WO2025120583A2 - ANTICORPS CD33, ANTICORPS MULTISPÉCIFIQUES CD33/Vδ2 ET LEURS UTILISATIONS - Google Patents

ANTICORPS CD33, ANTICORPS MULTISPÉCIFIQUES CD33/Vδ2 ET LEURS UTILISATIONS Download PDF

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WO2025120583A2
WO2025120583A2 PCT/IB2024/062299 IB2024062299W WO2025120583A2 WO 2025120583 A2 WO2025120583 A2 WO 2025120583A2 IB 2024062299 W IB2024062299 W IB 2024062299W WO 2025120583 A2 WO2025120583 A2 WO 2025120583A2
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seq
antibody
antigen
heavy chain
binding fragment
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WO2025120583A3 (fr
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Sara Mohamed A ELASHKAR
Lorena A KALLAL
Steven A. Jacobs
Ulrike Philippar
Patrick John DOONAN
Paul Willem Henri Ida PARREN
Sabrina Julia Louisa MERAT
Robertus Cornelis ROOVERS
Johannes Jelle VAN DER VLIET
Paula Maria Wilhelmina Van Helden
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Janssen Biotech Inc
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Janssen Biotech Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • CD33 antibodies multispecific CD33/V82 antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies.
  • Methods of making the antibodies, and methods of using the antibodies to treat diseases, including hematological cancers, are also provided.
  • AML Acute Myeloid Leukemia
  • CD33 is restricted to hematopoietic cells (Paul, Taylor, Stansbury, & McVicar, 2000; Ulyanova, Blasioli, Woodford- Thomas, & Thomas, 1999) but is absent on normal hematopoietic stem cells (Andrews, Torok-Storb, & Bernstein, 1983; Griffin, Linch, Sabbath, Larcom, & Schlossman, 1984; Jilani et al., 2002).
  • CD33 chimeric antigen receptor
  • CAR-T and CAR-NK cell therapies challenges have emerged. Specifically, therapies involving CD3 T-cell engagers targeting CD33 have encountered issues related to efficacy, tolerability, and an undesirable safety profile. Therefore, addressing the unmet medical needs of AML and MDS patients whose condition does not respond to traditional chemotherapy is imperative.
  • antibodies, or antigen-binding fragments thereof that bind CD33 of cancer cells, in particular of AML or Myelodysplastic Syndrome (MDS) cells.
  • MDS Myelodysplastic Syndrome
  • the antibodies or antigen-binding fragments thereof bind membrane-bound CD33 (mCD33).
  • multispecific antibodies, or antigen-binding fragments thereof that bind to CD33 of cancer cells and to V62 chain of a T cell receptor (TCR) expressed on a Vy9V82 T cell.
  • the antibodies, multispecific antibodies, or antigen-binding fragments thereof bind mCD33.
  • the CD33 antibodies, CD33/V62 multispecific antibodies, or antigen-binding fragments thereof bind the C2 domain of CD33.
  • the CD33 antibodies, CD33/V62 multispecific antibodies, or antigen-binding fragments thereof bind specifically to CD33, preferably human CD33.
  • the CD33 antibodies, CD33/V62 multispecific antibodies, or antigen-binding fragments thereof do not significantly bind to soluble human CD33 (sCD33).
  • sCD33 soluble human CD33
  • the antibody or antigen-binding fragment thereof comprises: a.
  • CDR1 heavy chain complementarity determining region 1
  • CDR2 heavy chain complementarity determining region 2
  • CDR3 heavy chain complementarity determining region 3
  • a light chain complementarity determining region 1 (CDR1), a light chain complementarity determining region 2 (CDR2), and a light chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 15, 16, and 17, respectively;
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 18, 19, and 14, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 15, 16, and 17, respectively; c. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 20, 21, and 14, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 15, 16, and 17, respectively; or d.
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 22, 23, and 24, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 25, 26, and 17, respectively.
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 27, 28, and 29, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 30, 31, and 32, respectively; f.
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 33, 34, and 29, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 30, 31, and 32, respectively; g. a heavy chain CDR1 , a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 35, 36, and 29, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 30, 31, and 32, respectively; or h.
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 37, 38, and 39, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 40, 41, and 32, respectively.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 42 or 44, or a light chain variable region (VL), comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43 or 45.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody, or antigen-binding fragment thereof comprises a VH comprising SEQ ID NO: 42, and a VL comprising SEQ ID NO: 43.
  • the antibody, or antigen-binding fragment thereof comprises a VH comprising SEQ ID NO: 44, and a VL comprising SEQ ID NO: 45.
  • the antigen-binding fragment of a CD33 antibody is a single heavy chain variable region (VHH).
