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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • A—HUMAN NECESSITIES
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  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• anti-CD38 antigen-binding proteins such as fully human anti-CD38 IgG class antibodies each having an altered amino acid sequence in their heavy chain variable region and/or light chain variable region compared to their wild type parent antibody.
• Disclosed variant antibodies exhibit improved antigen binding, cell binding and cytotoxicity capabilities compared to their parent antibody.
• Disclosed variant antibodies also exhibit the same species cross reactivity as their parent antibody.
• CD38 is a 45 kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain.
• the CD38 protein is a bifunctional ectoenzyme that can catalyze the conversion of NAD + into cyclic ADP-ribose (cADPR) and also hydrolyze cADPR into ADP-ribose.
• cADPR cyclic ADP-ribose
• CD38 appears on CD34 + committed stem cells and lineage-committed progenitors of lymphoid, erythroid and myeloid cells.
• CD38 expression persists mostly in the lymphoid lineage with varying expression levels at different stages of T and B cell development.
• CD38 is upregulated in many hematopoeitic malignancies and in cell lines derived from various hematopoietic malignancies, including non-Hodgkin's lymphoma (NHL), Burkitf s lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML).
• NHL non-Hodgkin's lymphoma
• BL Burkitf s lymphoma
• MM multiple myeloma
• B-CLL B chronic lymphocytic leukemia
• ALL acute lymphocytic leukemia
• TCL T cell lymphoma
• AML acute myeloid leukemia
• CD38 has been reported to be involved in Ca 2+ mobilization (Morra et ah, 1998,
• CD38 was proposed to be an important signaling molecule in the maturation and activation of lymphoid and myeloid cells during their normal development.
• CD38 - knockout mice which have a defect in their innate immunity and a reduced T-cell dependent humoral response due to a defect in dendritic cell migration (Partida-Sanchez et ak, 2004, Immunity, 20: 279-291; Partida-Sanchez et ak, 2001, Nat. Med., 7: 1209-1216).
• mice are identical to that in humans since the CD38 expression pattern during hematopoiesis differs greatly between human and mouse: a) unlike immature progenitor stem cells in humans, similar progenitor stem cells in mice express a high level of CD38 (Randall et ak, 1996, Blood, 87:4057- 4067; Dagher et ak, 1998, Biol. Blood Marrow Transplant, 4:69-74), b) while during the human B cell development, high levels of CD38 expression are found in germinal center B cells and plasma cells (Uckun, 1990, Blood, 76: 1908-1923; Kumagai et ak, 1995, J. Exp.
• a chimeric OKT10 antibody with mouse Fab and human IgGl Fc mediates antibody-dependent cell-mediated cytotoxicity (ADCC) very efficiently against lymphoma cells in the presence of peripheral blood mononuclear effector cells from either MM patients or normal individuals (Stevenson et al.,
• a CDR-grafted humanized version of the anti- CD38 antibody AT13/5 has been shown to have potent ADCC activity against CD38-positive cell lines.
• Human monoclonal anti-CD38 antibodies have been shown to mediate the in vitro killing of CD38- positive cell lines by ADCC and/or complement-dependent cytotoxicity (CDC), and to delay the tumor growth in SCID mice bearing MM cell line RPMI-8226 (W02005/103083 A2).
• CDC complement-dependent cytotoxicity
• several anti- CD38 antibodies, IB4, SUN-4B7, and OKT10, but not IB6, ATI, or AT2 induced the proliferation of peripheral blood mononuclear cells (PBMC) from normal individuals (Ausiello et al. 2000, Tissue Antigens, 56:539-547).
• PBMC peripheral blood mononuclear cells
• T16 Another anti-CD38 antibody, T16, induced apoptosis of immature lymphoid cells and leukemic lymphoblast cells from an ALL patient (Kumagai et al. 1995, J. Exp. Med, 181 : 1101-1110), and of leukemic myeloblast cells from AML patients (Todisco et al. 2000, Blood, 95:535-542), but T16 induced apoptosis only in the presence of stroma cells or stroma- derived cytokines (IL-7, IL-3, stem cell factor).
• IL-7 stroma- derived cytokines
• the present disclosure provides a n anti-CD38 antigen-binding protein or fully human anti-CD38 antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region 1 (CDR1) a heavy chain CDR2 and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3; and (a) the heavy chain CDR1 has the amino acid sequence of SEQ ID NO:29, the heavy chain CDR2 has the amino acid sequence of SEQ ID NO:30, the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:31, the light chain CDR1 has the amino acid sequence of SEQ ID NO:32, the light chain CDR2 has the amino acid sequence of SEQ ID NO:33, and the light chain CDR3 has the amino acid sequence of SEQ ID NO:34; (b) the heavy chain CDR1 has the amino acid sequence
• the antibody or antigen-binding fragment thereof comprises a heavy chain variable region that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and a light chain variable region that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a fully human anti-CD38 antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13; and the light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a fully human anti-CD38 antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences of SEQ ID NOS:3 and 4, respectively (e.g., herein called 3H10ml); SEQ ID NOS:5 and 4, respectively (e.g., herein called 3G8ml); SEQ ID NOS:6 and 4, respectively (e.g., herein called 3E3ml); SEQ ID NOS:7 and 2, respectively (e.g., herein called 3G3); SEQ ID NOS:9 and 2, respectively (e.g., herein called 3E11); SEQ ID NOS: 10 and 2, respectively (e.g., herein called 3H10); SEQ ID NOS: 11 and 12, respectively (e.g., herein called 3H10N);
• SEQ ID NOS: 13 and 12 respectively (e.g., herein called 3H10NS); SEQ ID NOS: l and 4, respectively (e.g., herein called 3E10); or SEQ ID NOS:3 and 12, respectively (e.g., herein called 3H10mlg).
• the present disclosure provides a fully human anti-CD38 antibody, or an antigen binding fragment thereof, wherein the antigen -binding fragment is a Fab fragment comprising a variable domain region from a heavy chain and a variable domain region from a light chain, wherein (a) the variable domain region from the heavy chain comprises: a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:29, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:30, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:31; and the variable domain region from the light chain comprises: a light chain CDR1 having the amino acid sequence of SEQ ID NO:32, a light chain CDR2 having the amino acid sequence of SEQ ID NO:33, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:34; wherein (b) the variable domain region from the heavy chain comprises: a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:35
• the present disclosure provides a fully human anti-CD38 antibody, or an antigen binding fragment thereof, wherein the antigen -binding fragment is a Fab fragment comprising a variable domain region from a heavy chain and a variable domain region from a light chain, wherein the variable domain region from the heavy chain comprises a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and wherein the variable domain region from the light chain comprises a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a fully human anti-CD38 antibody, or an antigen binding fragment thereof, wherein the antigen -binding fragment is a Fab fragment comprising a variable domain region from a heavy chain and a variable domain region from a light chain, wherein the variable domain region from the heavy chain and the variable domain region from the light chain are SEQ ID NOS:3 and 4, respectively (e.g., herein called 3H10ml); SEQ ID NOS:5 and 4, respectively (e.g., herein called 3G8ml); SEQ ID NOS:6 and 4, respectively (e.g., herein called 3E3ml); SEQ ID NOS:7 and 2, respectively (e.g., herein called 3G3); SEQ ID NOS:9 and 2, respectively (e.g., herein called 3E11); SEQ ID NOS: 10 and 2, respectively (e.g., herein called 3H10); SEQ ID NOS: 11 and 12, respectively (e.g., herein called 3H10N); SEQ ID NO
• the present disclosure provides a fully human anti-CD38 antibody, or an antigen binding fragment thereof, wherein the antigen-binding fragment is a single chain antibody comprising a variable domain region from a heavy chain and a variable domain region from a light chain joined together with a peptide linker, wherein (a) a heavy chain complementarity determining region 1 (CDR1) has the amino acid sequence of SEQ ID NO:29, a heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 30, a heavy chain CDR3 has the amino acid sequence of SEQ ID NO:31, a light chain CDR1 has the amino acid sequence of SEQ ID NO:32, a light chain CDR2 has the amino acid sequence of SEQ ID NO:33, and a light chain CDR3 has the amino acid sequence of SEQ ID NO:34 (e.g., herein called 3H10ml); (b) a heavy chain CDR1 has the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 has the amino acid sequence of
• the present disclosure provides a fully human anti-CD38 antibody, or an antigen binding fragment thereof, wherein the antigen-binding fragment is a single chain antibody comprising a variable domain region from a heavy chain and a variable domain region from a light chain joined together with a peptide linker, wherein the variable domain region from the heavy chain comprises a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and wherein the variable domain region from the light chain comprises a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a fully human anti-CD38 antibody, or the antigen binding fragment thereof, wherein the antigen-binding fragment is a single chain antibody comprising a variable domain region from a heavy chain and a variable domain region from a light chain joined together with a peptide linker, wherein the variable domain region from the heavy chain and the variable domain region from the light chain are SEQ ID NOS:3 and 4, respectively (e.g., herein called 3H10ml); SEQ ID NOS:5 and 4, respectively (e.g., herein called 3G8ml); SEQ ID NOS:6 and 4, respectively (e.g., herein called 3E3ml); SEQ ID NOS:7 and 2, respectively (e.g., herein called 3G3); SEQ ID NOS:9 and 2, respectively (e.g., herein called 3E11); SEQ ID NOS: 10 and 2, respectively (e.g., herein called 3H10); SEQ ID NOS: 11 and 12, respectively (e.g.,
• any of the fully human anti-CD38 antibodies disclosed herein, or any of the antigen-binding fragments thereof is an IgGl, IgG2, IgG3 or IgG4 class antibody.
• any of the fully human anti-CD38 antibodies disclosed herein, or any of the antigen-binding fragments thereof is an IgGl or IgG4 class antibody.
• any of the fully human anti-CD38 antibodies disclosed herein, or any of the antigen-binding fragments thereof bind to human CD38 protein (SEQ ID NO: 19) and cross-reacts with CD38 protein from any one or any combination of two or more of CD38 proteins from cynomolgus (SEQ ID NO:20), mouse (SEQ ID NO:21) and/or rat (SEQ ID NO: 19).
• any of the fully human anti-CD38 antibodies disclosed herein, or any of the antigen-binding fragments thereof bind to human CD38 protein (SEQ ID NO: 19) and do not cross-react with CD38 protein from cynomolgus (SEQ ID NO:20), mouse (SEQ ID NO:21) or rat (SEQ ID NO:22).
• any of the fully human anti-CD38 antibodies disclosed herein, or any of the antigen-binding fragments thereof bind to cells expressing or over-expressing CD38 protein including for example bind to human myeloma cells (e.g., human multiple myeloma cells), human B lymphoma cells, activated T cells, or cultured cell lines including RPMI8226, Raji or Ramos.
• the cells expressing CD38 protein include transgenic cells engineered to express CD38 protein using recombinant DNA technology.
• the present disclosure provides a pharmaceutical composition, comprising any one of the disclosed the human anti-CD38 antibodies, or any of the antigen-binding fragments thereof, and a pharmaceutically-acceptable excipient.
• the present disclosure provides a kit, comprising any one of the disclosed the human anti-CD38 antibodies, or any of the antigen-binding fragments thereof.
• the present disclosure provides one or more nucleic acids encoding an antigen binding protein, antibody or antigen-binding fragment described herein.
• the one or more nucleic acids are contained in one or more vectors.
• the one or more nucleic acids may be operably linked to one or more promoters.
• a host cell comprising the one or more nucleic acids or vectors.
• methods of producing an antigen binding protein, antibody or antigen-binding fragment described herein comprising culturing the host cell under conditions wherein the antigen-binding protein, antibody or antigen-binding fragment is produced.
• the present disclosure provides a first nucleic acid that encodes a first polypeptide comprising an antibody heavy chain variable region having a heavy chain complementarity determining region (CDR) of any one of the disclosed the human anti-CD38 antibodies, including (a) a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:29, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:30, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:31; (b) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 36, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:37; (c) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:41, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:42, a heavy chain CDR3 having the amino acid sequence of SEQ ID
• the present disclosure provides a first vector comprising a first promoter operably linked to a first nucleic acid which encodes a first polypeptide comprising an antibody heavy chain variable region having the CDRs 1, 2 and 3, of any one of the disclosed the human anti- CD38 antibodies disclosed herein.
