WO2024206820A1 - Ingénierie fc pour format d'anticorps hétérodimère - Google Patents

Ingénierie fc pour format d'anticorps hétérodimère Download PDF

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WO2024206820A1
WO2024206820A1 PCT/US2024/022229 US2024022229W WO2024206820A1 WO 2024206820 A1 WO2024206820 A1 WO 2024206820A1 US 2024022229 W US2024022229 W US 2024022229W WO 2024206820 A1 WO2024206820 A1 WO 2024206820A1
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amino acid
polypeptide
polypeptide chain
acid substitutions
comprises amino
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Mark Welch
Sridhar Govindarajan
Thomas Purcell
Jennifer CODDING-BUI
Renee SAVILLE
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DNA Twopointo Inc
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DNA Twopointo Inc
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    • 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
    • C07K16/36—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Antibodies, or immunoglobulins are proteins of the immune system that typically comprise four polypeptide chains: two heavy chains (HC) and two light chains (LC). These polypeptides associate with each other to form a “Y”-shaped, symmetrical protein, linked by disulfide bonds between a heavy chain dimer and two heavy/light chain heterodimers.
  • the stem of this “Y” is formed by the C-terminal portion of the heavy chains and is referred to as the Fc (fragment, crystallizable) region of the antibody.
  • the Fc region may further be divided into two or three “constant” domains, depending on the antibody class.
  • Immunoglobulins of class G, A, and D have two such constant domains (CH2 and CH3), whereas IgM and IgE have three (CH2, CH3, and CH4).
  • the Fc region has the same sequence for a given species.
  • the N-terminal fragment of the “Y,” termed the fragment antigen-binding (Fab) domain comprises paired light and heavy chains, each with two domains; another constant region (CHI on the heavy chain and CL or CK-Lambda or Kappa-on the light chain); and a sequencevariable region (VH or VL for heavy and light chains, respectively).
  • the variable domains determine the antibody’s antigen specificity, or the particular antigen to which an antibody will bind, whereas the Fc region interacts with various components of the immune system to mediate physiological effects and immune responses.
  • the Fc region comprises a homodimer of identical polypeptide chains bound together by disulfide bonds
  • the Fab region comprises a pair of identical heterodimers, resulting in a molecule with specialized binding to a singular epitope. That is, both arms of the antibody bind to the same molecular structure, and the antibody is divalent and monospecific.
  • Advances in the field of antibody engineering have given rise to heterodimeric molecules based upon the interaction between the Fc region monomers in which the Fc is a protein scaffold. In these molecules, the Fab fragment is not symmetrical, and in some applications, the Fab fragment has been replaced with a fusion to another protein at the N terminus.
  • the Fc region is fused at the C terminus to other protein domains with additional functionality.
  • heterodimeric molecules include, for example, monovalent or multivalent, bispecific and multi-specific antibodies, appendages of single-chain variable fragments (scFv) and single-chain Fab fragments (scFab), as well as fusions to cytokines and other proteins, and many combinations of these formats.
  • bispecific antibodies and multi-specific antibodies recognizing two or more unique targets are biomedically advantageous and of significant interest to the pharmaceutical industry.
  • a bsAb targeting two or more antigens e.g., a tumor antigen and a T-cell target CD3, can bring a cytotoxic T cell in proximity to its tumor target, increasing cytotoxicity and decreasing the amount of drug required to treat the patient.
  • Different formats of bispecific and multi-specific Abs exist as either IgG-like molecules or Ab fragments and fusions of various types.
  • heterodimerization Provided herein is a novel approach to the “heavy chain problem” for heterodimerization; that is, the pairing of two identical HCs in place of the heterodimerization required to make a heterodimeric molecule.
  • the approach at least one of: (i) promotes heterodimeric HC association; (ii) disfavors homodimeric HC association; (iii) enables mutations to be incorporated into the Fc domains of, e.g., biotherapeutic Ab candidates, to drive heterodimerization; and (iv) increases the amount of the desired heterodimeric molecule within the heterogeneous population of molecules, decreasing production and purification costs.
  • a method for making a heterodimeric molecule comprising: introducing into a cell a first heterologous polynucleotide, the first heterologous polynucleotide comprising a nucleotide sequence that encodes for a first polypeptide, the first polypeptide comprising a variant of a human IgGl Fc chain (a “first variant”); and introducing into the cell a second heterologous polynucleotide, the second heterologous polynucleotide comprising a nucleotide sequence that encodes for a second polypeptide, the second polypeptide comprising a variant of a human IgGl Fc chain (a “second variant”), wherein the first variant optionally comprises an amino acid substitution T366Y; wherein the second variant optionally comprises an amino acid substitution Y407T; wherein at least one of the first variant and the second variant comprises at least one amino acid substitution giving rise to an electrostatic attraction between the first variant and the second variant comprises at least one amino acid
  • the at least one amino acid substitution in the first variant may comprise D356K, D356R, or D356H and/or K439R or K439H
  • the at least one amino acid substitution in the second variant may comprise K439D or K439E and/or D356E.
  • the at least one amino acid substitution in the first variant may comprise K439D or K439E and/or D356E
  • the at least one amino acid substitution in the second variant may comprise D356K, D356R, or D356H and/or K439R or K439H.
  • the first variant may comprise the amino acid substitution T366Y, or it may not, independent of whether the second variant comprises the amino acid substitution Y407T; and that the second variant may comprise the amino acid substitution Y407T, or it may not, independent of whether the first variant comprises the amino acid substitution T366Y. This is true of each of the following aspects, as well, whether or not explicitly stated.