  • the antibody or antigen-binding fragment thereof comprises: a. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 46, 47, and 48, respectively; b. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 49, 50, and 48, respectively; c. a heavy chain CDR1 , a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 51, 52, and 48, respectively; d.
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 53, 54, and 76, respectively; e. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 53, 54, and 111, respectively; f. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 55, 56, and 57, respectively; g.
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 58, 59, and 57, respectively; h. a heavy chain CDR1 , a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 60, 61, and 57, respectively; or i. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 62, 63, and 64, respectively.
  • the antibody, or antigen-binding fragment thereof comprises a WH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 65 or 66.
  • the antibody, or antigen-binding fragment thereof comprises a VHH comprising SEQ ID NO: 65.
  • the antibody, or antigen-binding fragment thereof comprises a VHH comprising SEQ ID NO: 66.
  • the antibody, or the antigen-binding fragment thereof comprises an IgG Fc domain, preferably a human IgGl Fc domain.
  • the antibody or the antigen-binding fragment comprises a human IgGl Fc domain comprising one or more mutations selected from T366S, L368A, T366W and Y407V per the EU numbering system.
  • the antibody or the antigen-binding fragment comprises a first human IgGl Fc domain comprising the mutations T366S, L368A and Y407V per the EU numbering system and a second human IgGl Fc domain comprising the mutation T366W per the EU numbering system.
  • the antibody, or the antigen-binding fragment thereof comprises a human IgGl Fc domain comprising one or more mutations selected from L234A, L235A, and D265S per the EU numbering system.
  • the antibody, or the antigen-binding fragment thereof comprises a human IgGl Fc domain comprising the triple mutation L234A/L235A/ D265S per the EU numbering system.
  • the antibody or the antigen-binding fragment thereof comprises a human IgGl Fc domain comprising mutations H435R and/or Y436F per the EU numbering system.
  • the antibody, or the antigen-binding fragment thereof comprises a human IgGl Fc domain comprising the triple mutation M252Y/S254T/T256E per the EU numbering system.
  • the antibody, or antigen-binding fragment thereof is chimeric, partially humanized, or fully humanized.
  • CD33/V62 multispecific antibodies or antigen-binding fragments thereof.
  • the CD33/V62 multispecific antibodies comprise a CD33 antibody, or an antigen-binding fragment thereof, specifically binding human mCD33, and a V62 antibody, or an antigen-binding fragment thereof, specifically binding the V82 chain of the human Vy9V82 T cell receptor.
  • the CD33 antibody or antigen-binding fragment thereof in the CD33/V62 multispecific antibody is as described herein for CD33 antibodies or antigen-binding fragments thereof
  • the V62 antibody or antigen-binding fragment thereof in the CD33/V62 multispecific antibody is as described herein for V62 antibodies or antigen-binding fragments thereof.
  • the CD33/V62 multispecific antibody comprises a CD33 antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, comprising the amino sequences of: a. SEQ ID NOs: 12, 13, 14, 15, 16, and 17, respectively; b. SEQ ID NOs: 18, 19, 14, 15, 16, and 17, respectively; c. SEQ ID NOs: 20, 21, 14, 15, 16, and 17, respectively; or d.
  • the CD33/V62 multispecific antibody comprises a CD33 antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, comprising the amino sequences of: a. SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively; b. SEQ ID NOs: 33, 34, 29, 30, 31, and 32, respectively; c. SEQ ID NOs: 35, 36, 29, 30, 31, and 32, respectively; or d.
  • the CD33/V62 multispecific antibody comprises a CD33 antibody or, an antigen-binding fragment thereof, comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino sequences of: a. SEQ ID NOs: 46, 47, and 48, respectively; b. SEQ ID NOs: 49, 50, and 48, respectively; c. SEQ ID NOs: 51, 52, and 48, respectively; d. SEQ ID NOs: 53, 54, and 76, respectively; or e.
  • SEQ ID NOs: 53, 54, and 111 respectively; and a V62 antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: f. SEQ ID NOs: 77, 78, and 79, respectively; g. SEQ ID NOs: 80, 81, and 79, respectively; h. SEQ ID NOs: 82, 83, and 79, respectively; or i. SEQ ID NOs: 84, 85, and 86, respectively.
  • the CD33/V62 multispecific antibody comprises a CD33 antibody, or an antigen-binding fragment thereof, comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino sequences of: a. SEQ ID NOs: 55, 56, and 57, respectively; b. SEQ ID NOs: 58, 59, and 57, respectively; c. SEQ ID NOs: 60, 61, and 57, respectively; or d.