• the present disclosure provides a first host cell harboring the first vector which comprises a first promoter operably linked to the first nucleic acid which encodes the first polypeptide comprising an antibody heavy chain variable region having the CDRs 1, 2 and 3, of any one of the disclosed the human anti-CD38 antibodies disclosed herein.
• the first vector comprises a first expression vector.
• the first host cell expresses the first polypeptide comprising the antibody heavy chain variable region having the CDRs 1, 2 and 3, of any one of the disclosed the human anti-CD38 antibodies disclosed herein.
• the present disclosure provides a method for preparing a first polypeptide having an antibody heavy chain variable region comprising CDRs 1, 2 and 3, the method comprising: culturing a population (e.g., a plurality) of the first host cells harboring the first expression vector under conditions suitable for expressing the first polypeptide having the antibody heavy chain variable region comprising the CDRs 1, 2 and 3.
• the method further comprises: recovering from the population of the first host cell the expressed first polypeptide having an antibody heavy chain variable region comprising the CDRs 1, 2 and 3.
• the present disclosure provides a first nucleic acid that encodes a first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13.
• the present disclosure provides a first vector comprising a first promoter operably linked to the first nucleic acid which encodes the first polypeptide comprising the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13.
• the present invention provides a first host cell harboring the first vector which comprises a first promoter operably linked to the first nucleic acid which encodes the first polypeptide comprising the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13.
• the first vector comprises a first expression vector.
• the first host cell expresses the first polypeptide comprising the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13.
• the present disclosure provides a method for preparing a first polypeptide having an antibody heavy chain variable region, the method comprising: culturing a population (e.g., a plurality) of the first host cells harboring the first expression vector under conditions suitable for expressing the first polypeptide having the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13.
• the method further comprises: recovering from the population of the first host cells the expressed first polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13.
• the present disclosure provides a second nucleic acid that encodes a polypeptide comprising an antibody light chain variable region having a light chain complementarity determining region (CDR) of any one of the disclosed the human anti-CD38 antibodies, including (a) a light chain CDR1 having the amino acid sequence of SEQ ID NO:32, a light chain CDR2 having the amino acid sequence of SEQ ID NO:33, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:34; (b) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 38, a light chain CDR2 having the amino acid sequence of SEQ ID NO:39, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:40; (c) a light chain CDR1 having the amino acid sequence of SEQ ID NO:44, a light chain CDR2 having the amino acid sequence of SEQ ID NO:45, and a light chain CDR3 having the amino acid sequence of SEQ ID NO:46; (d
• the present disclosure provides a second vector comprising a second promoter operably linked to a second nucleic acid which encodes a second polypeptide comprising an antibody light chain variable region having the CDRs 1, 2 and 3, of any one of the disclosed the human anti-CD38 antibodies disclosed herein.
• the present disclosure provides a second host cell harboring the second vector which comprises a second promoter operably linked to the second nucleic acid which encodes the second polypeptide comprising an antibody light chain variable region having the CDRs 1, 2 and 3, of any one of the disclosed the human anti-CD38 antibodies disclosed herein.
• the second vector comprises a second expression vector.
• the second host cell expresses the second polypeptide comprising the antibody light chain variable region having the CDRs 1, 2 and 3, of any one of the disclosed the human anti-CD38 antibodies disclosed herein.
• the present disclosure provides a method for preparing a second polypeptide having an antibody light chain variable region comprising CDRs 1, 2 and 3, the method comprising:
• the method further comprises: recovering from the population of the second host cell the expressed second polypeptide having an antibody light chain variable region comprising the CDRs 1, 2 and 3.
• the present disclosure provides a second nucleic acid that encodes a second polypeptide comprising an antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a second vector comprising a second promoter operably linked to the second nucleic acid which encodes the second polypeptide comprising the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present invention provides a second host cell harboring the second vector which comprises a second promoter operably linked to the second nucleic acid which encodes the second polypeptide comprising the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the second vector comprises a second expression vector.
• the second host cell expresses the second polypeptide comprising the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a method for preparing a second polypeptide having an antibody light chain variable region, the method comprising: culturing a population (e.g., a plurality) of the second host cells harboring the second expression vector under conditions suitable for expressing the second polypeptide having the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the method further comprises: recovering from the population of the second host cells the expressed second polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a first nucleic acid that encodes a first polypeptide comprising an antibody heavy chain variable region having heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, and a second nucleic acid that encodes a second polypeptide comprising an antibody light chain variable region having light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, wherein (a) the heavy chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:29, 30 and 31, respectively, and the light chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:32, 33 and 34, respectively; or (b) the heavy chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:35, 36 and 37, respectively, and the light chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:38, 39 and 40,
• the present disclosure provides a vector operably linked to the first nucleic acid that encodes the first polypeptide comprising an antibody heavy chain variable region having heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, and the vector is operably linked to the second nucleic acid that encodes the second polypeptide comprising the antibody light chain variable region having light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies.
• the vector comprises a promoter which is operably linked to the first and second nucleic acids.
• the vector comprises a first promoter which is operably linked to the first nucleic acid, and the vector comprises a second promoter which is operably linked to the second nucleic acid.
• the present disclosure provides a host cell harboring the vector which is operably linked to the first nucleic acid that encodes the first polypeptide comprising the heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies and the vector comprises is operably linked to the second nucleic acid that encodes the second polypeptide comprising the light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies.
• the vector comprises an expression vector.
• the vector comprises a promoter which is operably linked to the first and second nucleic acids.
• the vector comprises a first promoter which is operably linked to the first nucleic acid, and the vector comprises a second promoter which is operably linked to the second nucleic acid.
• the host cell expresses the first polypeptide comprising the heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies and the second polypeptide comprising the light chain
• LC-CDRs 1, 2 and 3 complementarity determining regions of any one of disclosed the human anti-CD38 antibodies.
• the present disclosure provides a method for preparing the first and second
• the method comprising: culturing a population (e.g., a plurality) of the host cell harboring the expression vector under conditions suitable for expressing the first polypeptide comprising the heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies and the second polypeptide comprising the light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies.
• the method further comprises: recovering from the population of the host cells the expressed first and second polypeptides.
• the present disclosure provides a first nucleic acid that encodes a first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and a second nucleic acid that encodes a second polypeptide comprising an antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a vector operably linked to the first nucleic acid that encodes a first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the vector is operably linked to the second nucleic acid which encodes the second polypeptide comprising the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the vector comprises a promoter which is operably linked to the first and second nucleic acids.
• the vector comprises a first promoter which is operably linked to the first nucleic acid, and the vector comprises a second promoter which is operably linked to the second nucleic acid.
• the present invention provides a host cell harboring the vector which is operably linked to the first nucleic acid that encodes a first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the vector is operably linked to the second nucleic acid which encodes the second polypeptide comprising the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the vector comprises an expression vector.
• the vector comprises a promoter which is operably linked to the first and second nucleic acids.
• the vector comprises a first promoter which is operably linked to the first nucleic acid, and the vector comprises a second promoter which is operably linked to the second nucleic acid.
• the host cell expresses the first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the host cell expresses the second polypeptide comprising the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a method for preparing a first polypeptide having an antibody heavy chain variable region and a second polypeptide having an antibody light chain variable region, the method comprising: culturing a population (e.g., a plurality) of the host cells harboring the expression vector under conditions suitable for expressing the first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and for expressing the second polypeptide having the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the method further comprises: recovering from the population of the host cells the expressed first
• polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the expressed second polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a first nucleic acid that encodes a first polypeptide comprising an antibody heavy chain variable region having heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, and a second nucleic acid that encodes a second polypeptide comprising an antibody light chain variable region having light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, wherein (a) the heavy chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:29, 30 and 31, respectively, and the light chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:32, 33 and 34, respectively; or (b) the heavy chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:35, 36 and 37, respectively, and the light chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:38, 39 and 40,
• the present disclosure provides a first vector comprising a first promoter operably linked to the first nucleic acid that encodes the first polypeptide comprising an antibody heavy chain variable region having heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, and a second vector comprising a second promoter operably linked to the second nucleic acid that encodes the second
• polypeptide comprising the antibody light chain variable region having light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies.
• the present disclosure provides a host cell harboring the first vector which comprises the first promoter operably linked to the first nucleic acid that encodes the first polypeptide comprising the heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, and the host cell harbors the second vector which comprises a second promoter operably linked to the second nucleic acid that encodes the second polypeptide comprising the light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies.
• the first and second vectors are first and second expression vectors, respectively.
• the host cell expresses the first polypeptide comprising the heavy chain
• complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies and the host cell expresses the second polypeptide comprising the light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies.
• the present disclosure provides a method for preparing the first and second polypeptides, the method comprising: culturing a population (e.g., a plurality) of the host cell harboring the first and second expression vectors under conditions suitable for expressing the first polypeptide comprising the heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies and the second polypeptide comprising the light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies.
• the method further comprises: recovering from the population of the host cells the expressed first and second polypeptides.
• the present disclosure provides a first nucleic acid that encodes a first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and a second nucleic acid that encodes a second polypeptide comprising an antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a first vector comprising a first promoter operably linked to the first nucleic acid that encodes a first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and a second vector comprising a second promoter operably linked to the second nucleic acid which encodes the second polypeptide comprising the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present invention provides a host cell harboring the first vector which comprises a first promoter operably linked to the first nucleic acid that encodes a first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the host cell harbors the second vector which comprises a second promoter operably linked to the second nucleic acid which encodes the second polypeptide comprising the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the first and second vectors comprise a first and second expression vector, respectively.
• the host cell expresses the first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the host cell expresses the second polypeptide comprising the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a method for preparing a first polypeptide having an antibody heavy chain variable region and a second polypeptide having an antibody light chain variable region, the method comprising: culturing a population (e.g., a plurality) of the host cells harboring the first and second expression vectors under conditions suitable for expressing the first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and for expressing the second polypeptide having the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the method further comprises: recovering from the population of the host cells the expressed first polypeptide comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the expressed second polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a nucleic acid that encodes a polypeptide (e.g., a single chain antibody including an scFv) comprising an antibody heavy chain variable region having heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, and an antibody light chain variable region having light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, wherein (a) the heavy chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:29, 30 and 31, respectively, and the light chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:32, 33 and 34, respectively; or (b) the heavy chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:35, 36 and 37, respectively, and the light chain CDR 1, 2 and 3 regions comprise the amino acid sequences of SEQ ID NOS:38, 39 and 40, respectively
• the present disclosure provides a vector comprising a promoter operably linked to the nucleic acid that encodes the polypeptide (e.g., a single chain antibody including an scFv) comprising the antibody heavy chain variable region having heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, and the antibody light chain variable region having light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies.
• the polypeptide e.g., a single chain antibody including an scFv
• HC-CDRs 1, 2 and 3 heavy chain complementarity determining regions
• LC-CDRs 1, 2 and 3 the antibody light chain variable region having light chain complementarity determining regions
• the present disclosure provides a host cell harboring the vector which comprises a promoter operably linked to the nucleic acid that encodes the polypeptide (e.g., a single chain antibody including an scFv) comprising the antibody heavy chain variable region having heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, and the antibody light chain variable region having light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies.
• the vector comprises an expression vector.
• the host cell expresses the polypeptide comprising the antibody heavy chain variable region having heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, and the antibody light chain variable region having light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies.
• HC-CDRs 1, 2 and 3 the antibody heavy chain variable region having heavy chain complementarity determining regions
• LC-CDRs 1, 2 and 3 of any one of disclosed the human anti-CD38 antibodies.
• the present disclosure provides a method for preparing a polypeptide described herein, the method comprising: culturing a population (e.g., a plurality) of a host cell harboring an expression vector described herein under conditions suitable for expressing the polypeptide.