  • a heterodimeric molecule comprising: a first polypeptide chain comprising a first engineered human IgGl Fc polypeptide chain, the first engineered human IgGl Fc polypeptide chain optionally comprising an amino acid substitution T366Y; and a second polypeptide chain comprising a second engineered human IgGl Fc polypeptide chain, the second engineered human IgGl Fc polypeptide chain optionally comprising an amino acid substitution Y407T, wherein either: the first engineered human IgGl Fc polypeptide chain further comprises an amino acid substitution selected from D356K, D356R, or D356H and/or K439R or K439H, and the second engineered human IgGl Fc polypeptide chain further comprises an amino acid substitution selected from K439D or K439E and/or D356E; or the first engineered human IgGl Fc polypeptide chain further comprises an amino amino acid substitution T366Y; and a second polypeptide chain compris
  • a method for preparing a heteromultimer comprising a first engineered human IgGl Fc polypeptide chain and a second engineered human IgGl Fc polypeptide chain, wherein the polypeptide chains meet at an engineered interface, the method comprising the steps of: replacing at least one contact residue of the first polypeptide chain; replacing at least one contact residue of the second polypeptide chain, wherein the contact residue replacement on the first polypeptide chain optionally comprises T366Y; wherein the contact residue replacement on the second polypeptide chain optionally comprises Y407T; and wherein either: the first polypeptide chain further comprises a contact residue replacement selected from D356K, D356R, or D356H and/or K439R or K439H, and the second polypeptide chain further comprises a contact residue replacement selected from K439D or K439E and/or D356E; or the first polypeptide chain further comprises a contact residue replacement selected from K439D or K439E
  • an isolated heteromultimer comprising a first polypeptide and a second polypeptide corresponding to a human IgGl residue, each comprising an antibody constant domain, the heteromulti m er having the following characteristics: the first and second polypeptides meet at an engineered interface within the domain; the first polypeptide optionally comprises an altered contact residue comprising T366Y; the second polypeptide optionally comprises an altered contact residue comprising Y407T; and either: the first polypeptide further comprises an altered contact residue comprising D356K, D356R, or D356H and/or K439R or K439H, and the second polypeptide further comprises an altered contact residue comprising K439D or K439E and/or D356E; or the first polypeptide further comprises an altered contact residue comprising K439D or K439E and/or D356E, and the second polypeptide further comprises an altered contact residue comprising D356K, D356R,
  • a method for making a heterodimeric molecule comprising: introducing into a cell a first heterologous polynucleotide, the first heterologous polynucleotide comprising a nucleotide sequence that encodes for a first polypeptide, the first polypeptide comprising either a wildtype human IgGl Fc chain or a variant of a first human IgGl Fc chain (a “first variant”); and either: the first heterologous polynucleotide comprises a second nucleotide sequence that encodes for a second polypeptide, the second polypeptide comprising either a wildtype human IgGl Fc chain or a variant of a second human IgGl Fc chain (a “second variant”); or introducing into a cell a second heterologous polynucleotide, the second heterologous polynucleotide comprising a nucleotide sequence that encodes for a second polypeptide, the second polypeptid
  • the first variant optionally comprises an amino acid substitution T366Y and comprises an amino acid substitution at D356 carrying a positive charge, i.e., D356K, D356R, or D356H, and/or an amino acid substitution at K439 carrying a positive charge, i.e., K439R or K439H.
  • the second variant optionally comprises an amino acid substitution Y407T and comprises an amino acid substitution at K439 carrying a negative charge, i.e., K439D or K439E, and/or an amino acid substitution at D356 carrying a negative charge, i.e., D356E.
  • the first variant optionally comprises an amino acid substitution T366Y and comprises an amino acid substitution at K439 carrying a negative charge, i.e., K439D or K439E and/or an amino acid substitution at D356 carrying a negative charge, i.e., D356E.
  • the second variant optionally comprises an amino acid substitution Y407T and comprises an amino acid substitution at D356 carrying a positive charge, i.e., D356K, D356R, or D356H and/or an amino acid substitution at K439 carrying a positive charge, i.e.,
  • the second variant may comprise, consist essentially of, or consist of amino acid substitutions Y407T, I253L, Q311R, and Y349H; if the first variant comprises, consists essentially of, or consists of amino acid substitutions T366Y, A339T, N390D, and S408T, then the second variant may comprise, consist essentially of, or consist of amino acid substitution Y407T; if the first variant comprises, consists essentially of, or consists of amino acid substitution T366Y, then the second variant may comprise, consist essentially of, or consist of amino acid substitutions Y407T, I253L, Q311R, and Y349H; if the first variant comprises, consists essentially of, or
  • the second variant may comprise, consist essentially of, or consist of amino acid substitutions Y407T, Y349H, and K439D; if the first variant comprises, consists essentially of, or consists of amino acid substitutions T366Y, D356R, and K439R, then the second variant may comprise, consist essentially of, or consist of amino acid substitutions Y407T and K439D; and if the first variant comprises, consists essentially of, or consists of amino acid substitutions T366Y, Y349H, D356R, and K439R, then the second variant may comprise, consist essentially of, or consist of amino acid substitutions Y407T and K439D.
  • a heterodimeric molecule comprising: a first polypeptide chain comprising either a wildtype human IgGl Fc polypeptide chain or a first engineered human IgGl Fc polypeptide chain, the first engineered human IgGl Fc polypeptide chain comprising amino acid substitutions at positions selected from: (1) T366Y, E283K, N390D, and H435R; (2) T366Y, A339T, N390D, and S408T; (3) T366Y, D270K, N390D, and K439R; (4) T366Y, E258D, V305T, and A339T; (5) T366Y; (6) T366Y, D356R, and K439R;
  • T366Y, Y349H, D356R, and K439R (8) T366Y and D356K, D356R, or D356H and/or K439R or K439H; or (9) T366Y and K439D or K439E and/or D356E; and a second polypeptide chain comprising either a wildtype human IgGl Fc polypeptide chain or a second engineered human IgGl Fc polypeptide chain, the second engineered human IgGl Fc polypeptide chain comprising amino acid substitutions at positions selected from: (1) Y407T, I253L, Q311R, and Y349H; (2) Y407T, Y349H, K370R, andK439Q; (3) Y407T, L358M, T393S, and K439Q; (4) Y407T, Y300L, Y349H, and V397I; (5) Y407
  • the second polypeptide chain comprises amino acid substitutions selected from: (1) Y407T, I253L, Q311R, and Y349H; (2) Y407T, Y349H, K370R, andK439Q; (3) Y407T, L358M, T393S, and K439Q; (4) Y407T, Y300L, Y349H, and V397I; or (5) Y407T, V273T, Y349H, and L358M; if the second polypeptide chain amino acid
  • the second polypeptide chain may comprise amino acid substitutions Y407T and K439D or K439E and/or D356E.