  • SEQ ID NOs: 62, 63, and 64 respectively; and a V62 antibody, or the antigen-binding fragment thereof, comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: e. SEQ ID NOs: 77, 78, and 79, respectively; f. SEQ ID NOs: 80, 81, and 79, respectively; g. SEQ ID NOs: 82, 83, and 79, respectively; or h. SEQ ID NOs: 84, 85, and 86, respectively.
  • the CD33/V62 multispecific antibody comprises a heavy chain variable region (VH) having an amino acid sequence at least 95% identical to SEQ ID NO:42 or 44, and a light chain variable region (VL) having an amino acid sequence at least 95% identical to SEQ ID NO: 43 or 45; and a single heavy chain only variable region (VHH) having an amino acid sequence at least 95% identical to SEQ ID NO: 87.
  • VH heavy chain variable region
  • VL light chain variable region
  • VHH single heavy chain only variable region
  • the CD33/V62 multispecific antibody comprises: a. a VH comprising the amino acid sequence of SEQ ID NO:42, and a VL comprising the amino acid sequence of SEQ ID NO: 43; and a VHH comprising the amino acid sequence of SEQ ID NO: 87; or b. a VH comprising the amino acid sequence of SEQ ID NO: 44, and a VL comprising the amino acid sequence of SEQ ID NO: 45; and a VHH comprising the amino acid sequence of SEQ ID NO: 87.
  • the CD33/V62 multispecific antibody comprises a VHH having an amino acid sequence at least 95% identical to SEQ ID NO:65 or 66, and a VHH having an amino acid sequence at least 95% identical to SEQ ID NO: 87.
  • the CD33/V62 multispecific antibody comprises: a. a VHH comprising the amino acid sequence of SEQ ID NO: 65; and a VHH comprising the amino acid sequence of SEQ ID NO: 87; or b. a VHH comprising the amino acid sequence of SEQ ID NO: 66; and a VHH comprising the amino acid sequence of SEQ ID NO: 87.
  • the CD33/V62 multispecific antibody comprises: a. a heavy chain comprising the amino acid sequence of SEQ ID NO:67; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 70, and a light chain comprising the amino acid sequence of SEQ ID NO:71; b. a heavy chain comprising the amino acid sequence of SEQ ID NO: 67; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 72, and a light chain comprising the amino acid sequence of SEQ ID NO:73; c. a heavy chain comprising the amino acid sequence of SEQ ID NO: 67; and a heavy chain comprising the amino acid sequence of SEQ ID NO:68; d.
  • a heavy chain comprising the amino acid sequence of SEQ ID NO: 74; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 75 or e. a heavy chain comprising the amino acid sequence of SEQ ID NO:91; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 92.
  • the CD33/V62 multispecific antibody is a bispecific antibody.
  • the CD33/V62 multispecific antibody, or an antigen-binding fragment thereof comprises an IgGFc domain, preferably a human IgGl Fc domain.
  • the CD33/V62 multispecific antibody, or an antigen-binding fragment thereof comprises a human IgGl Fc domain comprising one or more mutations selected from T366S, L368A, T366W and Y407V per the EU numbering system.
  • the CD33/V62 multispecific antibody comprises a first human
  • IgGl Fc domain comprising the triple mutations T366S/L368A/Y407V per the EU numbering system, and a second human IgGl Fc domain comprising the mutation T366W per the EU numbering system.
  • the CD33/V62 multispecific antibody, or an antigen-binding fragment thereof comprises a human IgGl Fc domain comprising one or more mutations selected from L234A, L235A, and D265S per the EU numbering system.
  • the CD33/V62 multispecific antibody, or the antigen-binding fragment thereof comprises a human IgGl Fc domain comprising the triple mutations L234A/ L235A/ D265S per the EU numbering system.
  • the CD33/V62 multispecific antibody, or an antigen-binding fragment thereof comprises a human IgGl Fc domain comprising mutations H435R and/or Y436F per the EU numbering system.
  • the CD33/V62 multispecific antibody, or an antigen-binding fragment thereof comprises a human IgGl Fc domain comprising the triple mutation M252Y/S254T/T256E per the EU numbering system.
  • CD33/V62 multispecific the antibody, or an antigen-binding fragment thereof, is chimeric, partially humanized, or fully humanized.
  • host cells comprising (i) one or more polynucleotides encoding the CD33 and/or CD33/V62 multispecific antibodies, or antigen-binding fragments thereof, described herein, or (ii) the one or more vectors comprising the polynucleotides encoding the CD33 and/or CD33/V62 multispecific antibodies, or antigen-binding fragments thereof, described herein.