• the polypeptide comprises the antibody heavy chain variable region having heavy chain complementarity determining regions (HC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies, and the antibody light chain variable region having light chain complementarity determining regions (LC-CDRs 1, 2 and 3) of any one of disclosed the human anti-CD38 antibodies.
• the method further comprises: recovering from the population of the host cells the expressed polypeptide.
• the present disclosure provides a nucleic acid that encodes a polypeptide (e.g., a single chain antibody including an scFv) comprising an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and a an antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4 or 12.
• a polypeptide e.g., a single chain antibody including an scFv
• an antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and a an antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 4 or 12.
• the present disclosure provides a vector comprising a promoter operably linked to the nucleic acid that encodes the polypeptide (e.g., a single chain antibody including an scFv) comprising the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• a promoter operably linked to the nucleic acid that encodes the polypeptide (e.g., a single chain antibody including an scFv) comprising the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present invention provides a host cell harboring the vector which comprises a promoter operably linked to the nucleic acid that encodes the polypeptide (e.g., a single chain antibody including an scFv) comprising the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the vector comprises an expression vector.
• the host cell expresses the polypeptide (e.g., a single chain antibody including an scFv) comprising the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the polypeptide e.g., a single chain antibody including an scFv
• the polypeptide comprising the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a method for preparing the polypeptide (e.g., a single chain antibody including an scFv), the method comprising: culturing a population (e.g., a plurality) of the host cells harboring the expression vector under conditions suitable for expressing the polypeptide (e.g., a single chain antibody including an scFv) comprising the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the method further comprises: recovering from the population of the host cells the expressed the polypeptide (e.g., a single chain antibody including an scFv) comprising the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, and the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the polypeptide e.g., a single chain antibody including an scFv
• the antibody heavy chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13
• the antibody light chain variable region having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or 12.
• the present disclosure provides a method (e.g., an in vitro method) for inhibiting growth or proliferation of CD38-expressing cells, comprising: contacting (i) a population (e.g., a plurality) of effector cells with (ii) a population (e.g., a plurality) of target cells which express CD38 (iii) in the presence of any one or any combination of 2-3 of the human anti-CD38 antibodies described herein, under conditions that are suitable for inhibiting growth or proliferation of the CD38-expressing cells.
• the population of effector cells comprises PBMCs or NK cells.
• the population of target cells comprise multiple myeloma (MM) cells expressing CD38 or transgenic cells expressing CD38.
• the ratio of the effector-to-target cells is 1 : 1, 2: 1, 3: 1, 4: 1 or 5: 1. In one
• the ratio of the effector-to-target cells is 5-10: 1, 10-20: 1, or 20-30: 1.
• the present disclosure provides a method (e.g., an in vitro method) for killing CD38- expressing cells, comprising: contacting (i) a population (e.g., a plurality) of effector cells with (ii) a population (e.g., a plurality) of target cells which express CD38 (iii) in the presence of any one or any combination of 2-3 of the human anti-CD38 antibodies described herein, under conditions that are suitable for killing the CD38-expressing cells.
• the population of effector cells comprises PBMCs or NK cells.
• the population of target cells comprise a multiple myeloma (MM) cells expressing CD38 or transgenic cells expressing CD38.
• the ratio of the effector-to-target cells is 1 : 1, 2: 1, 3 : 1, 4: 1 or 5: 1. In one embodiment, the ratio of the effector-to-target cells is 5-10: 1, 10-20: 1, or 20-30: 1.
• the present disclosure provides a method for treating a subject having a disease associated with CD38 over-expression, the method comprising: administering to the subject an effective amount of a therapeutic composition comprising any one or any combination of 2-3 of the human anti-CD38 antibodies described herein.
• the disease associated with CD38 over-expression comprises: a B-cell leukemia, B-cell lymphoma or B-cell myeloma.
• the disease associated with CD38 over-expression is selected from a group consisting of multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) including Burkitf s lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), systemic lupus erythematosus (SLE), B and T acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma, chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), follicular lymphoma, Waldenstrom's Macroglobulinemia, mantle cell lymphoma, Hodgkin's Lymphoma (HL), plasma cell myeloma, precursor B cell lymphoblastic leukemia/lymphoma, plasmacytoma, giant cell myeloma, plasma cell myeloma
• Figure 1 shows an SPR sensorgram of binding kinetics of an anti-CD38 antibody (Daratumumab).
• Figure 2 shows an SPR sensorgram of binding kinetics of an anti-CD38 antibody (parent antibody A2).
• Figure 3 shows an SPR sensorgram of binding kinetics of an anti-CD38 antibody (variant antibody 3H10ml).
• Figure 4 shows an SPR sensorgram of binding kinetics of an anti-CD38 antibody (variant antibody 3G3).
• Figure 5 shows an SPR sensorgram of binding kinetics of an anti-CD38 antibody (variant antibody 3E10).
• Figure 6 shows a table that summarizes binding kinetics obtained from SPR data of parent antibody A2, and variant antibodies 3E10 and 3H10ml, compared to Daratumumab.
• Figure 7 shows histograms of flow cytometry data of non-activated T cells stained with various anti-CD38 antibodies including parent antibody A2, variant antibodies 3H10ml and 3E1, and Daratumumab.
• Figure 8A shows histograms of flow cytometry data of activated T cells stained with various anti-CD38 antibodies including parent antibody A2, variant antibodies 3H10ml and 3E1, and Daratumumab.
• Figure 8B shows flow cytometry data of activated T cells stained with anti-CD47 H3D4 antibody.
• Figure 9 shows a bar graph of a binding titration assay of RPMI 8226 cells stained with various anti-CD38 antibodies including parent antibody A2, variant antibodies 3H10ml and 3G3 and 3E1, and Daratumumab.
• Each data set includes three different antibody concentrations (from left to right) of 10 ug/mL, 1 ug/mL and 0.1 ug/mL.
• Figure 10 shows a graph of a cell binding assay of various anti-CD38 antibodies binding to human B lymphoma cell line Raji, including anti-CD38 scFv-Fc (line A), parent antibody A2 (line B), variant antibody 3G3 (line C), variant antibody 3H10ml (line D) and Daratumumab (line E).
• Figure 11 shows a graph of a cell binding assay of various anti-CD38 antibodies binding to human B lymphoma cell line Ramos, including anti-CD38 scFv-Fc (line A), parent antibody A2 (line B), Daratumumab (line C), variant antibody 3G3 (line D) and variant antibody 3H10ml (line E).
• Figure 12 shows a graph of a cell binding assay of various anti-CD38 antibodies binding to human primary T cells, including anti-CD38 scFv-Fc (line A), parent antibody A2 (line B), variant antibody 3G3 (line C), Daratumumab (line D), and variant antibody 3H10ml (line E).
• Figure 13 shows a bar graph of a species cross-reactivity assay of Daratumumab compared to anti-CD38-A2 parent antibody.
• Figure 14 shows flow cytometry data of a species cross-reactivity assay of parent antibody A2 compared to variant antibodies 3G3 and 3H10ml, and Daratumumab.
• Figure 15 shows a bar graph of an antibody-dependent cellular phagocytosis (ADCP) assay comparing killing activity of A2 parent antibody, with variant antibodies 3G3 and
• Figure 16 shows a graph of an antibody-dependent cellular cytotoxicity (ADCC) assay comparing killing activity of various anti-CD38 antibodies, including parent antibody A2 (line A), Daratumumab (line B), anti-CD38 scFv-Fc (line C), variant antibody 3H10ml (line D) and variant antibody 3G3 (line E).
• ADCC antibody-dependent cellular cytotoxicity
• Figure 17 shows an SPR sensorgram of ranked affinities of variant antibodies 3G3, 3H10, 3H10ml, 3E11, 3E3ml and 3G8ml.
• Enzymatic reactions and enrichment/purification techniques are also well known and are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein.
• the terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
• the term“and/or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other.
• the term“and/or” as used in a phrase such as“A and/or B” herein is intended to include“A and B,”“A or B,”“A” (alone), and“B” (alone).
• the term“and/or” as used in a phrase such as“A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
• the term“about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example,“about” or
• “approximately” can mean within one or more than one standard deviation per the practice in the art.
• “about” or“approximately” can mean a range of up to 10% (i.e., ⁇ 10%) or more depending on the limitations of the measurement system.
• about 5 mg can include any number between 4.5 mg and 5.5 mg.
• the terms can mean up to an order of magnitude or up to 5-fold of a value.
• Polypeptide refers to a polymer of amino acids and are not limited to any particular length.
• Polypeptides may comprise natural and non-natural amino acids.
• Polypeptides include recombinant or chemically-synthesized forms.
• Polypeptides also include precursor molecules that have not yet been subjected to cleavage, for example cleavage by a secretory signal peptide or by non-enzymatic cleavage at certain amino acid residues.
• Polypeptides include mature molecules that have undergone cleavage.
• polypeptides comprising amino acid sequences of binding proteins that bind CD38 (e.g., anti-CD38 variant antibodies or variant antigen-binding portions thereof) prepared using recombinant procedures are described herein.
• nucleic acid refers to polymers of nucleotides and are not limited to any particular length.
• Nucleic acids include recombinant and chemically-synthesized forms. Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecule can be single-stranded or double-stranded.
• nucleic acid molecules of the disclosure comprise a contiguous open reading frame encoding an antibody, or a fragment or scFv, derivative, mutein, or variant thereof.
• nucleic acids comprise a one type of polynucleotides or a mixture of two or more different types of polynucleotides. Nucleic acids encoding the antibody variant light chains, antibody variant heavy chains, anti-CD38 variant antibodies or variant antigen-binding portions thereof, are described herein.
• nucleic acids encode a heavy chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13.
• nucleic acids encode a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• the term“recover” or“recovery” or“recovering”, and other related terms refers to obtaining a protein (e.g., an antibody or an antigen binding portion thereof), from host cell culture medium or from host cell lysate or from the host cell membrane.
• the protein is expressed by the host cell as a recombinant protein fused to a secretion signal peptide sequence which mediates secretion of the expressed protein.
• the secreted protein can be recovered from the host cell medium.
• the protein is expressed by the host cell as a recombinant protein that lacks a secretion signal peptide sequence which can be recovered from the host cell lysate.
• the protein is expressed by the host cell as a membrane-bound protein which can be recovered using a detergent to release the expressed protein from the host cell membrane.
• the protein can be subjected to procedures that remove cellular debris from the recovered protein.
• the recovered protein can be subjected to chromatography, gel electrophoresis and/or dialysis.
• the chromatography comprises any one or any combination or two or more procedures including affinity chromatography,
• affinity chromatography comprises protein A or G (cell wall components from Staphylococcus aureus).
• isolated refers to a protein (e.g., an antibody or an antigen binding portion thereof) or polynucleotide that is substantially free of naturally associated components (e.g., other cellular material, or components associated with cellular expression system).
• a protein may be rendered substantially free of naturally associated components (or components associated with a cellular expression system or chemical synthesis methods used to produce the antibody) by isolation, using protein purification techniques well known in the art.
• isolated also refers in some embodiments to protein or polynucleotides that are substantially free of other molecules of the same species, for example other protein or polynucleotides having different amino acid or nucleotide sequences, respectively.
• the purity of homogeneity of the desired molecule can be assayed using techniques well known in the art, including low resolution methods such as gel electrophoresis and high resolution methods such as HPLC or mass spectrophotometry.
• low resolution methods such as gel electrophoresis and high resolution methods such as HPLC or mass spectrophotometry.
• HPLC high resolution methods
• any of the antibody variant light chains, antibody variant heavy chains, anti-CD38 variant antibodies or variant antigen binding protein described herein can be isolated.
• an "antigen binding protein” and related terms used herein refers to a protein comprising a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen.
• antigen binding proteins include antibodies, antibody fragments (e.g., an antigen binding portion of an antibody), antibody derivatives, and antibody analogs.
• the antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives.
• Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1 : 121-129; Roque et al., 2004, Biotechnol. Prog. 20:639-654.
• PAMs peptide antibody mimetics
• Antigen binding proteins that bind CD38 are described herein.
