat# PR-AC002) from a 384 well sample plate (ThermoNunc, Cat# 264573), with a heating ramp of 1 °C/minute between 20 ⁇ 95 °C using the Prometheus NT.48 instrument (NanoTemper Technologies GmbH). Each sample was measured at 0.5 mg/ml in phosphate buffer saline (PBS) in duplicates. The intrinsic fluorescence of each sample at 330 and 350 nm was used to monitor unfolding during temperature ramp and recorded as changes in fluorescence intensity over time. Data were collected and saved as projects, processed using the PR.STABILITYANALYSIS v1.0.2 software.
• the processed data contained integrated thermal melting profiles, first derivatives for fluorescence at 330 nm, 350 nm, ratio 330/350, and light scattering data for all the samples.
• Thermal melting mid-point (Tm) values as well as onset of aggregation (Tagg) were identified and reported.
• Bispecific samples were loaded to antigen coated SA sensors at 7 antibody concentrations starting at 100 nM in 2-fold dilution (100 nM ⁇ 1.5 nM), diluted in 1xDPBS with 0.05% tween-20 to prevent non-specific interactions. Association and dissociation times were 900 s, respectively.
• anti-human IgG Fc (AHC) capture biosensors (Sartorius) were loaded with 3 ⁇ g/mL bispecific sample of interest in PBS. After loading sensor tips were washed in 1 ⁇ DPBS + 0.02% Tween 20 + 1 mg/mL Bovine Serum Albumin (BSA) for blocking.
• AHC anti-human IgG Fc
• Recombinant CD3 antigen were loaded to antibody coated sensors at 7 concentrations, starting at 100 nM in 2-fold dilutions (100 nM ⁇ 1.5 nM), diluted in 1 ⁇ DPBS with 0.02% tween 20 and 1 mg/mL BSA to prevent non-specific interactions. Association was monitored for 1800 s and dissociation for 900 s, respectively. All measurements were performed at 30 °C with agitation at 1,200 rpm. Sensorgrams were referenced for buffer effects and then analyzed using the ForteBio Data Analysis HT Software (V.12.0.1.55).
• BM Chemilluminesence ELISA Substrate (Millipore) and plates were immediately read on an ENVISION plate reader (Perkin Elmer) using ultrasensitive illuminesence.
• Raw data were exported to GRAPHPAD PRISM, where curves were generated and analyzed with a nonlinear regression curve fit. 6.3.1.14 High concentration and heat stress study [00891] Concentratability studies were performed using Amicon Ultra 4 centrifugal filtration devices with 30 kDa MWCO membrane (Catalog# UFC803096. MilliporeSigma, Burlington MA). First, the spin columns were filled with water and spun at 4200 g for 6 min to equilibrate the membrane.
• TD01B46 (2 mg/ml) and TD01B49 proteins (0.5 mg/ml) were each loaded in pre-washed spin columns and centrifuged at 4200 g at 15-minute time intervals. At the end of each 15-minute centrifugation step, the concentrators were removed from the centrifuge and a visual estimate of the remaining sample volume was recorded. The concentration step was repeated until enough volume of the concentrated sample was available for profiling by SEC for purity analysis. At the end of the centrifugation process, the concentrated samples were recovered, and the protein concentration was determined using NANODROP ND1000 (ThermoFisher Scientific, Waltham MA).
• the final concentrations were 68 mg/ml and 54 mg/ml for TD01B46 (stapled) and TD01B49 (unstapled) bispecific proteins, respectively.
• the concentrated samples were split into two equal parts, and one stored at 4 °C and the other stored at 40 °C. Aliquots from the incubated samples were taken at different time points (t0, t-1wk, t-2wk and t-6wk) and diluted to 1 mg/mL with 1x dPBS for purity analysis by SEC.
• Cytotoxicity assay [00892] To determine the effect of BCMA targeting test molecules upon T cell activation and killing potential of tumor cells, the INTELLICYT IQUE3 and FORECYT software (Sartorius) was utilized. H929-Fluc-GFP cells served as target cells for two human donor Pan T cells (Hemacare). The assay was set up in a 96-well plate at a T cell to target ratio of 3:1. Test molecules were added at a starting concentration of 10 nM and serially diluted at 1:4 in complete media. All molecules were tested in duplicate at minimum. Detection of killing and T cell activation status was assessed 72 hrs. later by flow cytometry.
• Endogenous GFP expressed in H929 was used to separate T cells from target cells. Cytotoxicity was measured using Near-IR Live/Dead stain (ThermoFisher), while activation in CD4 and CD8 T cells was assessed with anti-human CD25-BV650 (BD Biosciences), anti-human CD4-BV510 (Biolegend) and anti-human CD8-PE/Cy7 (Biolegend). Using PRISM software (GRAPHPAD), cytotoxicity and CD25 MFI data was exported, log-transformed, and 4 parameter logistically fit to generate regression curves for reporting of EC50.