  • provided is a pharmaceutical composition comprising the CD33 antibody, or antigen-binding fragment thereof, described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising the CD33/V62 multispecific antibody, or antigen-binding fragment thereof, described herein and a pharmaceutically acceptable carrier.
  • the hematologic cancer can be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low or high risk), acute lymphocytic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • ALL acute lymphocytic leukemia
  • ALL diffuse large B-cell lymphoma
  • CML chronic myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic cell neoplasm
  • the hematological cancer is AML or MDS.
  • the methods comprise culturing a cell comprising one or more polynucleotides encoding the CD33 or CD33/V62 multispecific antibody, or antigen-binding fragment(s) thereof, under conditions to produce the CD33 or CD33/V62 multispecific antibody, or antigen-binding fragment(s) thereof, and recovering the CD33 or CD33/V62 multispecific antibody, or antigen-binding fragment(s) thereof, from the cell or culture.
  • Also provided are methods of producing a pharmaceutical composition comprising the CD33 and/or CD33/V62 multispecific antibody, or an antigen-binding fragment thereof, described herein, comprising combining CD33 and/or CD33/V62 multispecific antibody, or an antigen-binding fragment thereof, with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
  • FIG. 1 shows a CD33xV82 bispecific antibody (GD33B273) binding to Vy9V82 T cells.
  • Vy9V82 T cells isolated and expanded from healthy donors were incubated with a dose range of the CD33xV82 bispecific antibody (GD33B273) or nullxV82 bispecific antibody (GD33B73) negative control.
  • NullxV82 bispecific antibody (GD33B73) has no CD33 binding arm. Binding was detected by flow cytometry. Data is shown from 5 different T-cell donors.
  • MFI Mean Fluorescence Intensity.
  • FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, and FIG. 2E A LC-MS assay was developed to measure the amount of free soluble CD33 (sCD33) in AML patients’ sera (AML1-AML30) after spike-in with the indicated antibody.
  • CD33xV82 bispecific antibodies GD33B112, GD33B116, GD33B139, and GD33B134 show minimal binding to sCD33.
  • the binding results of IL5 to sCD33 are also shown using sera from different AML patients (Indiv 517-Indiv 532) (FIG. 2E).
  • FIG. 3 shows the results of an experiment demonstrating that the tested CD33xV82 bispecific antibodies (GD33B112, GD33B116, and GD33B134) induce T-cell mediated cytotoxicity against THP1 cancer cells in vitro.
  • THP1 cells are a human monocytic cell line derived from an acute monocytic leukemia patient.
  • pan T cells effectors
  • CFSE labelled THP1 cells
  • FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D depict the cell binding of CD33xV82 bispecific antibodies GD33B273 (FIG. 4A), GD33B112 (FIG. 4B), GD33B116 (FIG. 4C), and GD33B139 (FIG. 4D), to CD33 positive THP1 cancer cells, and to CD33 negative THP1 CD33 knock-out cell lines.
  • FIG. 4E depicts the cell binding of CD33xV82 bispecific antibody GD33B273 to an isogenic THP1 C2 cell line that specifically expresses the IgC2 domain of CD33.
  • the IgC2 domain of CD33 is a conserved region of the CD33 cell surface receptor protein.
  • FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D present the efficacy of representative bispecific CD33x82 antibodies, GD33B134 (FIG. 5A and FIG. 5B) and GD33B273 (FIG. 5C and FIG. 5D) in the presence of recombinant CD33 (rCD33).
  • the presence of rCD33 does not impact the cytotoxicity and T cell activation effects of GD33B134 and GD33B273.
  • FIG. 6 presents the efficacy of bispecific antibody GD33B273 using AML derived V82+ T cells.
  • V82+ T cells were expanded from AML patient-derived PBMCs and tested in a T cell cytotoxicity assay with GD33B273 and THP-1 target cells at an E:T ratio of 1 : 1. Cytotoxicity of THP-1 cells was evaluated after 24 hours.
  • FIG.7A, FIG. 7B, FIG. 7C, FIG. 7D, and FIG. 7E present the preferential cancer cell cytotoxicity of representative bispecific CD33x82 antibodies GD33B134, GD33B273, and NullxV82 antibody (GD33B73), showing activity against MOLM-13 AML model cells (FIG. 7 A) and no activity against monocyte (FIG. 7B) and NK immune cells (FIG. 7C).
  • Preferential cancer cell cytotoxicity was compared between representative bispecific CD33x82 antibody GD33B134 and representative CD33x82 antibody GD33B273 in flow-cytometry based assays at 96 hours with MOLM-13 as target cancer cells (FIG.
  • FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D show how bispecific CD33x82 antibody GD33B273 mediates selective cytotoxicity of CD33+ cancer cells (MOLM-13 and THP-1) and selective activation of V82 T cells when compared to the antibody lacking the CD33 arm (NullxV82, aka GD33B73).