  • the second polypeptide chain may comprise amino acid substitutions Y407T and D356K, D356R, or D356H and/or K439R or K439H.
  • a molecule comprising an engineered human IgGl Fc polypeptide comprising, consisting essentially of, or consisting of amino acid substitutions, numbered according to EU convention relative to the wildtype human IgGl Fc chain, at positions selected from: (1) Y407T, I253L, Q311R, and Y349H; (2) Y407T, Y349H, K370R, and K439Q; (3) Y407T, L358M, T393S, and K439Q; (4) Y407T, Y300L, Y349H, and V397I; (5) Y407T, V273T, Y349H, and L358M; (6) Y407T; (7) Y407T and K439D; (8) Y407T, Y349H, and K439D; (9) Y407T and
  • the engineered human IgGl Fc polypeptide binds to a second molecule comprising a second engineered human IgGl Fc polypeptide.
  • the second engineered human IgGl Fc polypeptide may comprise amino acid substitutions at T366Y and D356K, D356R, or D356H and/or K439R or K439H.
  • the second engineered human IgGl Fc polypeptide may comprise amino acid substitutions at T366Y and K439D or K439E and/or D356E.
  • a heterodimeric molecule comprising: (A) a first engineered human IgGl Fc polypeptide chain, the first engineered human IgGl Fc polypeptide chain comprising amino acid substitutions, numbered according to EU convention relative to the wildtype human IgGl Fc chain, at positions selected from: (1) T366Y, E283K, N390D, and H435R; (2) T366Y, A339T, N390D, and S408T; (3) T366Y, D270K, N390D, and K439R; (4) T366Y, E258D, V305T, and A339T; (5) T366Y; (6) T366Y, D356R, and K439R;
  • T366Y, Y349H, D356R, and K439R (8) T366Y and D356K, D356R, orD356H and/or K439R or K439H; or (9) T366Y and K439D orK439E and/or D356E; and (B) a second engineered human IgGl Fc polypeptide chain, the second engineered human IgGl Fc polypeptide chain comprising amino acid substitutions, numbered according to EU convention relative to the wildtype human IgGl Fc chain, at positions selected from: (1) Y407T, I253L, Q311R, and Y349H; (2) Y407T, Y349H, K370R, and K439Q; (3) Y407T, L358M, T393S, and K439Q; (4) Y407T, Y300L, Y349H, and V397I; (5) Y407T,
  • Y407T, Y349H, and K439D Y407T and K439D or K439E and/or D356E; or (10) Y407T and D356K, D356R, or D356H and/or K439R or K439H, with the provisos that: (i) if the first engineered human IgGl Fc polypeptide chain amino acid substitution consists of T366Y, then the second engineered human IgGl Fc polypeptide chain comprises amino acid substitutions selected from: (1) Y407T, I253L, Q311R, and Y349H; (2) Y407T, Y349H, K370R, and K439Q; (3) Y407T, L358M, T393S, and K439Q; (4) Y407T, Y300L, Y349H, and V397I; or (5) Y407T, V273T, V273T,
  • a heterodimeric molecule comprising: (A) an engineered human IgGl Fc polypeptide chain, the engineered human IgGl Fc polypeptide chain comprising amino acid substitutions, numbered according to EU convention relative to the wildtype human IgGl Fc chain, at positions selected from: (1) T366Y, E283K, N390D, and H435R; (2) T366Y, A339T, N390D, and S408T; (3) Y407T, L358M, T393S, and K439Q; or (4) Y407T, V273T, Y349H, and L358M; and (B) a wildtype human IgGl Fc polypeptide chain.
  • a pharmaceutical composition comprising a heterodimeric molecule, the heterodimeric molecule comprising: (A) a first engineered human IgGl Fc polypeptide chain, the first engineered human IgGl Fc polypeptide chain comprising amino acid substitutions, numbered according to EU convention relative to the wildtype human IgGl Fc chain, at positions selected from: (1) T366Y, E283K, N390D, and H435R; (2) T366Y, A339T, N390D, and S408T; (3) T366Y, D270K, N390D, and K439R; (4) T366Y, E258D, V305T, and A339T; (5) T366Y; (6) T366Y, D356R, and K439R; (7) T366Y, Y349H, D356R, and K439R; (8) T366Y and D356K, D
  • IgGl Fc polypeptide chain the second engineered human IgGl Fc polypeptide chain comprising amino acid substitutions, numbered according to EU convention relative to the wildtype human IgGl Fc chain, at positions selected from: (1) Y407T, I253L, Q311R, and Y349H; (2) Y407T, Y349H, K370R, and K439Q; (3) Y407T, L358M, T393S, and K439Q; (4) Y407T, Y300L, Y349H, and V397I; (5) Y407T, V273T, Y349H, and L358M; (6) Y407T; (7) Y407T and K439D; (8) Y407T, Y349H, and K439D; (9) Y407T and K439D or K439E and/or D356E; or (10) Y407T and D35
  • a pharmaceutical composition comprising a heterodimeric molecule, the heterodimeric molecule comprising: (A) an engineered human IgGl Fc polypeptide chain, the engineered human IgGl Fc polypeptide chain comprising amino acid substitutions, numbered according to EU convention relative to the wildtype human IgGl Fc chain, at positions selected from: (1) T366Y, E283K, N390D, and H435R; (2) T366Y, A339T, N390D, and S408T; (3) Y407T, L358M, T393S, and K439Q; or (4) Y407T, V273T, Y349H, and L358M; and (B) a wildtype human IgGl Fc polypeptide chain; and a pharmaceutically acceptable excipient.