• An antigen binding protein can have, for example, the structure of an
• an "immunoglobulin” refers to a tetrameric molecule composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
• the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
• the carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains.
• Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
• the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).
• the variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two antigen binding sites.
• an antigen binding protein can be a synthetic molecule having a structure that differs from a tetrameric immunoglobulin molecule but still binds a target antigen or binds two or more target antigens.
• a synthetic antigen binding protein can comprise antibody fragments, 1-6 or more polypeptide chains, asymmetrical assemblies of polypeptides, or other synthetic molecules. Antigen binding proteins having immunoglobulin-like properties that bind specifically to CD38 are described herein.
• variable regions of immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
• One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein.
• An antigen binding protein may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently.
• the CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest.
• an "antibody” and“antibodies” and related terms used herein refers to an intact immunoglobulin or to an antigen binding portion thereof that binds specifically to an antigen.
• Antigen binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
• Antigen binding portions include, inter alia , Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
• Antibodies include recombinantly produced antibodies and antigen binding portions.
• Antibodies include non-human, chimeric, humanized and fully human antibodies.
• Antibodies include monospecific, multispecific (e.g., bispecific, trispecific and higher order specificities).
• Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, heavy chain dimers.
• Antibodies include F(ab’)2 fragments, Fab’ fragments and Fab fragments.
• Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragment (scFv), camelized antibodies, affibodies, disulfide- linked Fvs (sdFv), anti-idiotypic antibodies (anti-id), minibodies.
• Antibodies include monoclonal and polyclonal populations. Anti-CD38 variant antibodies, comprising variant light and/or heavy chains are described herein.
• An“antigen binding domain,”“antigen binding region,” or“antigen binding site” and other related terms used herein refer to a portion of an antigen binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least part of at least one of its CDR domains. Antigen binding domains from anti-CD38 variant antibodies are described herein.
• telomere binding refers to non-covalent or covalent preferential binding to an antigen relative to other molecules or moieties (e.g., an antibody specifically binds to a particular antigen relative to other available antigens).
• an antibody specifically binds to a target antigen if it binds to the antigen with a dissociation constant KD of 10 5 M or less, or 10 6 M or less, or 10 7 M or less, or 10 8 M or less, or 10 9 M or less, or 10 10 M or less.
• Anti-CD38 variant antibodies that specifically bind CD38 are described herein.
• a dissociation constant can be measured using a BIACORE surface plasmon resonance (SPR) assay.
• SPR surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).
• An "epitope" and related terms as used herein refers to a portion of an antigen that is bound by an antigen binding protein (e.g., by an antibody or an antigen binding portion thereof).
• An epitope can comprise portions of two or more antigens that are bound by an antigen binding protein.
• An epitope can comprise non-conti guous portions of an antigen or of two or more antigens (e.g., amino acid residues that are not contiguous in an antigen’s primary sequence but that, in the context of the antigen’s tertiary and quaternary structure, are near enough to each other to be bound by an antigen binding protein).
• the variable regions, particularly the CDRs, of an antibody interact with the epitope.
• Anti-CD38 variant antibodies, and variant antigen binding proteins thereof, that bind an epitope of a CD38 polypeptide (antigen) are described herein.
• an "antibody fragment”, “antibody portion”, “antigen-binding fragment of an antibody”, or “antigen-binding portion of an antibody” and other related terms used herein refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
• antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAb; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.
• Antigen binding portions of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
• Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer antigen binding properties to the antibody fragment.
• Antigen-binding fragments of anti-CD38 variant antibodies are described herein.
• the terms“Fab”,“Fab fragment” and other related terms refers to a monovalent fragment comprising a variable light chain region (VL), constant light chain region (CL), variable heavy chain region (VH), and first constant region (CHI).
• a Fab is capable of binding an antigen.
• An F(ab')2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
• a F(Ab’)2 has antigen binding capability.
• An Fd fragment comprises VH and CHI regions.
• An Fv fragment comprises VL and VH regions.
• An Fv can bind an antigen.
• a dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain
• U.S. Patents 6,846,634 and 6,696,245 U.S. published Application Nos. 2002/02512, 2004/0202995, 2004/0038291, 2004/0009507, 2003/0039958; and Ward et al., Nature 341 :544- 546, 1989.
• Fab fragments comprising antigen binding portions from anti-CD38 variant antibodies are described herein.
• a single-chain antibody is an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain.
• a linker e.g., a synthetic sequence of amino acid residues
• the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).
• Single chain antibodies comprising antigen binding portions from anti-CD38 variant antibodies are described herein.
• Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994, Structure 2: 1121-23). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites.
• Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites.
• tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.
• Diabody, tribody and tetrabody constructs can be prepared using antigen binding portions from any of the anti- CD38 variant antibodies described herein.
• variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody).
• human immunoglobulin sequences e.g., a fully human antibody.
• These antibodies may be prepared in a variety of ways, examples of which are described below, including through recombinant methodologies or through immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes. Fully human anti-CD38 antibodies and antigen binding proteins thereof that are variant antibodies are described herein.
• A“humanized” antibody refers to an antibody having a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject.
• certain amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody.
• the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species.
• one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the likely immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues either are not critical for immunospecific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.
• chimeric antibody refers to an antibody that contains one or more regions from a first antibody and one or more regions from one or more other antibodies.
• one or more of the CDRs are derived from a human antibody.
• all of the CDRs are derived from a human antibody.
• the CDRs from more than one human antibody are mixed and matched in a chimeric antibody.
• a chimeric antibody may comprise a CDR1 from the light chain of a first human antibody, a CDR2 and a CDR3 from the light chain of a second human antibody, and the CDRs from the heavy chain from a third antibody.
• the CDRs originate from different species such as human and mouse, or human and rabbit, or human and goat.
• the framework regions may be derived from one of the same antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody.
• a portion of the heavy and/or light chain is identical with, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with, homologous to, or derived from an antibody (-ies) from another species or belonging to another antibody class or subclass.
• fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind a target antigen).
• Chimeric antibodies can be prepared from portions of any of the anti-CD38 variant antibodies described herein.
• variant polypeptides and“variants” of polypeptides refers to a polypeptide comprising an amino acid sequence with one or more amino acid residues inserted into, deleted from and/or substituted into the amino acid sequence relative to a reference polypeptide sequence.
• Polypeptide variants include fusion proteins.
• a variant polynucleotide comprises a nucleotide sequence with one or more nucleotides inserted into, deleted from and/or substituted into the nucleotide sequence relative to another
• Polynucleotide sequence includes fusion polynucleotides.
• the term“derivative” of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., via conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin),
• antibody includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below.
• the term“Fc” or“Fc region” as used herein refers to the portion of an antibody heavy chain constant region beginning in or after the hinge region and ending at the C-terminus of the heavy chain.
• the Fc region comprises at least a portion of the CH and CH3 regions and may, or may not, include a portion of the hinge region.
• Two polypeptide chains each carrying a half Fc region can dimerize to form a full Fc domain.
• An Fc domain can bind Fc cell surface receptors and some proteins of the immune complement system.
• An Fc domain exhibits effector function, including any one or any combination of two or more activities including complement- dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), opsonization and/or cell binding.
• An Fc domain can bind an Fc receptor, including FcyRI (e.g., CD64), FcyRI I (e.g, CD32) and/or FcyRI 11 (e.g.,
• labeled antibody refers to antibodies and their antigen binding portions thereof that are unlabeled or joined to a detectable label or moiety for detection, wherein the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A.
• detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A.
• a variety of labels can be employed, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens). Any of the anti-CD38 variant antibodies described herein can be unlabeled or can be joined to
• The“percent identity” or“percent homology” and related terms used herein refers to a quantitative measurement of the similarity between two polypeptide or between two
• the percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions that are shared between the two polypeptide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polypeptide sequences.
• the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions that are shared between the two
• polynucleotide sequences taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polynucleotide sequences.
• a comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between two polynucleotide sequences, may be accomplished using a
• the "percent identity” or “percent homology” of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3
• Expressions such as“comprises a sequence with at least X% identity to Y” with respect to a test sequence mean that, when aligned to sequence Y as described above, the test sequence comprises residues identical to at least X% of the residues of Y.
• the amino acid sequence of a test antibody may be similar but not identical to any of the amino acid sequences of the light chain and/or heavy chain polypeptides that make up any of the anti-CD38 variant antibodies, or variant antigen binding protein thereof, described herein.
• the similarities between the test antibody and the polypeptides can be at least 95%, or at or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, to any of the light chain and/or heavy chain polypeptides that make up any of the anti-CD38 variant antibodies, or variant antigen binding protein thereof, described herein.
• similar polypeptides can contain amino acid substitutions within a heavy and/or light chain.
• the amino acid substitutions comprise one or more conservative amino acid substitutions.
• a "conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).
• R group side chain
• a conservative amino acid substitution will not substantially change the functional properties of a protein.
• a skilled artisan can introduce up to 5% conservative and/or non-conservative amino acid substitutions in a heavy chain variable region and/or light chain variable region without negatively impacting the physical structure, binding capability or cell killing capability of an antibody.
• Well known methods for identifying and making conservative amino acid substitutions in a variable region that are designed to retain or improve antibody binding characteristics are described in:
• aliphatic side chains glycine, alanine, valine, leucine and isoleucine
• aliphatic-hydroxyl side chains serine and threonine
• amide-containing side chains
• Antibodies can be obtained from sources such as serum or plasma that contain immunoglobulins having varied antigenic specificity. If such antibodies are subjected to affinity purification, they can be enriched for a particular antigenic specificity. Such enriched preparations of antibodies usually are made of less than about 10% antibody having specific binding activity for the particular antigen. Subjecting these preparations to several rounds of affinity purification can increase the proportion of antibody having specific binding activity for the antigen. Antibodies prepared in this manner are often referred to as "monospecific.”
• Monospecfic antibody preparations can be made up of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% antibody having specific binding activity for the particular antigen.
• Antibodies can be produced using recombinant nucleic acid technology as described below.
• a "vector" and related terms used herein refers to a nucleic acid molecule (e.g., DNA or RNA) which can be operably linked to foreign genetic material (e.g., nucleic acid transgene).
• Vectors can be used as a vehicle to introduce foreign genetic material into a cell (e.g., host cell).
• Vectors can include at least one restriction endonuclease recognition sequence for insertion of the transgene into the vector.
• Vectors can include at least one gene sequence that confers antibiotic resistance or a selectable characteristic to aid in selection of host cells that harbor a vector-transgene construct.
• Vectors can be single-stranded or double-stranded nucleic acid molecules.
• Vectors can be linear or circular nucleic acid molecules.
• a donor nucleic acid used for gene editing methods employing zinc finger nuclease, TALEN or CRISPR/Cas can be a type of a vector.
• One type of vector is a "plasmid," which refers to a linear or circular double stranded extrachromosomal DNA molecule which can be linked to a transgene, and is capable of replicating in a host cell, and transcribing and/or translating the transgene.
• a viral vector typically contains viral RNA or DNA backbone sequences which can be linked to the transgene. The viral backbone sequences can be modified to disable infection but retain insertion of the viral backbone and the co-linked transgene into a host cell genome.
• viral vectors examples include retroviral, lentiviral, adenoviral, adeno-associated, baculoviral, papovaviral, vaccinia viral, herpes simplex viral and Epstein Barr viral vectors.
• Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors).
• Other vectors e.g., non-episomal mammalian vectors
• An "expression vector” is a type of vector that can contain one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers. Expression vectors can include ribosomal binding sites and/or polyadenylation sites. Regulatory sequences direct transcription, or transcription and translation, of a transgene linked to the expression vector which is transduced into a host cell.
• the regulatory sequence(s) can control the level, timing and/or location of expression of the transgene.
• the regulatory sequence can, for example, exert its effects directly on the transgene, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid). Regulatory sequences can be part of a vector.
• An expression vector can comprise nucleic acids that encode at least a portion of any of the light chain, heavy chain or anti-CD38 variant antibodies described herein.