  • T cells from healthy donors were evaluated in T-cell cytotoxicity and activation assays with GD33B273 and NullxV82 antibodies and target cell lines. Cancer cells cytotoxicity was determined for GD33B273 in flow-cytometry based assays at 72 hours with MOLM-13 and THP-1 as target cancer cells (FIG. 8 A and 8C).
  • FIG. 8E and 8F show how bispecific CD33x82 antibody GD33B273 mediates selective cytotoxicity of THP-1 C2 cells expressing CD33 IgC2 domain (FIG. 8E) and selective activation of V82 T cells (FIG. 8F) when compared to the antibody lacking the CD33 arm (NullxV82, aka GD33B73).
  • FIG. 8G and 8H show how bispecific CD33x82 antibody GD33B273 does not mediate selective cytotoxicity of OCI-LylO / CD33negative cells, i.e., not expressing CD33 (FIG. 8G), and does not mediate or shows minimal selective activation of V82 T cells (FIG. 8H) when compared to the antibody lacking the CD33 arm (NullxV82, aka GD33B73).
  • OCI-LylO cells are a human-derived cell line and are used as target cancer cells.
  • FIGs. 9A and 9B the bispecific CD33x82 antibody GD33B273 shows no on-target off- tumor toxicities.
  • healthy cells like monocytes, B cells, and NK cells
  • the bispecific CD33x82 antibody GD33B273 show no antibody effect on cytotoxicity of these healthy cells.
  • the presence of the bispecific CD33x82 antibody GD33B273 results in no or minimal cytokine production (IFN-y, TNF-a, IL1-P).
  • Whole blood from healthy donors was tested in a T-cell cytotoxicity assay with GD33B273 for 24 or 96 hours in the absence of cancer cell cocultures.
  • the percentage cytotoxicity of monocytes, B cells and NK cells (FIG. 9A) and secreted cytokines IFN-y, TNF-a, and IL1-0 (FIG. 9B) were determined and shown.
  • FIG. 10 shows how bispecific CD33x82 antibody GD33B273 induces potent cytotoxicity of AML bone marrow (BM) blast cells from 3 different human patients.
  • T cells from healthy donors were tested in T-cell cytotoxicity assays with GD33B273 and AML patient-derived BM cells, performed at a relative E:T ratio of 2: 1. Cytotoxicity of CD33+ blast was assessed 24 hours.
  • FIG. 11 shows how bispecific CD33x82 antibody GD33B273 represents low risk of hematopoietic toxicity while showing cytotoxicity for the cancer cells THP-1 in a dose response fashion.
  • CD34+ HSPC are stem cells from healthy donors, and the presence of surviving robust colonies (light grey bars) shows that the antibody does not impact them. In contrast, THP-1 cancer cells colonies (black bars) decrease survival in a dose-dependent relation to the antibody concentration.
  • Naive V82+ T cells were isolated from healthy donor PBMCs and used as effectors at 1 : 1 relative E:T ratio with CD34+ HSPC cells or THP-1 cells as target cells in a CFU assay.
  • HSPC hematopoietic stem and progenitor cells; CD, cluster of differentiation; CFU, colony forming unit; E:T, effector to target; PBMC, peripheral blood mononuclear cell.
  • FIG. 12 presents the effect of treatment with a representative bispecific CD33x82 antibody GD33B134 on growth of MOLM-13 xenografts admixed with pan-T cells in NSG mice.
  • the size of the tumor increases in absence of antibody (with just buffer DPBS) and the size of tumor decreases in the presence of the antibody at different doses (1, 3, and 10 mg/Kg).
  • T-cell-humanized NSG mice were injected SC with MOLM-13 tumors admixed with pan-T cells and dosed IP with indicated doses on Days 1, 4, 7, 10, 13, 16, 20, 23, 27, 30, and 35 (the dosing period is indicated by the solid bar under the X-axis, i.e., from 0 to 35 days).
  • FIG. 13A and FIG. 13B presents the efficacy of a representative bispecific CD33x82 antibody GD33B134 in a MOLM-13 regression mice model with enriched V82 T cells as effectors.
  • hIL-15 NOG mice were injected IV with MOLM-13 cells, humanized with enriched V82 T cells (IV) on Day 3 and 16, and dosed IP with DPBS or antibody at the indicated doses (1, 3, and 10 mg/Kg) on Days 4, 7, 10, 14, 17, 21, 24, and 28 (the dosing period is indicated by the solid bar under the X-axis, i.e. from day 5 to day 28 for both FIGs. 13A and 13B). From Day 7 onward, body weight and disease-related clinical signs were observed daily, and results are presented as (FIG. 9A) percent survival or (FIG. 9B) mean body weight change ⁇ SEM for each group. Data are only displayed for the period during which two thirds of animals were still alive in a group.