  • a method for preparing a heteromultimer comprising a first engineered human IgGl Fc polypeptide chain and a second engineered human IgGl Fc polypeptide chain, wherein the polypeptide chains meet at an engineered interface, the method comprising the steps of: replacing at least one contact residue of the first polypeptide chain; replacing at least one contact residue of the second polypeptide chain, wherein the contact residue replacement on the first polypeptide chain comprises: (1) T366Y, E283K, N390D, and H435R; (2) T366Y, A339T, N390D, and S408T; (3) T366Y, D270K, N390D, and K439R; (4) T366Y, E258D, V305T, and A339T; (5) T366Y; (6) T366Y, D356R, and K439R; (7) T366Y, Y349H, D356R, and K4
  • the contact residue replacement on the first polypeptide chain comprises T366Y and D356K, D356R, orD356H and/or K439R or K439H
  • the contact residue replacement on the second polypeptide chain may comprise Y407T and K439D or K439E and/or D356E.
  • the contact residue replacement on the second polypeptide chain may comprise Y407T and D356K, D356R, or D356H and/or K439R or K439H.
  • a method for preparing a heteromultimer comprising an engineered human IgGl Fc polypeptide chain and a wildtype human IgGl Fc polypeptide chain is provided, wherein the polypeptide chains meet at an engineered interface, the method comprising the steps of: (A) replacing at least one contact residue of the first polypeptide chain; and wherein the contact residue replacement on the first polypeptide chain comprises: (1) T366Y, E283K, N390D, and H435R; (2) T366Y, A339T, N390D, and S408T; (3) Y407T, L358M, T393S, and K439Q; or (4) Y407T, V273T, Y349H, and L358M; (B) expressing the first and second polypeptides in a host cell to obtain the heteromultimer; (C) recovering the heteromultimer from the host cell culture; wherein the replacing at least one contact residue of the first
  • an isolated heteromultimer comprising a first polypeptide and a second polypeptide, each comprising an antibody constant domain, the heteromultimer having the following characteristics: the first and second polypeptides meet at an engineered interface within the domain; the first polypeptide comprises at least one altered contact residue; the second polypeptide comprises at least one altered contact residue; the at least one altered contact residue of the first polypeptide corresponds to a human IgGl residue and comprises: (1) T366Y, E283K, N390D, and H435R; (2) T366Y, A339T, N390D, and S408T; (3) T366Y, D270K, N390D, and K439R; (4) T366Y, E258D, V305T, and A339T; (5) T366Y; (6) T366Y, D356R, and K439R; (7) T366Y, Y349H, D356R,
  • the at least one altered contact residue of the second polypeptide chain comprises: (1) Y407T, I253L, Q311R, and Y349H; (2) Y407T, Y349H, K370R, and K439Q; (3) Y407T, L358M, T393S, and K439Q; (4) Y407T, Y300L, Y349H, and V397I; or (5) Y407T,
  • the at least one altered contact residue of the second polypeptide chain comprises: (1) T366Y, E283K, N390D, and H435R; (2) T366Y, A339T, N390D, and S408T; (3) T366Y, D270K, N390D, and K439R; or (4) T366Y, E258D, V305T, and A339T.
  • the altered contact residues of the first polypeptide comprise T366Y and D356K, D356R, or D356H and/or K439R or K439H
  • the altered contact residues of the second polypeptide may comprise Y407T and K439D or K439E and/or D356E.
  • the altered contact residues of the second polypeptide may comprise Y407T and D356K, D356R, or D356H and/or K439R or K439H.
  • an isolated heteromultimer comprising a first polypeptide and a second polypeptide, each comprising an antibody constant domain, the heteromulti mer having the following characteristics: the first and second polypeptides meet at an engineered interface within the domain; the first polypeptide comprises at least one altered contact residue; the at least one altered contact residue of the first polypeptide corresponds to a human IgGl residue and comprises: (1) T366Y, E283K, N390D, and H435R; (2) T366Y, A339T, N390D, and S408T; (3) T366Y, D356R, and K439R; (4) T366Y, Y349H, D356R, and K439R; (4) Y407T, L358M, T393S, and K439Q; (5) Y407T, V273T, Y349H, and L358M; (6) Y407T and
  • a host cell comprising a nucleic acid encoding a heteromultimer or heterodimeric molecule as described herein.
  • the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are present in a single vector.
  • the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are present in separate vectors.
  • a method of making a heteromultimer is provided, the method comprising culturing the host cell so that the nucleic acid is expressed and recovering the heteromultimer or heterodimeric antibody from the cell culture.
  • the host cell is a mammalian cell.
  • the heteromultimer or heterodimeric molecule is recovered from the cell culture media.
  • Figure 1 is a schematic diagram of example Fc variant chains A and B, including N- and C-terminal segments.
  • Figures 2A and 2B show examples of SDS PAGE and micro capillary electrophoresis SDS analyses, respectively, of example Fc variant chains.
  • Figure 3 shows uCE chromatograms of the purified expression products of a gene encoding for Fc Chains A and B each having wild type Fc (SEQ ID NO: 1) as the Fc domain (“Wt Fc”) compared to the purified expression products of a gene encoding for Fc chain A having one amino acid substitution in the human IgGl Fc domain consisting of T366Y (SEQ ID NO: 2) and chain B having one amino acid substitution in the human IgGl Fc domain consisting of Y407T (SEQ ID NO: 3) (together, the “Knob-Hole Parent”).
  • Figures 4A, 4B, and 4C show standard curves derived from the uCE chromatograms described with respect to Figure 3.
  • Figure 5 is a graph of measured vs. predicted mol percent of heterodimerized example Fc variant chains A and B.