• Vectors operably linked to a nucleic acid encoding the antibody variant light chains, antibody variant heavy chains, anti-CD38 variant antibodies or variant antigen-binding portions thereof, are described herein.
• a vector is operably linked a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13.
• nucleic acids encode a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• a transgene is“operably linked” to a vector when there is linkage between the transgene and the vector to permit functioning or expression of the transgene sequences contained in the vector.
• a transgene is "operably linked” to a regulatory sequence when the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene.
• transfected or “transformed” or “transduced” or other related terms used herein refer to a process by which exogenous nucleic acid (e.g., transgene) is transferred or introduced into a host cell.
• a "transfected” or “transformed” or “transduced” host cell is one which has been transfected, transformed or transduced with exogenous nucleic acid (transgene).
• the host cell includes the primary subject cell and its progeny.
• Exogenous nucleic acids encoding at least a portion of any of the light chain, heavy chain or anti-CD38 variant antibodies described herein can be introduced into a host cell.
• Expression vectors comprising at least a portion of any of the light chain, heavy chain or anti-CD38 variant antibodies described herein can be introduced into a host cell, and the host cell can express polypeptides comprising at least a portion of the light chain, heavy chain or anti-CD38 variant antibody.
• the terms "host cell” or“or a population of host cells” or related terms as used herein refer to a cell (or a population thereof) into which foreign (exogenous or transgene) nucleic acids have been introduced.
• the foreign nucleic acids can include an expression vector operably linked to a transgene, and the host cell can be used to express the nucleic acid and/or polypeptide encoded by the foreign nucleic acid (transgene).
• a host cell (or a population thereof) can be a cultured cell or can be extracted from a subject.
• the host cell (or a population thereof) includes the primary subject cell and its progeny without any regard for the number of passages. Progeny cells may or may not harbor identical genetic material compared to the parent cell.
• Host cells encompass progeny cells.
• a host cell describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and/or manipulated in any way to express an antibody, as disclosed herein.
• the host cell (or population thereof) can be introduced with an expression vector operably linked to a nucleic acid encoding the desired antibody, or an antigen binding portion thereof, described herein.
• Host cells and populations thereof can harbor an expression vector that is stably integrated into the host’s genome or can harbor an extrachromosomal expression vector.
• host cells and populations thereof can harbor an extrachromosomal vector that is present after several cell divisions or is present transiently and is lost after several cell divisions.
• Transgenic host cells can be prepared using non-viral methods, including well-known designer nucleases including zinc finger nucleases, TALENS or CRISPR/Cas.
• a transgene can be introduced into a host cell’s genome using genome editing technologies such as zinc finger nuclease.
• a zinc finger nuclease includes a pair of chimeric proteins each containing a non specific endonuclease domain of a restriction endonuclease (e.g., Fokl ) fused to a DNA-binding domain from an engineered zinc finger motif.
• the DNA-binding domain can be engineered to bind a specific sequence in the host’s genome and the endonuclease domain makes a double- stranded cut.
• the donor DNA carries the transgene, for example any of the nucleic acids encoding a CAR or DAR construct described herein, and flanking sequences that are
• Transgenic mammalian host cells have been prepared using zinc finger nucleases (U.S. patent Nos. 9,597,357, 9,616,090, 9,816,074 and 8,945,868).
• a transgenic host cell can be prepared using TALEN (Transcription Activator-Like Effector Nucleases) which are similar to zinc finger nucleases in that they include a non-specific endonuclease domain fused to a DNA- binding domain which can deliver precise transgene insertion.
• TALEN Transcription Activator-Like Effector Nucleases
• TALEN Like zinc finger nucleases, TALEN also introduce a double-strand cut into the host’s DNA.
• Transgenic host cells can be prepared using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats).
• CRISPR employs a Cas endonuclease coupled to a guide RNA for target specific donor DNA integration.
• the guide RNA includes a conserved multi-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region in the target DNA and hybridizes to the host cell target site where the Cas endonuclease cleaves the double-stranded target DNA.
• the guide RNA can be designed to hybridize to a specific target site. Similar to zinc finger nuclease and TALEN, the CRISPR/Cas system can be used to introduce site specific insertion of donor DNA having flanking sequences that have homology to the insertion site. Examples of
• transgenic host cells can be prepared using zinc finger nuclease, TALEN or CRISPR/Cas system, and the host target site can be a TRAC gene (T Cell Receptor Alpha Constant).
• the donor DNA can include for example any of the nucleic acids encoding a CAR or DAR construct described herein. Electroporation, nucleofection or lipofection can be used to co-deliver into the host cell the donor DNA with the zinc finger nuclease, TALEN or CRISPR/Cas system.
• a host cell can be a prokaryote, for example, E. coli , or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma.
• a prokaryote for example, E. coli
• a eukaryote for example, a single-celled eukaryote (e.g., a yeast or other fungus)
• a plant cell e.g., a tobacco or tomato plant cell
• an mammalian cell e.g., a human cell, a monkey cell, a hamster cell, a rat cell,
• a host cell can be introduced with an expression vector operably linked to a nucleic acid encoding a desired antibody thereby generating a transfected/transformed host cell which is cultured under conditions suitable for expression of the antibody by the transfected/transformed host cell, and optionally recovering the antibody from the transfected/transformed host cells (e.g., recovery from host cell lysate) or recovery from the culture medium.
• host cells comprise non-human cells including CHO, BHK, NSO, SP2/0, and YB2/0.
• host cells comprise human cells including HEK293, HT-1080, Huh-7 and PER.C6.
• host cells examples include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et ah, 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum- free media (see Rasmussen et ah, 1998, Cytotechnology 28:31) or CHO strain DX-B 11, which is deficient in DHFR (see Urlaub et ah, 1980, Proc. Natl. Acad. Sci.
• COS-7 line of monkey kidney cells ATCC CRL 1651
• L cells C127 cells
• 3T3 cells ATCC CCL 163
• CHO Chinese hamster ovary
• HeLa cells include lymphoid cells such as Y0, NSO or Sp20.
• a host cell is a mammalian host cell, but is not a human host cell.
• a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell.
• the phrase“transgenic host cell” or "recombinant host cell” can be used to denote a host cell that has been transformed or transfected with a nucleic acid to be expressed.
• a host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid.
• host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
• a host cell, or a population of host cells, harboring a vector (e.g., an expression vector) operably linked to a nucleic acid encoding the antibody variant light chains, antibody variant heavy chains, anti-CD38 variant antibodies or variant antigen-binding portions thereof, are described herein.
• a host cell harbors a vector operably linked a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13.
• nucleic acids encode a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• Polypeptides of the present disclosure can be produced using any methods known in the art.
• the polypeptides are produced by recombinant nucleic acid methods by inserting a nucleic acid sequence (e.g., DNA) encoding the polypeptide into a recombinant expression vector which is introduced into a host cell and expressed by the host cell under conditions promoting expression.
• a nucleic acid sequence e.g., DNA
• nucleic acid e.g., DNA
• the nucleic acid encoding the polypeptide is operably linked to an expression vector carrying one or more suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes.
• Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation.
• the expression vector can include an origin or replication that confers replication capabilities in the host cell.
• the expression vector can include a gene that confers selection to facilitate recognition of transgenic host cells (e.g., transformants).
• the recombinant DNA can also encode any type of protein tag sequence that may be useful for purifying the protein.
• protein tags include but are not limited to a histidine tag, a FLAG tag, a myc tag, an HA tag, or a GST tag.
• Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).
• the expression vector construct can be introduced into the host cell using a method appropriate for the host cell.
• a variety of methods for introducing nucleic acids into host cells are known in the art, including, but not limited to, electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; viral transfection; non-viral transfection; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent).
• Suitable host cells include prokaryotes, yeast, mammalian cells, or bacterial cells.
• Suitable bacteria include gram negative or gram positive organisms, for example, E. coli or Bacillus spp. Yeast, preferably from the Saccharomyces species, such as S. cerevisiae, may also be used for production of polypeptides.
• Saccharomyces species such as S. cerevisiae
• Various mammalian or insect cell culture systems can also be employed to express recombinant proteins. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, (Bio/Technology, 6:47, 1988).
• suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines.
• Purified polypeptides are prepared by culturing suitable host/vector systems to express the recombinant proteins. For many applications, the small size of many of the polypeptides disclosed herein would make expression in E. coli as the preferred method for expression. The protein is then purified from culture media or cell extracts. Any of the light chain, heavy chain or anti-CD38 variant antibodies, or variant antigen binding protein thereof, can be expressed by transgenic host cells.
• Antibodies and antigen binding proteins disclosed herein can also be produced using cell-translation systems.
• the nucleic acids encoding the polypeptide must be modified to allow in vitro transcription to produce mRNA and to allow cell-free translation of the mRNA in the particular cell-free system being utilized (eukaryotic such as a mammalian or yeast cell-free translation system or prokaryotic such as a bacterial cell-free translation system.
• Nucleic acids encoding any of the various polypeptides disclosed herein may be synthesized chemically. Codon usage may be selected so as to improve expression in a cell. Such codon usage will depend on the cell type selected. Specialized codon usage patterns have been developed for A. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See for example: Mayfield et ak, Proc. Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002 (1):96-105; Connell N D. Curr. Opin. Biotechnol. 2001 12(5):446-9; Makrides et al. Microbiol. Rev. 1996 60(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.
• Antibodies and antigen binding proteins described herein can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications to the protein can also be produced by chemical synthesis.
• Antibodies and antigen binding proteins described herein can be purified by isolation/purification methods for proteins generally known in the field of protein chemistry.
• Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reversed-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution or any combinations of these.
• polypeptides may be exchanged into different buffers and/or concentrated by any of a variety of methods known to the art, including, but not limited to, filtration and dialysis.
• the purified antibodies and antigen binding proteins described herein are preferably at least 65% pure, at least 75% pure, at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% pure. Regardless of the exact numerical value of the purity, the polypeptide is sufficiently pure for use as a pharmaceutical product. Any of the light chain, heavy chain or anti-CD38 variant antibodies, or variant antigen binding protein thereof, described herein can be expressed by transgenic host cells and then purified to about 65-98% purity or high level of purity using any art-known method.
• the antibodies and antigen binding proteins herein can further comprise post-translational modifications.
• post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation,
• the modified polypeptides may contain non-amino acid elements, such as lipids, poly- or mono-saccharide, and phosphates.
• a preferred form of glycosylation is sialylation, which conjugates one or more sialic acid moieties to the polypeptide. Sialic acid moieties improve solubility and serum half-life while also reducing the possible immunogenicity of the protein. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76.
• the antibodies and antigen binding proteins described herein can be modified to become soluble polypeptides which comprises linking the Antibodies and antigen binding proteins to non-proteinaceous polymers.
• the non-proteinaceous polymer comprises polyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes, in the manner as set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
• PEG is a water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161).
• the term“PEG” is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented by the formula: X—
• n 20 to 2300 and X is H or a terminal modification, e.g., a Ci-4 alkyl.
• the PEG terminates on one end with hydroxy or methoxy, i.e., X is H or CEE (“methoxy PEG”).
• a PEG can contain further chemical groups which are necessary for binding reactions; which results from the chemical synthesis of the molecule; or which is a spacer for optimal distance of parts of the molecule.
• such a PEG can consist of one or more PEG side-chains which are linked together. PEGs with more than one PEG chain are called multiarmed or branched PEGs.
• Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol.
• a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide.
• Branched PEG are described in, for example, EP-A 0 473 084 and U.S. Pat. No. 5,932,462.
• One form of PEGs includes two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini et ah, Bioconjugate Chem. 6 (1995) 62-69).
• the serum clearance rate of PEG-modified polypeptide may be modulated (e.g., increased or decreased) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative to the clearance rate of the unmodified antibodies and antigen binding proteins binding polypeptides.
• the PEG-modified antibodies and antigen binding proteins may have a half-life (ti/ 2 ) which is enhanced relative to the half-life of the unmodified polypeptide.
• the half-life of PEG-modified polypeptide may be enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by 1000% relative to the half-life of the unmodified antibodies and antigen binding proteins.