  • hIL-15 human interleukin- 15; IP, intraperitoneal; IV, intravenous; NOG, non-obese diabetic (NOD)/Shi-severe combined immunodeficiency (scid) IL2rgamma(null); SEM, standard error of the mean.
  • FIG. 14A and FIG. 14B presents the efficacy of the bispecific CD33x82 antibody GD33B273 in a MOLM-13 regression mice model.
  • the MOLM-13 cancer cells were labelled with luciferase. This labeled MOLM-13 cancer cells were implanted on Day 0 in the hIL-15 NOG mice. The same mice were injected with the T cells on Day 3 and 16, and dosed with antibody at the indicated doses (0, 0.3, and 3 mg/Kg) on Days 4, 7, 10, 14, 17, 21, and 24 (the dosing period is indicated by the solid bar under the X-axis, i.e. from day 4 to day 24 for both FIGs. 14A and 14B).
  • FIG. 14A shows an increase in average radiance for the untreated mice (0 mg/Kg) when compared to the treated mice with 0.3 and 3 mg/Kg antibody.
  • FIG. 14A also shows 61% and 54.9% of tumor growth inhibition at day 17 for treated mice with antibody at 0.3 and 3 mg/Kg, respectively, when compared to untreated mice (0 mg/Kg).
  • FIG. 14B shows an increase in the survival of the mice treated with 0.3 and 3 mg/Kg of antibody when compared to untreated mice (0 mg/Kg).
  • ILS increased life span
  • TGI tumor growth inhibition.
  • the present disclosure relates to antibodies, multispecific antibodies or antigen-binding fragments thereof that specifically target CD33, a critical target for hematological cancers.
  • the multispecific antibodies or antigen binding fragments thereof may additionally bind V62.
  • polypeptide and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
  • polypeptides containing one or more analogs of an amino acid including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a “polypeptide” can occur as a single chain or as two or more associated chains.
  • An “antigen” is a structure to which an antibody can specifically and/or selectively bind.
  • a target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound.
  • the target antigen is a polypeptide.
  • an antigen is associated with a cell, for example, is present on or in a cell.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the term refers to at least 70% identity between two or more sequences, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, and more preferably at least 99% or greater identity.
  • the percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.
  • inventions include CD33 specific antibodies, or antigen-binding fragments, that have framework, scaffold, or other non-binding regions that do not share significant identity with the antibodies and antigen-binding fragments described herein, but do incorporate one or more CDRs or other sequences needed to confer binding that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences described herein.
  • a “vector” is a replicon, such as plasmid, phage, cosmid, or virus in which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
  • the terms “subject” and “patient” may be used interchangeably.
  • a subject is a mammal, such as a non-primate or a primate (e.g., human).
  • the subject is a human.
  • the subject is a mammal, e.g., a human, diagnosed with a disease or disorder.
  • the subject is a mammal, e.g., a human, at risk of developing a disease or disorder.
  • administer refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A and B and C; A and B or C; B and A or C; C and A or B; A and B; A and C; B and C; A or B or C; A or B; A or C; B or C; A (alone); B (alone); and C (alone).
  • CD33 refers to a 67 kD single pass transmembrane (i.e. membrane-bound) glycoprotein, a member of the sialic acid-binding immunoglobulin-like lectins (Siglecs) family. Also recognized as Siglec-3, gp67, or p67, CD33 comprises an amino-terminal V-set Ig-like domain (exon 2 of CD33), responsible for sialic acid binding, and a C2-set Ig-like domain (exon 4) in its extracellular portion (Laszlo GS et al. Expression and functional characterization of CD33 transcript variants in human acute myeloid leukemia. Oncotarget.
  • CD33 is primarily considered a myeloid differentiation antigen. It exhibits low expression in myeloid progenitors, neutrophils, and macrophages, yet is highly expressed in circulating monocytes and dendritic cells. Notably, CD33 is detected on blasts and leukemic stem cells in 85-90% of AML patients. Remarkably, its expression is confined to hematopoietic cells but absent on normal hematopoietic stem cells (Paul SP, Taylor LS, Stansbury EK, McVicar DW. Myeloid specific human CD33 is an inhibitory receptor with differential ITIM function in recruiting the phosphatases SHP-1 and SHP-2. Blood.
  • the sialoadhesin CD33 is a myeloid-specific inhibitory receptor. Eur J Immunol. 1999;29(11):3440-3449). This distinctive expression pattern suggests CD33 as a promising target for antibody-based therapy in AML and MDS.