  • Figure 6 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, E283K, N390D, and H435R (SEQ ID NO: 4/A062) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, I253L, Q311R, and Y349H (SEQ ID NO: 8/B089), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, E283K, N390D, and H435R
  • Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, I253L, Q311R, and Y349H
  • Figure 7 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, A339T, N390D, and S408T (SEQ ID NO: 5/A004) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T (SEQ ID NO: 3/B000), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 8 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y (SEQ ID NO: 2/A000) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, I253L, Q311R, and Y349H (SEQ ID NO: 8/B089), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 9 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y (SEQ ID NO: 2/A000) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, Y349H, K370R, and K439Q (SEQ ID NO: 9/B077), compared to
  • Figure 10 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, D270K, N390D, and K439R (SEQ ID NO: 6/A001) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, L358M, T393S, and K439Q (SEQ ID NO: 10/B056), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 11 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, A339T, N390D, and S408T (SEQ ID NO: 5/A004) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, Y349H, K370R, and K439Q (SEQ ID NO: 9/B077), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, A339T, N390D, and S408T
  • Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, Y349H, K370R, and K439Q
  • Figure 12 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, D270K, N390D, and K439R (SEQ ID NO: 6/A001) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T (SEQ ID NO: 3/B000), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 13 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y (SEQ ID NO: 2/A000) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, L358M, T393S, and K439Q (SEQ ID NO: 10/B056), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 14 shows a chromatogram of the expression products of a gene encoding for (i)
  • Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, A339T, N390D, and S408T (SEQ ID NO: 5/A004) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, I253L, Q311R, and Y349H (SEQ ID NO: 8/B089), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 15 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, A339T, N390D, and S408T (SEQ ID NO: 5/A004) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, L358M, T393S, and K439Q (SEQ ID NO: 10/B056), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, A339T, N390D, and S408T
  • Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, L358M, T393S, and K439Q
  • Figure 16 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, E258D, V305T, and A339T (SEQ ID NO: 7/A016) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, Y300L, Y349H, and V397I (SEQ ID NO: 11/B016), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 17 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, A339T, N390D, and S408T (SEQ ID NO: 5/A004) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, V273T, Y349H, and L358M (SEQ ID NO: 12/B033), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 18 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, D270K, N390D, and K439R (SEQ ID NO: 6/A001) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, V273T, Y349H, and L358M (SEQ ID NO: 12/B033), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, D270K, N390D, and K439R
  • Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, V273T, Y349H, and L358M (SEQ ID NO: 12/B03
  • Figure 19 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y (SEQ ID NO: 2/A000) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, V273T, Y349H, and L358M (SEQ ID NO: 12/B033), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 20 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, D270K, N390D, and K439R (SEQ ID NO: 6/A001) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, Y349H, K370R, and K439Q (SEQ ID NO: 9/B077), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 21 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, E283K, N390D, and H435R (SEQ ID NO: 4/A062) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, L358M, T393S, and K439Q (SEQ ID NO: 10/B056), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, E283K, N390D, and H435R
  • Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, L358M, T393S, and K439Q
  • Figure 22 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, D270K, N390D, and K439R (SEQ ID NO: 6/A001) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, I253L, Q311R, and Y349H (SEQ ID NO: 8/B089), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 23 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y,
  • E283K, N390D, and H435R (SEQ ID NO: 4/A062) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, V273T, Y349H, and L358M (SEQ ID NO: 12/B033), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 24 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, E283K, N390D, H435R (SEQ ID NO: 4/A062) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T (SEQ ID NO: 3/B000), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 25 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, E283K, N390D, H435R (SEQ ID NO: 4/A062) and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, Y349H, K370R, K439Q (SEQ ID NO: 9/B077), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Figure 26 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, E283K, N390D, H435R (SEQ ID NO: 4/A062) and (ii) a wild-type Fc chain B (that is, Fc Chain B having no amino acid substitutions in the human IgGl Fc domain; SEQ ID NO: 1), compared to Wt Fc and compared to the Knob-Hole Parent.
  • Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, E283K, N390D, H435R (SEQ ID NO: 4/A062)
  • a wild-type Fc chain B that is, Fc Chain B having no amino acid substitutions in the human IgGl Fc domain; SEQ ID NO: 1
  • Figure 27 shows a chromatogram of the expression products of a gene encoding for (i) Fc chain A having amino acid substitutions in the human IgGl Fc domain consisting of T366Y, A339T, N390D, S408T (SEQ ID NO: 5/A004) and (ii) a wild-type Fc chain B (that is, Fc Chain B having no amino acid substitutions in the human IgGl Fc domain; SEQ ID NO: 1), compared to Wt Fc and compared to a control comprising Fc Chain A having amino acid substitution T366Y and Fc chain B comprising wild-type Fc (the “Knob-WT Parent”).
  • Figure 28 shows a chromatogram of the expression products of a gene encoding for (i) a wild-type Fc chain A (that is, Fc Chain A having no amino acid substitutions in the human IgGl Fc domain; SEQ ID NO: 1) and (ii) an Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, V273T, Y349H, L358M (SEQ ID NO: 12/B033), compared to Wt Fc and compared to the Knob-WT Parent.
  • a wild-type Fc chain A that is, Fc Chain A having no amino acid substitutions in the human IgGl Fc domain; SEQ ID NO: 1
  • an Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, V273T, Y349H, L358M (SEQ ID NO: 12/B033)
  • Figure 29 shows a chromatogram of the expression products of a gene encoding for (i) a wild-type Fc chain A (that is, Fc Chain A having no amino acid substitutions in the human IgGl Fc domain; SEQ ID NO: 1) and (ii) an Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, L358M, T393S, K439Q (SEQ ID NO: 10/B056), compared to Wt Fc and compared to a control comprising Fc Chain A comprising wild-type Fc and Fc chain B having amino acid substitution Y407T (the “WT-Hole Parent”).
  • a wild-type Fc chain A that is, Fc Chain A having no amino acid substitutions in the human IgGl Fc domain
  • SEQ ID NO: 10/B056 an Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T, L358M,
  • Figure 30 shows LC/MS traces from the homo- and heterodimers formed when expressing engineered Fc protein domains as described herein with Fabs from Emicizumab.
  • Figure 31 shows example chromatograms of Emicizumab-like “wild type” homo- and heterodimer antibodies, separated by cation exchange chromatography. The composition of the heterodimer pair, as determined by peak area percentage, is indicated at right.
  • Figure 32 shows the cIEX chromatograms of each heterodimer combination for the Emicizumab Fabs referred to in Figures 30 and 31. Percent heterodimer is indicated at the right.
  • Figure 33 shows SAX chromatograms of certain heterodimer combinations of the Emicizumab Fabs referred to in Figures 30-32. Percent heterodimer is indicated at the right.
  • Figure 34 shows LC/MS traces from the homo- and heterodimers formed when expressing engineered Fc protein domains as described herein with Fabs from CD3-CD20.