• the protein half-life is determined in vitro , such as in a buffered saline solution or in serum.
• the protein half-life is an in vivo half-life, such as the half-life of the protein in the serum or other bodily fluid of an animal.
• compositions comprising any of the light chain, heavy chain or anti-CD38 variant antibodies, or variant antigen binding protein thereof, described herein in and a pharmaceutically-acceptable excipient.
• An excipient encompasses carriers, stabilizers and excipients.
• Excipients of pharmaceutically acceptable excipients includes for example inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Additional examples include buffering agents, stabilizing agents, preservatives, non-ionic detergents, anti-oxidants and isotonifiers.
• Therapeutic compositions and methods for preparing them are well known in the art and are found, for example, in“Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.).
• Therapeutic compositions can be formulated for parenteral administration may, and can for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
• Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the antibody (or antigen binding protein thereof) described herein.
• Nanoparticulate formulations may be used to control the biodistribution of the antibody (or antigen binding protein thereof).
• Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
• concentration of the antibody (or antigen binding protein thereof) in the formulation varies depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.
• any of the anti-CD38 variant antibodies may be administered as a pharmaceutically acceptable salt, such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry.
• acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like.
• Metal complexes include zinc, iron, and the like.
• the antibody (or antigen binding portions thereof) is formulated in the presence of sodium acetate to increase thermal stability.
• variant anti-CD38 antibodies may be formulated for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients.
• Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.
• subject refers to human and non-human animals, including vertebrates, mammals and non-mammals.
• the subject can be human, non human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.
• murine e.g., mice and rats
• bovine porcine
• equine canine
• feline feline
• caprine caprine
• lupine ranine or piscine.
• administering refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the
• parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
• the formulation is administered via a non- parenteral route, e.g., orally.
• non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
• Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
• Any of the anti-CD38 variant antibodies described herein (or variant antigen binding protein thereof) can be administered to a subject using art-known methods and delivery routes.
• an antigen binding protein e.g., any of the anti-CD38 variant antibodies described herein or variant antigen binding protein thereof
• Therapeutically effective amounts of antibodies provided herein, when used alone or in combination, will vary depending upon the relative activity of the antibodies and combinations (e.g. , in inhibiting cell growth) and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition in the subject, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
• a therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated and may be ascertained by one skilled in the art using known techniques.
• the polypeptide is administered at about 0.01 g/kg to about 50 mg/kg per day, preferably 0.01 mg/kg to about 30 mg/kg per day, most preferably 0.1 mg/kg to about 20 mg/kg per day.
• the polypeptide may be administered daily (e.g., once, twice, three times, or four times daily) or preferably less frequently (e.g., weekly, every two weeks, every three weeks, monthly, or quarterly).
• adjustments for age as well as the body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the disease may be necessary.
• the present disclosure provides methods for treating a subject having a disease associated with expression of CD38.
• the disease comprises cancer or tumor cells expressing the tumor-associated antigens.
• the cancer or tumor includes cancer of the prostate, breast, ovary, head and neck, bladder, skin, colorectal, anus, rectum, pancreas, lung (including non-small cell lung and small cell lung cancers), leiomyoma, brain, glioma, glioblastoma, esophagus, liver, kidney, stomach, colon, cervix, uterus, endometrium, vulva, larynx, vagina, bone, nasal cavity, paranasal sinus, nasopharynx, oral cavity, oropharynx, larynx, hypolarynx, salivary glands, ureter, urethra, penis and testis.
• the cancer comprises hematological cancers, including leukemias, lymphomas, myelomas and B cell lymphomas.
• Hematologic cancers include multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) including Burkitf s lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), systemic lupus erythematosus (SLE), B and T acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma, chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), follicular lymphoma, Waldenstrom's Macroglobulinemia, mantle cell lymphoma, Hodgkin's Lymphoma (HL), plasma cell myeloma, precursor B cell lymphoblastic leukemia/lymphom
• An anti-CD38 parent antibody is disclosed in U.S. patent application publication No. US 2016/0297888 Al, published 13 October 2016 (the disclosure of which is incorporated by reference herein in its entirety), granted as US Patent No. 10,059,774 on August 28, 2018.
• This antibody referred to herein as the“parent” and/or“wild type” antibody, comprises a parent heavy chain having a heavy variable region comprising the amino acid sequence of SEQ ID NO: 1, and a parent light chain having a light variable region comprising the amino acid sequence of SEQ ID NO:2.
• the parent antibody disclosed herein is designated“A2”.
• the present disclosure provides anti-CD38 antigen-binding proteins, including anti- CD38 variant antibodies, or antigen-binding portions thereof, that specifically bind CD38 and uses thereof.
• the anti-CD38 variant antibodies can exhibit improved characteristics compared to the parent antibody A2, where the improved characteristics include improved binding to CD38 antigen, improved binding to CD38-expressing cells and/or higher levels of cytotoxicity.
• the anti-CD38 variant antibodies like parent antibody A2, can cross-react (bind) with cynomolgus CD38 antigen.
• the present disclosure provides an antigen-binding protein, such as a fully human antibody of an IgG class, that binds to an epitope of a CD38 polypeptide (e.g., target antigen) or fragment of a CD38 polypeptide, wherein the antibody is a variant antibody having a heavy chain variable region and/or light chain variable region that differs from the parent antibody A2.
• the CD38 target antigen comprises a naturally- occurring polypeptide (e.g., UniProtKB accession number P28907 (NP 0017766.2)) having a wild-type or polymorphic or mutant amino acid sequence.
• the CD38 target antigen can be prepared by recombinant methods or can be chemically synthesized.
• the CD38 target antigen can be in soluble form or membrane-bound form (e.g., expressed by a cell or phage).
• the CD38 target antigen comprises an extracellular portion of a cell surface CD38 antigen.
• the CD38 target antigen is expressed by a cell, for example a cancer or non-cancer cell line that naturally expresses CD38 such as Raji, Ramos, Daudi, MOLT-4, Karpas-707, REH, U-266/70, U-698, RPMI-8226, A549, or expressed by a cell line that is engineered to express CD38 such as CHO, HeLa, HEK293 or PanoplyTM (from Creative Biogene, Shirley, New York).
• CD38 target antigen can be a fusion protein or conjugated for example with a detectable moiety such as a fluorophore.
• the CD38 target antigen can be a recombinant polypeptide with or without a histidine-tag.
• the CD38 target antigen can be a CD38 his-tagged protein from human, mouse or cynomolgus (e.g., from Sino Biological, catalog # 10818-H08H, 50191-M08H, or 90050-C08H, respectively).
• the CD38 polypeptide comprises the amino acid sequence of SEQ ID NO: 19.
• wild type and/or mutated human CD38 antigen can be used in an assay comparing binding capabilities of any of the anti-CD38 variant antibodies described herein compared to the anti-CD38 parent antibody (A2), and/or in an epitope mapping assay comparing binding capabilities of any of the anti-CD38 variant antibodies described herein compared to the anti-CD38 parent antibody (A2).
• the present disclosure provides an antigen-binding protein, such as a fully human antibody of an IgG class, that binds to an epitope of a CD38 polypeptide (target antigen), wherein the antibody is a variant antibody comprising a heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the anti- CD38 variant antibody comprises a light chain variable region having 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof; or wherein the antibody is a variant antibody comprising a heavy
• the present disclosure provides an antigen-binding protein, such as a fully human antibody of an IgG class, that binds to an epitope of a CD38 polypeptide (target antigen), wherein the antibody is a variant antibody comprising a heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the anti-CD38 variant antibody comprises a light chain variable region having 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:
• the anti-CD38 variant antibody is an isolated antibody. In one embodiment, the anti-CD38 variant antibody is a recombinant antibody. In one embodiment, the anti-CD38 variant antibody comprises an IgGl, IgG2, IgG3 or IgG4 class antibody. In one embodiment, the anti-CD38 variant antibody comprises an IgGl or IgG4 class antibody. In one embodiment, the hinge region of an anti-CD38 antibody can be mutated to alter the number of potential disulfide bond formation. In one embodiment, the anti-CD38 variant antibody comprises a hinge region having the amino acid sequence CPPC, CPSC, SPPC or SPSC.
• the anti-CD38 variant antibody comprises a heavy chain constant region having a hinge region wherein the amino acid sequence CPSC, SPPC or SPSC replaces the sequence CPPC (e.g., see bold and underlined sequence at positions 109-112 of SEQ ID NO: 14, 15 or 16).
• the heavy chain of an anti-CD38 antibody can be mutated to eliminate one or more NG motifs (e.g., as part of an NGR motif) that are known to isomerize.
• the isomerized site can bind integrin.
• the anti-CD38 variant antibody comprises a heavy chain that includes an SGR motif that replaces an NGR motif.
• the anti-CD38 variant antibody comprises a heavy chain variable region wherein an SGR motif replaces an NGR motif (e.g., see bold and underlined sequence at positions 54-56 of SEQ ID NO:3, 5, 6, 7,
• the anti-CD38 variant antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 17 or 18.
• the heavy and/or light chain of an anti-CD38 antibody can be mutated, and the mutated antibody exhibits the same or similar binding capabilities to CD38 antigen and/or CD38-expressing cells.
• the anti-CD38 variant antibody, or fragment thereof comprises an antigen binding portion that binds an epitope of a CD38 polypeptide (target antigen) with a binding affinity (KD) of 10 6 M or less, 10 7 M or less, 10 8 M or less, 10 9 M or less, or 10 10 M or less (see Figures 1-6).
• binding between the anti-CD38 variant antibody, or fragment thereof can be detected and measured using surface plasmon resonance, flow cytometry and/or ELISA.
• the present disclosure provides an anti-CD38 variant antibody which binds an epitope of a CD38 polypeptide from a human, and can bind (e.g., cross-react) with an epitope of a CD38 polypeptide (e.g., homologous antigen) from at least one of a non-human animal such as mouse, rat, goat, rabbit, hamster and/or monkey (e.g., cynomolgus).
• the anti-CD38 variant antibody binds mouse CD38 with a binding affinity KD of 10 5 M or less, or 10 6 M or less, or 10 7 M or less, or 10 8 M or less, or 10 9 M or less, or 10 10 M or less.
• the anti-CD38 variant antibody binds cynomolgus CD38 with a binding affinity KD of 10 5 M or less, or 10 6 M or less, or 10 7 M or less, or 10 8 M or less, or 10 9 M or less, or 10
• the non-human CD38 comprises a mouse CD38 polypeptide (e.g., from Sino Biological, catalog # 50191-M08H), or a cynomolgus CD38 polypeptide (e.g, from Sino Biological, catalog # 90050-C08H).
• a mouse CD38 polypeptide e.g., from Sino Biological, catalog # 50191-M08H
• a cynomolgus CD38 polypeptide e.g, from Sino Biological, catalog # 90050-C08H.
• the present disclosure provides a fully human antibody that binds a CD38 polypeptide, wherein the antibody is a variant antibody comprising both heavy and light chains, wherein the heavy /light chain variable region amino acid sequences have at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to any of the following amino acid sequence sets: SEQ ID NOS:3 and 4 (called 3H10ml herein), SEQ ID NOS:5 and 4 (called 3G8ml herein), SEQ ID NOS:6 and 4 (called 3E3ml herein), SEQ ID N0S:7 and 2 (called 3G3 herein), SEQ ID N0S:9 and 2 (called 3E11 herein), SEQ ID NOS: 10 and 2 (called 3H10 herein), SEQ ID NOS: 11 and 12 (called 3H10N herein), SEQ ID NOS: 13 and 12 (called 3H10NS herein), SEQ ID NOS: 3
• the present disclosure provides a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain.