  • soluble CD33 protein or “sCD33” refer to a form of a CD33 protein, e.g., a human CD33 protein, that is not associated or bound to a cell membrane.
  • Soluble CD33 protein or “sCD33” encompass the extracellular domain (ECD) of full length CD33 proteins, and variants thereof, that are shed from cells, i.e., not associated or bound to a cell membrane.
  • Soluble CD33 protein or “sCD33” also encompass the ECD of any fragment of a CD33 protein, or variants thereof, that are shed from cells; i.e., not associated or bound to a cell membrane.
  • Soluble CD33 protein” or “sCD33” also encompass the ECD of CD33 proteins that are shed from cells, i.e., not associated or bound to a cell membrane and that lack one or more domains, such as a transmembrane domain and/or a cytoplasmic domain, for example, as a result of proteolysis of the CD33 protein (e.g., in an endosome in a cell).
  • a “soluble CD33 protein” or “sCD33” comprises the IgV domain of a CD33 protein, or a fragment thereof.
  • a “soluble CD33 protein” or “sCD33” comprises the C2 domain of a CD33 protein, or a fragment thereof.
  • a “soluble CD33 protein” or “sCD33” comprises the IgV domain of a CD33 protein, or a fragment thereof, and the IgC2 domain of a CD33 protein, or a fragment thereof.
  • the extracellular domain (ECD) of CD33 is shed from cells; therefore, normal and AML patient samples contain a soluble form of CD33 (i.e., sCD33).
  • sCD33 could compete for the bispecific antibody in the blood and impact its efficacy due to sink effects.
  • the inventors have recognized that antibodies which bind sCD33 may suffer from “antigen sink effect”, whereby the antibody bound to sCD33 in the blood is removed from the system diminishing the amount of antibody that can reach the target cells, decreasing its efficacy.
  • Described herein are antibodies or antigen-binding fragments capable of specifically binding membrane-bound CD33.
  • antibody immunoglobulin
  • immunoglobulin immunoglobulin
  • Ig immunoglobulin
  • monoclonal antibodies including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies
  • antibody compositions with polyepitopic or monoepitopic specificity polyclonal or monovalent antibodies
  • multivalent antibodies multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) formed from at least two intact antibodies, single chain antibodies, and fragments thereof (e.g., domain antibodies), as described below.
  • an antibody can be human, humanized, chimeric and/or affinity matured, as well as an antibody from other species, for example, mouse, rabbit, llama, etc.
  • the term “antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region.
  • Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, antibodies including from Camelidae species (e.g., llama or alpaca), including VHH or nanobodies, or their humanized variants, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen binding fragments) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • Camelidae species e.g., llama or alpaca
  • VHH or nanobodies e.g., VHH or nanobodies
  • functional fragments e.g., antigen binding fragments
  • Non-limiting examples of functional fragments include singlechain Fvs (scFv) (e.g., including monospecific, bispecific, etc. , Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, VHH, diabody, triabody, tetrabody, and minibody.
  • scFv singlechain Fvs
  • dsFv disulfide-linked Fvs
  • Fv fragments e.g., VHH, diabody, triabody, tetrabody, and minibody.
  • antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.
  • the antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.
  • Antibodies may be agonistic antibodies or antagonistic antibodies.
  • Antibodies may be neither agonistic nor antagonistic.
  • Antigen-binding fragment refers to a portion of the protein that binds an antigen.
  • Antigen binding fragments may be synthetic, enzymatically obtainable or genetically engineered polypeptides and include portions of an immunoglobulin that bind an antigen, such as VH, the VL, the VH and the VL, Fab, Fab’, F(ab’)2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, VHH domains, minimal recognition units consisting of the amino acid residues that mimic the CDRs of an antibody, such as FR3-CDR3-FR4 portions, the HCDR1, the HCDR2 and/or the HCDR3 and the LCDR1, the LCDR2 and/or the LCDR3, alternative scaffolds that bind an antigen, and multispecific proteins comprising the antigen binding fragments.
  • Antigen binding fragments may be linked together via a synthetic linker to form various types of single antibody designs where the VH/VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chains, to form a monovalent antigen binding domain, such as single chain Fv (scFv), stapled single chain Fv (spFv), or diabody.
  • Antigen binding fragments may also be conjugated to other antibodies, proteins, antigen-binding fragments or alternative scaffolds which may be monospecific or multispecific to engineer bispecific and multispecific proteins.
  • binding refers to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope.
  • the ratio of dissociation rate (k o rr) to association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen (k o ff/k on ) is the dissociation constant KD, which is inversely related to affinity.