  • Figure 35 shows example chromatograms of CD3-CD20 “wild type” homo- and heterodimer antibodies, separated by cation exchange chromatography. The composition of the heterodimer pair, as determined by peak area percentage, is indicated at right.
  • Figure 36 shows the chromatograms of each heterodimer combination for the CD3- CD20 Fabs referred to in Figures 34 and 35. Percent heterodimer is indicated at the right.
  • the disclosure refers to several genes and proteins for which it provides an example “SEQ ID NO:.” Unless otherwise apparent from the context, reference to a gene or protein should be understood as including the specific SEQ ID NO, as well as allelic and species variants thereof having at least 90, 95, or 99% identity thereto. Examples of allelic and species variants can be found in the SwissProt and other databases.
  • a “cavity” refers to at least one amino acid side chain that is recessed from the interface of a first polypeptide and therefore accommodates a corresponding protuberance on the adjacent interface of a second polypeptide.
  • the cavity may exist in the original interface or may be introduced synthetically (e.g., by altering nucleic acid encoding the interface). To achieve this, the nucleic acid encoding at least one original amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one replacement amino acid residue that has a smaller side chain volume than the original amino acid residue.
  • contact residue or “contact amino acid residue” or the like means any residue that influences the structure or interactions at the interface between two Fc chains. Contact residues may directly form covalent, ionic, or hydrogen bonds, van der Waals interactions, electrostatic interactions, or other interactions with residues in the complimentary chain. A contact residue may also be one that influences such interactions by altering, for example, interactions mediated by salt bridges, solvent molecules, or those involving other amino acid residues at the interface.
  • heteromultimer and “heterodimeric molecule” are interchangeable and mean a molecule comprising at least a first polypeptide and a second polypeptide, wherein the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue.
  • the heteromultimer has binding specificity for at least two different ligands or binding sites.
  • Example structures for the heteromultimer include fusions of antibody binding domains, enzymes, Ab domains with kappa or lambda LCs, receptor binding domains, peptides, and chemically-linked proteins, peptides, and non-protein molecules.
  • Fused antibody domains may comprise, for example, antibody binding domains, an Ab domain with a kappa or lambda light chain, a single domain antibody, an antibody fragment, a fragment variable (Fv) domain, an N-terminal domain, a C-terminal domain, CL, CHI, CH2, CH3, CL, a light chain variable domain (VH), VHH domains, VHHS domains, a heavy chain variable domain (VL), VH + CHI, CL + VL, CL and CHI, a single chain Fv antibody (scFv), a flanking scFv, a trimeric scFv, an scFv-Fc, a bisFv, a multimer, a VH-VL pair, a Fab, a Fab-fragment, a F(ab’)2, a diabody, intrabodies, bi- and multifunctional antibodies,
  • Receptor binding domains may include, for example, viral spike protein domains, tumor necrosis factor receptor 2 (TNFR2), cytokines, proinflammatory cytokines, biased payload immunocytokines, signal modulating immunocytokines, interleukins, masked cytokines, IL-2, monokines, chemokines, lymphokines, interferons, muteins, hormones, insulin, growth factors, integrins, cadherins, spike protein receptor binding domain, and other receptor binding domains as known in the art.
  • TNFR2 tumor necrosis factor receptor 2
  • Non-limiting examples of fused DNA, genes, proteins, and enzymes include cell-free DNA (cfDNA) fragments, a gene, cytosolic enzymes, secretory enzymes, post-translational enzymes, glycoproteins, recombinant proteins, protein modifying enzymes, oxidoreductases, cytotoxins, recombinant immunotoxins (RITs), effector proteins, carboxypeptidases, phosphatases, oxidases, glucose oxidases, galactose oxidases, lactate oxidases, sulfhydryl oxidases, sulfatases, isomerases, a peptide, a chemically-linked protein or peptide, kinases, tyrosine kinases, caspases, caspase 9, targeted enzyme replacement fusion proteins, enzyme linked receptors, neuroprotective payload fusion protein, and soluble factor trap fusion protein formats, and the like.
  • cfDNA cell-free DNA
  • cfDNA
  • Two elements are “heterologous” to one another if not naturally associated.
  • a nucleic acid is heterologous to a cell if not naturally found in the cell or if naturally found in the cell but in a different location (e.g., episomal or different genomic location) than the location described.
  • the term “host” means any prokaryotic or eukaryotic organism that can be a recipient of a nucleic acid.
  • a “host” includes prokaryotic or eukaryotic organisms that can be genetically engineered.
  • the terms “host,” “host cell,” “host system,” and “expression host” can be used interchangeably.
  • an “interface” between two Fc chains comprises those contact amino acid residues (or other non-amino acid groups) of the first polypeptide that interact with one or more contact amino acid residues (or other non-amino acid groups) of the second polypeptide.
  • the interface may be a domain of an immunoglobulin such as a variable domain or constant domain (or regions thereof).
  • the interface comprises the CH2 or CH3 domain of an immunoglobulin derived from an IgG antibody, e.g., a human IgGl antibody.
  • Such an interface may capitalize on a protuberance/cavity motif, such as in the knob-hole relationship demonstrated with the amino acid substition T366Y in one polypeptide and Y407T in the other.
  • the electrostatic attraction exists irrespective of which polypeptide chain of the heterodimer carries the positively charged amino acid(s), so long as the heterodimer counterpart carries the negatively charged amino acid(s) in the corresponding positions.
  • the protuberance/cavity motif may be especially effectively combined with the electrostatic attraction motif to drive heterodimerization and/or disfavor homodimerization.
  • An “isolated” polypeptide or polynucleotide means a polypeptide or polynucleotide that has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. Polypeptides or polynucleotides may be purified, that is, essentially free from any other polypeptide or polynucleotide and associated cellular products or other impurities.
  • nucleoside and nucleotide include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, for example, where one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like.
  • nucleotidic unit is intended to encompass nucleosides and nucleotides.
  • An “Open Reading Frame” or “ORF” means a portion of a polynucleotide that, when translated into amino acids, contains no stop codons.