• the sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof
• the sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides a Fab fully human antibody fragment which is a variant antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy /light chain variable region amino acid sequences are at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the following amino acid sequence sets: SEQ ID NOS:3 and 4 (called 3H10ml herein), SEQ ID NOS:5 and 4 (called 3G8ml herein), SEQ ID NOS:6 and 4 (called 3E3ml herein), SEQ ID NOS:7 and 2 (called 3G3 herein), SEQ ID NOS:9 and 2 (called 3E11 herein), SEQ ID NOS: 10 and 2 (called 3H10 herein), SEQ ID NOS: 11 and 12 (called 3H10N herein), SEQ ID NOS: 13 and 12 (called 3H10NS herein), SEQ ID NOS: l and 4 (called 3E
• the present disclosure provides a single chain fully human antibody which is a variant single chain antibody comprising a polypeptide chain having a variable region from a fully human heavy chain and a variable region from a fully human light chain, and optionally a linker (e.g., peptide linker) joining the variable heavy and variable light chain regions.
• a linker e.g., peptide linker
• variable heavy region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof
• variable light region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• variable heavy region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof
• variable light region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides a single chain fully human antibody which is a variant single chain antibody comprising a polypeptide chain having heavy chain variable region and a light chain variable region, wherein the heavy /light chain variable region amino acid sequence sets are at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the following amino acid sequence sets: SEQ ID NOS:3 and 4 (called 3H10ml herein), SEQ ID NOS:5 and 4 (called 3G8ml herein),
• SEQ ID NOS:6 and 4 (called 3E3ml herein), SEQ ID NOS:7 and 2 (called 3G3 herein), SEQ ID NOS:9 and 2 (called 3E11 herein), SEQ ID NOS: 10 and 2 (called 3H10 herein), SEQ ID NOS:
• the single chain fully human antibody comprises an optional linker (e.g., peptide linker) joining the variable heavy and variable light chain regions.
• an excipient encompasses carriers and stabilizers.
• the therapeutic compositions comprise an anti-CD38 variant antibody, or variant antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy /light chain variable region amino acid sequences are at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the following amino acid sequence sets: SEQ ID NOS:3 and 4 (called 3H10ml herein), SEQ ID NOS:5 and 4 (called 3G8ml herein), SEQ ID NOS:6 and 4 (called 3E3ml herein), SEQ ID NOS:7 and 2 (called 3G3 herein), SEQ ID NOS:9 and 2 (called 3E11 herein), SEQ ID NOS: 10 and 2 (called 3H10 herein), SEQ ID NOS: 11 and 12 (called 3H10N herein), SEQ ID NOS: 13 and 12 (called 3H10NS herein), SEQ ID NOS: l and 4 (called 3H10
• the present disclosure provides nucleic acids encoding an antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof.
• the present disclosure provides nucleic acids encoding an antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides nucleic acids encoding an antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof.
• the present disclosure provides nucleic acids encoding an antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides nucleic acids encoding an antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the nucleic acids encode an antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides nucleic acids encoding an antibody heavy chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the nucleic acids encode an antibody light chain variable region having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides nucleic acids encoding a variant antibody comprising both heavy and light chains, wherein the heavy /light chain variable region amino acid sequences have at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to any of the following amino acid sequence sets: SEQ ID NOS:3 and 4 (called 3H10ml herein), SEQ ID NOS:5 and 4 (called 3G8ml herein), SEQ ID NOS:6 and 4 (called 3E3ml herein), SEQ ID NOS:7 and 2 (called 3G3 herein), SEQ ID NOS:9 and 2 (called 3E11 herein), SEQ ID NOS: 10 and 2 (called 3H10 herein), SEQ ID NOS: 11 and 12 (called 3H10N herein), SEQ ID NOS: 13 and 12 (called 3H10NS herein), SEQ ID NOS: l and 4 (called 3E10 herein) or SEQ
• the present disclosure provides nucleic acids encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof.
• the present disclosure provides nucleic acids encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides nucleic acids encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof.
• the present disclosure provides nucleic acids encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides nucleic acids encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the nucleic acids encode the variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides nucleic acids encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the nucleic acids encode the variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides nucleic acids encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy /light chain variable region amino acid sequences are at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the following amino acid sequence sets: SEQ ID NOS: SEQ ID NOS:3 and 4 (called 3H10ml herein), SEQ ID NOS:5 and 4 (called 3G8ml herein), SEQ ID NOS:6 and 4 (called 3E3ml herein), SEQ ID NOS:7 and 2 (called 3G3 herein), SEQ ID NOS:9 and 2 (called 3E11 herein), SEQ ID NOS: 10 and 2 (called 3H10 herein), SEQ ID NOS: 11 and 12 (called 3H10N herein), SEQ ID NOS: 13 and 12 (called 3H10NS herein), SEQ ID NO
• the present disclosure provides nucleic acids encoding a single chain fully human antibody comprising a polypeptide chain having a variable region from a fully human heavy chain and a variable region from a fully human light chain, and optionally a linker (e.g., peptide linker) joining the variable heavy and variable light chain regions.
• a linker e.g., peptide linker
• variable heavy region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the variable light region which comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides nucleic acids encoding a single chain fully human antibody comprising a polypeptide chain having a variable region from a fully human heavy chain and a variable region from a fully human light chain, and optionally a linker (e.g., peptide linker) joining the variable heavy and variable light chain regions.
• a linker e.g., peptide linker
• variable heavy region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the variable light region which comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the present disclosure provides nucleic acids encoding a single chain fully human antibody which is a variant single chain antibody comprising a polypeptide chain having heavy chain variable region and a light chain variable region, wherein the heavy /light chain variable region amino acid sequence sets are at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the following amino acid sequence sets: SEQ ID NOS: SEQ ID NOS: SEQ ID NOS: SEQ ID NOS: SEQ ID NOS:3 and 4 (called 3H10ml herein), SEQ ID NOS:5 and 4 (called 3G8ml herein), SEQ ID NOS:6 and 4 (called 3E3ml herein), SEQ ID NOS:7 and 2 (called 3G3 herein), SEQ ID NOS:9 and 2 (called 3E11 herein), SEQ ID NOS: 10 and 2 (called 3H10 herein), SEQ ID NOS: 11 and 12 (called 3H10N herein
• the present disclosure provides a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, and a second vector (e.g., second expression vector) operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• a first vector e.g., a first expression vector
• a second vector operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• the present disclosure also provides a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, and a second vector (e.g., second expression vector) operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• a first vector e.g., a first expression vector
• a second vector operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• the present disclosure provides a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13.
• the first vector is also operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• the present disclosure also provides a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, which is also operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• a first vector e.g., a first expression vector
• the present disclosure provides a vector (e.g., an expression vector) encoding a nucleic acid encoding a variant antibody comprising both heavy and light chains, wherein the heavy /light chain variable region amino acid sequences have at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to any of the following amino acid sequence sets: SEQ ID NOS:3 and 4 (called 3H10ml herein), SEQ ID NOS:5 and 4 (called 3G8ml herein), SEQ ID NOS:6 and 4 (called 3E3ml herein), SEQ ID NOS:7 and 2 (called 3G3 herein), SEQ ID NOS:9 and 2 (called 3E11 herein), SEQ ID NOS: 10 and 2 (called 3H10 herein), SEQ ID NOS: 11 and 12 (called 3H10N herein), SEQ ID NOS: 13 and 12 (called 3H10NS herein), SEQ ID NOS:
• the present disclosure provides a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof.
• the present disclosure also a second vector (e.g., a second expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• a second vector e.g., a second expression vector
• the present disclosure provides a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof.
• the present disclosure also a second vector (e.g., a second expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• a second vector e.g., a second expression vector
• the present disclosure provides a vector (e.g., an expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the vector is operably linked to nucleic acids encoding the variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid
• the present disclosure provides a vector (e.g., an expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the vector is operably linked to nucleic acids encoding the variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid
• the present disclosure provides a vector (e.g., an expression vector) operably linked to nucleic acids encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy /light chain variable region amino acid sequences are at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the following amino acid sequence sets: SEQ ID NOS: SEQ ID NOS: 3 and 4 (called 3H10ml herein), SEQ ID NOS:5 and 4 (called 3G8ml herein), SEQ ID NOS:6 and 4 (called 3E3ml herein), SEQ ID NOS:7 and 2 (called 3G3 herein), SEQ ID NOS:9 and 2 (called 3E11 herein), SEQ ID NOS: 10 and 2 (called 3H10 herein), SEQ ID NOS: 11 and 12 (called 3H10N herein), SEQ
• the present disclosure provides a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a single chain fully human antibody comprising a polypeptide chain having a variable region from a fully human heavy chain and a variable region from a fully human light chain, and optionally a linker (e.g., peptide linker) joining the variable heavy and variable light chain regions, wherein the variable heavy region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and wherein the variable light region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least
• the present disclosure also provides a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a single chain fully human antibody comprising a polypeptide chain having a variable region from a fully human heavy chain and a variable region from a fully human light chain, and optionally a linker (e.g., peptide linker) joining the variable heavy and variable light chain regions, wherein the variable heavy region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and wherein the variable light region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least
• the present disclosure provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, and the host cell or individual host cells from the population of host cells harbors a second vector (e.g., a second expression vector ) operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• the first expression vector directs expression of the heavy chain variable region
• the second expression vector directs expression of the light chain variable region in the host cell or the population
• the present disclosure also provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, and the host cell or individual host cells from the population of host cells harbors a second vector (e.g., a second expression vector ) operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• the first expression vector directs expression of the heavy chain variable region
• the second expression vector directs expression of the light chain variable region in the host cell or the
• the present disclosure provides a first host cell, or a first population of host cells, wherein the first host cell or individual host cells from the first population of host cells harbors a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, and a second host cell or a second population of host cells, wherein the second host cell or individual host cells from the second population of host cells harbors a second vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• the first expression vector directs expression of the heavy chain variable region
• the present disclosure also provides a first host cell, or a first population of host cells, wherein the first host cell or individual host cells from the first population of host cells harbors a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, and a second host cell or a second population of host cells, wherein the second host cell or individual host cells from the second population of host cells harbors a second vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• the first expression vector directs expression of the heavy chain variable
• the present disclosure provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a vector (e.g., an expression vector) operably linked to a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, and the vector in the host cell is also operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• the expression vector directs expression of the heavy chain variable region and the light chain variable region in the host cell.
• the present disclosure also provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a vector (e.g., an expression vector) operably linked to a nucleic acid encoding a heavy chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: l l or SEQ ID NO: 13, and the vector in the host cell is also operably linked to a nucleic acid encoding a light chain variable region comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12.
• the expression vector directs expression of the heavy chain variable region and the light chain variable region in the host cell.
• the present disclosure provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof.
• a first vector e.g., a first expression vector
• the host cell or individual host cells from the population of host cells harbors also harbors a second vector (e.g., a second expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the first expression vector directs expression of the heavy chain variable region and the second expression vector directs expression of the light chain variable region in the host cell.
• the present disclosure provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof.
• a first vector e.g., a first expression vector
• the host cell or individual host cells from the population of host cells harbors also harbors a second vector (e.g., a second expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 12, or combinations thereof.
• the first expression vector directs expression of the heavy chain variable region and the second expression vector directs expression of the light chain variable region in the host cell.
• the present disclosure provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the first vector is operably linked to nucleic acids encoding the variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least
• the present disclosure provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a variable region from a heavy chain and a variable region from a light chain, wherein the amino acid sequence of the variable region from the heavy chain is at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and the first vector is operably linked to nucleic acids encoding the variable region from a light chain, wherein the amino acid sequence of the variable region from the light chain is at least
• the present disclosure provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a vector (e.g., an expression vector) operably linked to nucleic acids encoding a Fab fully human antibody fragment which is a variant antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy /light chain variable region amino acid sequences are at least 95% identical, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to any of the following amino acid sequence sets: SEQ ID NOS: SEQ ID NOS: 3 and 4 (called 3H10ml herein), SEQ ID NOS:5 and 4 (called 3G8ml herein), SEQ ID NOS:6 and 4 (called 3E3ml herein), SEQ ID NOS:7 and 2 (called 3G3 herein), SEQ ID NOS:9 and 2 (called 3E11 herein), SEQ ID NOS: 10
• the present disclosure provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding any of the following amino acid sequence sets: SEQ ID NOS:3 and 4 (called 3H10ml herein), SEQ ID NOS:5 and 4 (called 3G8ml herein), SEQ ID NOS:6 and 4 (called 3E3ml herein), SEQ ID NOS:7 and 2 (called 3G3 herein), SEQ ID NOS:9 and 2 (called 3E11 herein), SEQ ID NOS: 10 and 2 (called 3H10 herein), SEQ ID NOS: 11 and 12 (called 3H10N herein), SEQ ID NOS: 13 and 12 (called 3H10NS herein), SEQ ID NOS: l and 4 (called 3E10 herein) or SEQ ID NOS:3 and 12 (called 3H10ml
• the present disclosure provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a single chain fully human antibody comprising a polypeptide chain having a variable region from a fully human heavy chain and a variable region from a fully human light chain, and optionally a linker (e.g., peptide linker) joining the variable heavy and variable light chain regions, wherein the variable heavy region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and wherein the variable light region comprises at least 95% sequence identity
• the expression vector directs expression of the single chain antibody in the host cell.