• H929 WT and KO cells were counted and stained with CFSE (BD Pharmingen, cat# C34554) and/or CELL TRACE violet proliferation dyes (BD Pharmingen, cat# C34557) as well as near IR live/dead stain (Thermofisher, cat# L10119).
• CFSE BD Pharmingen, cat# C34554
• CELL TRACE violet proliferation dyes BD Pharmingen, cat# C34557
• near IR live/dead stain Thermofisher, cat# L10119.
• FBS Gibco, cat# 16000-036
• Fc-blocked with Human TRUSTAIN FcX blocking reagent Biolegend, cat# 422301
• the cells were then incubated with serial dilutions of test molecules for 1 hr at 37 °C (1/2 log serial dilutions starting at 2 ⁇ M).
• the cells were washed 2x in FACS buffer (Becton Dickinson, cat# 554657) and then incubated with 1 ⁇ g/mL AF647-labelled goat anti human Fc (Jackson IR, cat# 109-606-098) detection reagent for 30 minutes at 4 °C.
• the cells were again washed 2 ⁇ in FACS buffer and then analyzed on the INTELLICYT IQUE 3 (Sartorius) high throughput flow cytometer.
• the raw data was exported and analyzed in GRAPHPAD PRISM.
• FIG.13A The scFv “stapling” strategy to minimize aggregation due to scFv instability and “breathing” is shown in FIG.13A.
• the stapling design is schematically illustrated in FIG. 13B.
• SSs disulfide bonds
• the scheme of linker-anchor points disulfide bonds mimics the effects of a staple and was thus termed “stapling” (FIG.13A and FIG.13B).
• the two domain Cys residues (anchor positions) be selected such that the distance (d AP ) between them had a narrow distribution but also have a low probability of forming inter-VL/VH disulfide bond directly;
• the distance between the two linker Cys residues (d staple ) was compatible with (d AP ) and these Cys residues have a low probability of forming a disulfide loop;
• the linker Cys residues be positioned close to the respective anchor Cys simultaneously, a condition that was the result of the linker segments L1 (from C terminus of the leading domain to first linker Cys) and L2 (second linker Cys to trailing domain N terminus).
• FIG.19A illustrated one choice of the positions for the two different orientations of the spFv.
• the chosen anchor points were position 42 for VL and position 105 for VH (FIG. 19A) when the sequences were numbered according to the Chothia scheme (Chothia and Lesk, Journal of molecular biology 196, 901-917 (1987)); for HL orientation, the chosen anchor points were Chothia position 43 for VH and position 100 for VL (FIG.19A).
• the anchor positions were also illustrated in several antibody sequences (FIG.19B).
• the distances and geometry of the anchor positions and domain termini were illustrated in FIG. 13C.
• the distances between both C ⁇ (d AP,C ⁇ ) and C ⁇ (d AP, C ⁇ ) atoms of these positions were determined from a survey of 2501 Fvs currently in the PDB (data shown in FIGS.20B-20E). Both distances had a rather narrow and similar distribution in antibody structure.
• the LH and HL C ⁇ distances had an average of 8.2 ⁇ (range of 7-9 ⁇ ) and 6.9 ⁇ (range of 6-8 ⁇ ), respectively (FIG.20B, orange and green curves).
• the corresponding C ⁇ distances were 8.2 ⁇ (range of 7-10.0 ⁇ ) and 8.7 ⁇ (range of 7-10.0 ⁇ ), respectively (FIG.20C, orange and green curves). These distances were much wider than the typical C ⁇ and C ⁇ distances (4-6.8 ⁇ in FIG.20B, and 3.5-4.8 ⁇ in FIG.20C, respectively as shown by black curves) of two positions that form direct disulfide bonds. These distances indicated low probability of the two anchor positions effectively forming disulfide bonds with each other as their distances, on average, were too far from each other. Thus, these two anchor residues were likely available for stapling.
• the CPPC staple would likely be of the right geometry for stapling, i.e., forming proper disulfide bonds correctly to the anchor point Cys residues.
• the placement of the stapling motif (CPPC) within the linker was not only important to allow proper stapling disulfide formation as designed, but also to prevent scrambling, i.e., formation of SS between unintended Cys residues.
• the first Cys residue must disulfide bond to the anchor point on the leading domain (VL domain in LH or VH in HL) and the second Cys residue to the anchor point on the trailing domain (VH domain in LH or VL in HL).
• Optimal L1 and L2 lengths would bring a linker Cys to within a short distance (C ⁇ atom distance of ⁇ 5-6 ⁇ ) of the intended anchor Cys, while keeping the distance between the linker Cys and the opposing anchor Cys residue as distant as possible.
• these two distances were determined by L1, dAP, d1 and d2, the distances between the C terminus of the leading domain and the two anchor Cys positions (FIG.13C and FIG.21A).
• FIG.19B shows the proposed first set of linker designs with the CPPC staple.
• 6.3.2.2 Model and Therapeutic spFv Molecules were Significantly More Stable [00899]
• several antibodies were selected to generate scFv and corresponding spFv: two antibodies with kappa light chains (GLk1 and GLk2) from the synthetic phage antibody libraries (Shi et al. Journal of molecular biology 397, 385-396 (2010)) and a lambda-containing antibody (CAT2200) obtained from a publication (Gerhardt et al.