  • the genetic code reads DNA sequences in groups of three base pairs, which means that a double-stranded DNA molecule can read in any of six possible reading frames-three in the forward direction and three in the reverse.
  • An ORF typically also includes an initiation codon at which translation may start.
  • operably linked refers to functional linkage between two sequences such that one sequence modifies the behavior of the other.
  • a first polynucleotide comprising a nucleic acid expression control sequence such as a promoter, IRES sequence, enhancer, or array of transcription factor binding sites
  • a second polynucleotide are operably linked if the first polynucleotide affects transcription and/or translation of the second polynucleotide.
  • a first amino acid sequence comprising a secretion signal i.e., a subcellular localization signal
  • a second amino acid sequence are operably linked if the first amino acid sequence causes the second amino acid sequence to be secreted or localized to a subcellular location.
  • original residue or “original nucleic acid” is meant the nucleic acid encoding a polypeptide of interest which can be “altered” (i.e., genetically engineered or mutated) to encode, e.g., a protuberance or cavity and/or a relatively positively charged residue or a relatively negatively charged residue.
  • the original or starting nucleic acid may be a naturally occurring nucleic acid or may comprise a nucleic acid which has been subjected to prior alteration.
  • altering the nucleic acid is meant that the original nucleic acid is mutated by inserting, deleting, or replacing at least one codon encoding an amino acid residue of interest.
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • nucleotide nucleotide
  • gene genetic code
  • polynucleotide examples include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (for example, peptide nucleic acids (“PNAs”)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration that
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • these terms include, for example, 3 ’-deoxy -2’, 5 ’-DNA, oligodeoxyribonucleotide N3’ P5’ phosphoramidates, 2’-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, and hybrids thereof including for example hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, “caps,” substitution of one or more of the nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (for example, methyl phosphonates, phosphotri esters, phosphoramidates, carbamates,
  • a “protuberance” refers to at least one amino acid side chain which projects from the interface of a first polypeptide and is therefore positionable in a compensatory cavity in the adjacent interface (i.e., the interface of a second polypeptide) so as to stabilize the heteromultimer, and thereby favor heteromultimer formation over homomultimer formation, for example.
  • the protuberance may exist in the original interface or may be introduced synthetically (e.g., by altering nucleic acid encoding the interface). To achieve this, the nucleic acid encoding at least one original amino acid residue in the interface of the first polypeptide is replaced with nucleic acid encoding at least one import amino acid residue which has a larger side chain volume than the original amino acid residue.
  • a protuberance is “positionable” in the cavity, which means that the spatial location of the protuberance and cavity on the interface of the first polypeptide and second polypeptide and the sizes of the protuberance and cavity are such that the protuberance can be located in the cavity without significantly perturbing the normal association of the first and second polypeptides at the interface.
  • Sequence identity can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.), using default gap parameters, or by inspection, and the best alignment (i.e., resulting in the highest percentage of sequence similarity over a comparison window).
  • algorithms such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.), using default gap parameters, or by inspection, and the best alignment (i.e., resulting in the highest percentage of sequence similarity over a comparison window).
  • Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of matched and mismatched positions not counting gaps in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the window of comparison between two sequences is defined by the entire length of the shorter of the two sequences.
  • Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal, or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.
  • Mutations are sometimes referred to in the form XnY, wherein X is a wildtype amino acid, n is an amino acid position of X in a wildtype sequence, and Y is a replacement amino acid. If the mutation occurs in a sequence having a different number of amino acids than the wildtype sequence, it is present at the position in the sequence aligned with position n in the wildtype sequence when the respective sequences are maximally aligned.
  • the numbering is according to EU convention and is relative to wildtype human IgGl Fc having sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQ VYTLPP SRDELTKNQ VSLTCLVKGF YP SDIA VEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGK (SEQ ID NO: 1), wherein the N-Terminus D is numbered D221 and the C-Terminus K is numbered K447.
  • Example 1 Demonstration of increased heterodimerization and decreased homodimerization using engineered Fc protein domains in a model system
  • the Fc variant chains A and B were designed to have significantly different molecular weights.
  • chain A comprised an FHVH33 (fully-human heavy chain variable domain) binding domain (e.g. Lam et al, 2020), a GS linker, an antibody upper hinge domain, and human IgGl Fc (“huIgGl Fc”) as modified, followed by a second GS linker, and FLAG and His tags; and chain B comprised an upper hinge region, huIgGl Fc as modified, a GS linker, and C myc and C tags (see Figure 1).
  • Wt Fc chains A and B are 40.87 kDa and 27.36 kDa, respectively, and they and the variants described herein were easily resolved via SDS PAGE and/or micro capillary electrophoresis SDS (see Figure 2).
  • the Fc variant chain A N-terminal FHVH33 binding domain and GS linker segments are collectively represented by SEQ ID NO: 22
  • the Fc variant chain A C-Terminal second GS linker and FLAG and His tags segments are collectively represented by SEQ ID NO: 23 (GGGSDYKDDDDKGHHHHHH).
  • the Fc variant chain B C-Terminal GS linker and C myc and C tags segments are collectively represented by SEQ ID NO: 24 (GGGSEQKLISEEDLEPEA).
  • HEK 293 cells were cultured in suspension in serum-free medium and transiently transfected with plasmids containing genes encoding for pairwise combinations of Fc variant chains A and B.
  • Fc chains A and B with either wild type Fc domain (SEQ ID NO: 1) or “knob into hole” sequences without additional substitutions (SEQ ID NOs: 2 and 3) in equivalent ratios to the experimental library variants were included as controls.
  • the cultures were harvested and clarified 7 days after transfection. If applicable, the expression products were purified from the supernatant using protein A resin and buffer exchanged into DPBS, pH 7.0, either by repeated concentration and dilution in centrifugal filter units or by gel filtration.
  • Expression product that is, homodimers of Fc chain A, homodimers of Fc chain B, and the desired heterodimers of Fc chains A and B concentration was determined by measuring the absorbance at 280 nm.