• the present disclosure also provides a host cell, or a population of host cells, wherein the host cell or individual host cells from the population of host cells harbors a first vector (e.g., a first expression vector) operably linked to a nucleic acid encoding a single chain fully human antibody comprising a polypeptide chain having a variable region from a fully human heavy chain and a variable region from a fully human light chain, and optionally a linker (e.g., peptide linker) joining the variable heavy and variable light chain regions, wherein the variable heavy region comprises at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 13, or combinations thereof, and wherein the variable light region comprises at least 95%
• the present disclosure provides methods for inhibiting growth or proliferation of target cells, or methods for killing target cells (e.g., cytotoxicity), the method comprising:
• the population of effector cells comprises natural killer (NK) cells or peripheral blood mononuclear cells (PBMCs).
• NK natural killer
• PBMCs peripheral blood mononuclear cells
• the PBMCs can include lymphocytes, including T cells, B cells and/or NK cells.
• the population of target cells comprise cells that express CD38, including Raji, Ramos, Daudi, MOLT-4, Karpas-707, REH, U-266/70, U- 698, RPMI-8226, A549, B lymphocytes, CD4+ cells, CD8+ cells, or cells from a subject having a disease associated with CD38-expression.
• the population of target cells are any type of transgenic cells that are engineered to express CD38.
• the population of target cells comprise a cell line that is engineered to express CD38 such as CHO, HeLa, HEK293 or PanoplyTM (from Creative Biogene, Shirley, New York).
• the ratio of effector to target cells can be about 1 : 1, or about 2: 1, or about 3 : 1, or about 4: 1, or about 5:1, or about 5-10: 1, or about 10-20: 1, or about 20-30: 1.
• the present disclosure provides methods for promoting phagocytosis of target cells, the method comprising: contacting a population of macrophage cells with a population of target cells (e.g., target cells expressing CD38) in the presence of any of the anti-CD38 variant antibodies (or variant antibody fragments thereof) described herein under conditions that are suitable for promoting phagocytosis of target cells.
• the population of macrophage cells can be obtained by culturing monocytes with macrophage colony-stimulating factor (M-CSF) to promote proliferation and differentiation of the monocytes into macrophage cells.
• M-CSF macrophage colony-stimulating factor
• the population of macrophage cells express CD14.
• the population of target cells comprise cells that express CD38, including Raji, Ramos, Daudi, MOLT-4, Karpas-707, REH, U-266/70, U-698, RPMI-8226, A549, B
• the population of target cells are any type of transgenic cells that are engineered to express CD38.
• the population of target cells comprise a cell line that is engineered to express CD38 such as CHO, HeLa, HEK293 or PANOPLY.
• the ratio of macrophage cells to target cells can be about 1 : 1, or about 2: 1, or about 3: 1, or about 4: 1, or about 5: 1, or about 5-10: 1, or about 10-20: 1, or about 20-30: 1.
• the present disclosure provides methods for treating a subject having a disease associated with CD38 expression (e.g., over-expression), the method comprising: administering to the subject an effective amount of a therapeutic composition comprising an anti-CD38 variant antibody or a variant antigen binding fragment thereof, which is selected from a group consisting of any of the fully human anti-CD38 antibodies described herein, any of the Fab fully human anti-CD38 antibodies described herein, and any of the single chain human anti-CD38 antibodies described herein.
• the disease associated with CD38 expression is a hematological cancer, including leukemia, lymphoma, myeloma or B cell lymphoma.
• the hematologic cancers include multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) including Burkitf s lymphoma (BL), B chronic lymphocytic leukemia (B- CLL), systemic lupus erythematosus (SLE), B and T acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma, chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), follicular lymphoma, Waldenstrom's Macroglobulinemia, mantle cell lymphoma, Hodgkin's Lymphoma (HL), plasma cell myeloma, precursor B cell lymphoblastic leukemia/lymphoma, plasmacytoma, giant cell myeloma, plasma cell myeloma, heavy-chain myeloma
• thrombocytopenia purpura anti-phospholipid syndrome, Chagas' disease, Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, anti-phospholipid syndrome, ANCA associated vasculitis, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy-chain disease, primary or immunocyte-associated amyloidosis, and monoclonal gammopathy of undetermined significance.
• an anti-CD38 antigen-binding protein comprises a heavy chain variable region wherein an SGR motif replaces an NGR motif; for example, in any one of SEQ ID NO:3, 5, 6, 7, 9, 10, 11 or 13, the NGR motif at positions 54-56 (bold and underlined in Table 1) may be replaced with an SGR motif.
• Anti-CD38 IgGl heavy chain constant region: SEQ ID NO: 14
• Anti-CD38 IgGl-SPPC heavy chain constant region: SEQ ID NO: 15
• Anti-CD38 IgG4 heavy chain constant region: SEQ ID NO: 16
• Anti-CD38 lambda light chain constant region: SEQ ID NO: 17
• Anti-CD38 kappa light chain constant region: SEQ ID NO: 18
• CD38 antigen - human (UniProt P28907): SEQ ID NO: 19
• CD38 antigen - cynomolgus monkey (UniProt Q5VAN0: SEQ ID NO:20
• CD38 antigen - mouse (UniProt P56528: SEQ ID NO:21
• CD38 antigen - rat (UniProt Q64244: SEQ ID NO:22
• Example 1 Comparative antigen binding analysis of parent antibody A2 and variant antibodies.
• Binding kinetics of A2 parent antibody, variant antibodies and a commercially- available anti-CD38 antibody Daratumumab (Darzalex) was measured using surface plasm on resonance (SPR).
• SPR surface plasm on resonance
• Kinetic interaction between a his-tagged CD38 protein and the various anti- CD38 variant antibodies was measured at approximately 25 °C using a Biacore T200 surface plasmon resonance (GE Healthcare).
• Anti-human Fc antibody from Human Antibody Capture Kit (catalog # BR-1008-39, from GE Healthcare) was immobilized on a Series S Sensor Chip CM5 (catalog # BR-1005-30, GE Healthcare) to approximately 5000 resonance units (RU) using standard N- Hydroxysuccinimide/1 -ethyl-3 -(-3 -dimethylaminopropyl) carbodiimide hydrochloride
• the SPR senorgrams are shown in Figure 1 (Daratumumab, from Janssen Biotech), Figure 2 (parent antibody A2), Figure 3 (variant antibody 3H10ml), Figure 4 (variant antibody 3G3), and Figure 5 (variant antibody 3E10).
• the results of the binding assays, including binding kinetics, are summarized in the table shown in Figure 6.
• the binding assays show that the variant antibodies 3E10, 3G3 and 3H10ml exhibit improved affinity to CD38 protein compared to the parent antibody A2, and these variant antibodies exhibit higher affinity for CD38 protein compared to Daratumumab, as indicated by their measured KD values.
• Example 2 Comparative cell binding analysis of parent antibody A2 and variant antibodies.
• the cells were resuspended in a 100 pL solution of PBS + 2% FCS containing the APC-labeled goat anti-human IgG at a final concentration of 1 : 1,000. After 15 minutes at 4°C, the cells were washed with a solution of PBS + 2% FCS and then resuspended in 150 pL of PBS + 2% FCS for analysis by flow cytometry. Controls for these cell binding assays included (1) T cells incubated with an irrelevant isotype control antibody followed by incubation with the APC-labeled goat anti-human IgG, and (2) T cells stained with an antiCD47 antibody. The results shown in
• Figures 7, 8A and 8B demonstrate that variant antibodies 3H10ml, 3G3 and 3E10 exhibit improved cell binding capabilities compared to parent antibody A2 at all concentrations tested, and these variant antibodies have cell binding capabilities that are comparable to Daratumumab.
• Example 3 Comparative cell binding analysis of parent antibody A2 and variant antibodies.
• Example 4 Comparative cell binding analysis of parent antibody A2 and variant antibodies.
• Example 5 Comparative cell binding analysis of parent antibody A2 and variant antibodies.
• Example 6 Cross-reactivity analysis of parent antibody A2 and Daratumumab.
• Daratumumab bind human CD38 protein but not murine CD38 protein.
• the parent antibody A2 binds cynomolgus CD38 protein whereas Daratumumab does not. This result indicates that the parent antibody A2 can enable toxicology evaluation in non-human primates for CD38-based immunotherapy studies.
• Example 7 Cross-reactivity analysis of parent antibody A2 and variant antibodies.
• Example 8 ADCP analysis of parent antibody A2 and variant antibodies.
• ADCP antibody-dependent cellular phagocytic
• Macrophages were prepared by culturing purified human monocytes with
• M-CSF macrophage colony-stimulating factor
• lxlO 5 macrophages were cultured with 5xl0 3 Ramos cells in the wells of 96-well plate in the presence or absence of an anti-CD38 antibody at 10 pg per mL. After three hours of incubation at 37° C the cells were stained with phycoerythrin (PE)- conjugated anti-human CD1 lb to identify the macrophages. The cells were then analyzed by flow cytometry with phagocytic activity being detected as cells which were positive for both CD1 lb staining and CFSE labeling.
• PE phycoerythrin
• the bar graph shown in Figure 15 demonstrates that the parent antibody A2, and variant antibodies 3H10ml and 3G3, exhibited higher levels of ADCP activity compared to Daratumumab.
• Example 9 ADCC analysis of parent antibody A2 and variant antibodies.
• the capability of the parent antibody A2, variant antibodies 3H10ml and 3G3, and Daratumumab to exhibit antibody-dependent cellular cytotoxicity (ADCC) activity was analyzed.
• NK cells served as the effector population in the ADCC assay.
• the NK cells were prepared from peripheral blood mononuclear cells (PBMC) using a human NK cell enrichment kit (StemCell Technologies). Once prepared, the NK cells were cultured in RPMI + 10% FCS containing IL-2 (100 U/mL). After 1 day of culture at 37 °C and 5% CO2, the NK cells were harvested, washed by centrifugation at 500g for 5 minutes, then resuspended in RPMI + 10% FCS and counted using a hemocytometer
• the anti-CD38 antibodies were added at 10 pg/mL or indicated dilutions thereof to 5xl0 3 Raji or 5xl0 3 T cells in 100 pL RPMI + 10% FCS in the wells of a 96-well white plate. After incubating 20 minutes at 37 °C and 5% CO2, the cells were washed with RPMI + 2% FCS followed by the addition of 1 5xl0 5 NK cells to give an effector to target ratio of 10: 1. The cells were incubated at 37 °C and 5% CO2 for a minimum of 4 hours, followed by measurement of cytotoxicity using the CytoTox-GloTM kit per manufacturer’s instructions and determining the luminescence on a FlexStation. Controls consisted of cells treated as above except that no primary antibody was added, and cells treated as above except an irrelevant isotype-matched control antibody was used instead of an anti-CD38 antibody.
• the graph at Figure 16 shows that variant antibodies 3H10ml (line D) and 3G3 (line E) exhibit a greater ability to promote cytotoxicity compared to the parent antibody A2 (line A) and Daratumumab (line B).
• Example 10 Thermal stability of parent antibody A2 and variant antibodies.

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