• FIG.14 The overall structures of the unbound spFv and some scFv/spFv:antigen complexes are shown in FIG.14. The structures were consistent with the typical Fv structures with both VL and VH domains packing against each other. In all spFv structures, some residues of the linker were ordered and resolved in the electron density maps. The disulfide bonds between the “staple” and the anchor points were generally well ordered in both LH and HL orientations (FIGS.14A-14E). A representative electron density map of the stapling linker region for Glk2 is shown in FIG.14D.
• the pairwise C ⁇ rmsd between the two HL spFv molecules was 0.20 ⁇ .
• the pairwise C ⁇ rmsds were slightly higher (from 0.53 to 0.77 ⁇ , average 0.61 ⁇ ) excluding the linker for 195 to 219 C ⁇ atoms (average 213).
• the LH and HL spFv was also compared with the Fv fragment in its corresponding Fab (PDB ID 5I19) ((Teplyakov et al. mAbs 8, 1045- 1063 (2016))).
• the rmsds between LH spFv and Fab Fv were from 0.41 to 0.64 ⁇ (average 0.57 ⁇ ) for 203 to 215 C ⁇ atoms.
• the rmsds between HL spFv and Fab Fv were 0.56 to 0.71 ⁇ (average 0.64 ⁇ ) for ⁇ 210 C ⁇ atoms.
• CAT2200 scFv and spFv molecules were crystallized in complex with its cognate target, IL-17.
• scFv and spFv of CAT2200 variants crystallized, the structures were nearly identical with and without a bound target (FIGS.14E- 14G). They were also identical regardless of orientation or presence or absence of the staple.
• FIG.15 shows the ordered structures of the linkers superimposed on the CPPC motif. It was clear that the CPPC structures were very similar with main chain atom rmsd of 0.37 ⁇ (range 0.15 to 0.5 ⁇ ). The C ⁇ (Cys1)–C ⁇ (Cys2) distances ranged from 6-9 ⁇ and C ⁇ (Cys1)-C ⁇ (Cys2) distances were from ⁇ 6.5 ⁇ to 9 ⁇ . These distances were comparable to those in the IgG hinge structures. In the structures the two Pro residues adopted the trans conformation and there was a high degree of similarity between the CPPC motif structures in these different crystals. This was consistent with the notion that Pro-Pro motif was relatively rigid.
• BCMB749 One anti-BCMA arm, BCMB749, was derived from a mouse monoclonal antibody.
• the second anti-BCMA arm, BCMB749h was a humanized variant in which the complimentary-determining regions (CDRs) of BCMB749 were grafted onto a human VL and VH acceptor germline, with a small number of back mutations to the mouse parent sequence (Details in FIG.23). Pairing the two BCMA Fab arms with two different anti-CD3s each in 3 formats (scFv with Bird linker, scFv with (G 4 S) 4 linker and spFv) generated twelve distinct BCMA-targeting bispecific molecules (FIG.24). The constructs are listed in Table 15. Table 15: Construct design for bispecific proof of concept anti-BCMA targeting antibodies.
• Cris7b containing molecules were chosen – TD01B46 (BCMB749 x Cris7b spFv), TD01B48 (BCMB749 x Cris7b scFv G4S) and TD01B49 (BCMB749 x Cris7b scFv Bird).
• TD01B46 BCMB749 x Cris7b spFv
• TD01B48 BCMB749 x Cris7b scFv G4S
• TD01B49 BCMB749 x Cris7b scFv Bird
• Disulfide mapping LC/MS experiments were used to confirm expected disulfide formation and identify presence of scrambling and free thiol.
• the stapled bispecific (TD01B46) was digested with a two-enzyme combination as described in the method section and expected disulfide links were determined.
• FIG.16C shows all the expected disulfide links.
• a total ion chromatogram of the digested protein is shown in FIG.16D.
• peaks corresponding to the expected peptides connected by disulfides were identified based on peak intensity in the extracted ion chromatogram (XIC), mass of the disulfide complex (MS1) and peptide sequences (MS2). Representative XIC, MS1 and MS2 data for several of the disulfide peptide complexes are shown in FIG.25 and FIG.26. Complete coverage of every expected disulfide was confirmed by this LC/MS/MS approach and the expected disulfides represented > 99% of all cysteine containing peptides detected. In this study, SS scrambling was also observed at ⁇ 0.5%, which was lower than the level of a typical mAb.
• the concentrated samples were then incubated at 4 and 40 °C. At two-week intervals, a small aliquot of each sample was diluted to 1 mg/ml and then run on aSEC. The results are shown in FIG.17B and FIG.17C. Over a six-week incubation period at 4 °C, the spFv bispecific TD01B46 (FIG.17B and FIG.17C, left panels) remained a monomer, while at 40 °C it showed a modest increase to about 5% dimer species at 6 weeks.

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