  • Figure 6 shows a chromatogram of Protein A-purified samples demonstrating increased percent heterodimerization in the variant pair having Fc chain A comprising amino acid substitutions in the human IgGl Fc domain consisting of T366Y, E283K, N390D, and H435R and (ii) Fc chain B having amino acid substitutions in the human IgGl Fc domain consisting of Y407T,
  • Wt Fc, Knob-Hole, Knob-WT, and WT-Hole Parental controls as the case may be, and peak areas indicate the notable phenotype favoring heterodimerization (e.g., decreased monomer, homodimer, and/or increased heterodimer formation).
  • Example 2 Demonstration of increased heterodimerization and decreased homodimerization between antibody heavy chains using engineered Fc protein domains and Fabs from Emicizumab
  • Fabs from Emicizumab sold under the brand name Hemlibra, a bispecific antibody that binds to both coagulation factor IX and factor X for the treatment of hemophilia A, were fused with selected, engineered Fc domains that combined the knob-hole motif with the electrostatic attraction motif.
  • a standard IgGl hinge sequence (SEQ ID NO: 25) was used (rather than the IgG4 hinge sequence typically used in Emicizumab).
  • Fab 1 and Fab 2 of Emicizumab are labeled “Heml” (SEQ ID NO: 26) and “Hem2” (SEQ ID NO: 27) respectively, in Figures 30-32.
  • the Fc chains are labeled “WT” (or wild-type, corresponding to SEQ ID NO: 1), “K” (or Knob, corresponding to SEQ ID NO: 2, carrying an amino acid substitution numbered relative to SEQ ID NO: 1 of T366Y), “H” (or Hole, corresponding to SEQ ID NO: 3, carrying an amino acid substitution numbered relative to SEQ ID NO: 1 of Y407T), and in the case of further variants, are labeled “Kvl” (corresponding to SEQ
  • Emicizumab has two unique heavy chains and two unique light chains.
  • the Emicizumab light chains are represented by SEQ ID NOs: 50 and 51.
  • Purified proteins were evaluated for percent heterodimerization by cIEX separation with a sodium chloride salt gradient at pH 6. Chromatograms of purified homodimers were used in each case to assign identity to appropriate peaks in the heterodimer-expressing samples (Figure 31). The area under each peak was determined, and the percentage of the total peak area corresponding to each species (homodimer or heterodimer) was determined for each pair ( Figure 32; see also Figure 33, which was generated by diluting the normalized protein samples 1 :1 with SAX MPA, injecting onto an Agilent Bio SAX, NP5 4.6x 250 mm Strong Anion exchange column equilibrated at pH 9.5, and separating with a NaCl gradient).
  • variant heterodimer combinations in each case show improved percent heterodimerization vs. the original Knob and Hole Fc domains in the context of the Emicizumab Fabs.
  • Example 3 Demonstration of increased heterodimerization and decreased homodimerization between antibody heavy chains using engineered Fc protein domains and Fabs from CD3-C20
  • Fabs from “CD3-CD20” a bispecific antibody that binds to both the T-cell coreceptor CD3 and to the B-cell membrane protein CD20, were fused with selected, engineered Fc domains, again combining the knob-hole motif with the electrostatic attraction motif.
  • Each version was assessed for percent heterodimerization by cIEX separation.
  • Fab 1 is labeled “CD3” (SEQ ID NO: 48)
  • Fab 2 is labeled “CD20” (SEQ ID NO: 49) in Figures 34-36.
  • the Fc chains are labeled as described in Example 2.
  • the full polypeptide chains, including the hinge sequence, are represented by SEQ ID NOs: 40-47 (see Table 3).
  • SEQ ID NOs: 40-47 As an asymmetric bi specific IgG antibody, CD3-CD20 has two kinds of heavy chain and two kinds of light chain.
  • the CD3-CD20 light chains are represented by SEQ ID NOs: 52 and 53.
  • Plasmids carrying each of the half antibodies were transiently transfected into HEK293 cells alone or in pairs (see Table 3), the cells were cultured, supernatants collected, and the antibodies were pro-A purified as described above. Purified proteins were positively identified via intact LC/MS ( Figure 34). Purified proteins were evaluated for percent heterodimerization by cIEX separation with a sodium chloride salt gradient at pH 6. Chromatograms of purified homodimers were used in each case to assign identity to appropriate peaks in the heterodimer-expressing samples (Figure 35). The area under each peak was determined, and the percentage of the total peak area corresponding to each species (homodimer or heterodimer) was determined for each pair ( Figure 36).
  • variant heterodimer combinations in each case show improved percent heterodimerization vs. the original Knob and Hole Fc domains in the context of the CD3-CD20 Fabs.

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Abstract

L'invention concerne des molécules hétérodimères, ainsi que des procédés de fabrication et d'utilisation de celles-ci.
PCT/US2024/022229 2023-03-30 2024-03-29 Ingénierie fc pour format d'anticorps hétérodimère Ceased WO2024206820A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180273642A1 (en) * 2009-12-29 2018-09-27 Aptevo Research And Development Llc Heterodimer binding proteins and uses thereof
US20210221917A1 (en) * 2016-05-23 2021-07-22 Momenta Pharmaceuticals, Inc. COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc CONSTRUCTS
WO2021207207A2 (fr) * 2020-04-07 2021-10-14 Neoleukin Therapeutics, Inc. Leurres protéiques de novo de l'enzyme 2 de conversion de l'angiotensine (ace2)
WO2022117065A1 (fr) * 2020-12-03 2022-06-09 江苏恒瑞医药股份有限公司 Protéine de liaison à un antigène multispécifique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180273642A1 (en) * 2009-12-29 2018-09-27 Aptevo Research And Development Llc Heterodimer binding proteins and uses thereof
US20210221917A1 (en) * 2016-05-23 2021-07-22 Momenta Pharmaceuticals, Inc. COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc CONSTRUCTS
WO2021207207A2 (fr) * 2020-04-07 2021-10-14 Neoleukin Therapeutics, Inc. Leurres protéiques de novo de l'enzyme 2 de conversion de l'angiotensine (ace2)
WO2022117065A1 (fr) * 2020-12-03 2022-06-09 江苏恒瑞医药股份有限公司 Protéine de liaison à un antigène multispécifique

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