  • KD dissociation constant
  • the value of KD varies for different complexes of antibody and antigen and depends on both k on and k O ff.
  • the dissociation constant KD for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art.
  • the affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen.
  • antibodies or antigen-binding fragments described herein terms such as “specifically binding to,” and analogous terms are also used interchangeably herein and refer to antibodies or antigen-binding fragments that can specifically bind to an antigen, such as a polypeptide.
  • Antibodies or antigen-binding fragments that bind to or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those of skill in the art.
  • antibodies or antigen-binding fragments binds to or specifically binds to an antigen when it binds to an antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme linked immunosorbent assay
  • a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion regarding binding specificity.
  • the extent of binding of an antibody or antigen-binding fragment to a “non-target” protein is less than about 10% of the binding of the antibody or antigen-binding fragment to its particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA.
  • An antibody or antigen-binding fragment that binds to an antigen includes one that is capable of binding the antigen with sufficient affinity such that the antibody or antigen-binding fragment is useful, for example, as a therapeutic and/or diagnostic agent in targeting the antigen.
  • an antibody or antigen-binding fragment that binds to an antigen has a dissociation constant (KD) of less than or equal to 1 pM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM,
  • KD dissociation constant
  • an antibody or antigenbinding fragment binds to an epitope of an antigen that is conserved among the antigen from different species.
  • the antibodies or antigen-binding fragments can comprise portions of a “fully human antibody” or “human antibody,” which terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin. “Fully human” antibodies, in certain embodiments, can also encompass antibodies which bind polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Interest, Fifth Edition, U.S Department of Health and Human Services, NIH
  • Kabat reference defines a numbering scheme for the variable regions of immunoglobulins (antibodies), often referred to as the Kabat numbering system or Kabat index.
  • the EU numbering system also called the EU index, classifies and numbers amino acid residues in Ig-like domains is explained in Edelman GM et al., Proc Natl Acad Sci USA
  • a “human antibody” is one that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks etal., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao et al. , Nature Protocols 1: 755-68 (2006)).
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, Curr. Opin. Biotechnol.
  • the antibodies or antigen-binding fragments can comprise a portion of a “monoclonal antibody,” wherein the term as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts or well-known post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation, each monoclonal antibody will typically recognize a single epitope on the antigen.
  • a “monoclonal antibody,” as used herein is an antibody produced by a single hybridoma or other cell.
  • the term “monoclonal” is not limited to any particular method for making the antibody.
  • the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., Nature 256:495 (1975), or may be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991), for example.
  • a typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • the 4-chain unit is generally about 150,000 daltons.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and £ isotypes.
  • VH variable domain
  • CH constant domains
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end.
  • VL variable domain
  • CL constant domain
  • the VL is aligned with the VH
  • the CL is aligned with the first constant domain of the heavy chain (CHI).
  • Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • the pairing of a VH and VL together forms a single antigen-binding site. Lor the structure and properties of the different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites etal. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5 th ed. 2001).
  • the term “Lab” or ‘Tab region” refers to an antibody region that binds to antigens.
  • a conventional IgG usually comprises two Lab regions, each residing on one of the two arms of the Y-shaped IgG structure.
  • Each Lab region is typically composed of one variable region and one constant region of each of the heavy and the light chain. More specifically, the variable region and the constant region of the heavy chain in a Lab region are VH and CHI regions, and the variable region and the constant region of the light chain in a Lab region are VL and CL regions.
  • the VH, CHI, VL, and CL in a Lab region can be arranged in various ways to confer an antigen binding capability according to the present disclosure.
  • VH and CHI regions can be on one polypeptide, and VL and CL regions can be on a separate polypeptide, similarly to a Lab region of a conventional IgG.
  • VH, CHI, VL and CL regions can all be on the same polypeptide and oriented in different orders as described in more detail the sections below.
  • the term “variable region,” “variable domain,” “V region,” or “V domain” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen.
  • variable region of the heavy chain may be referred to as “VH.”
  • variable region of the light chain may be referred to as “VL.”
  • the term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long.
  • FRs framework regions
  • variable regions of heavy and light chains each comprise four FRs, largely adopting a 0 sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the 0 sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991))
  • the constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
  • the variable regions differ extensively in sequence between different antibodies.
  • the variable region is a human variable region.
  • the term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy -terminal portion includes a constant region.
  • the constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (a), delta (8), epsilon (s), gamma (y), and mu (p), based on the amino acid sequence of the heavy chain constant region.
  • the distinct heavy chains differ in size: a, 8, and y contain approximately 450 amino acids, while p and £ contain approximately 550 amino acids.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgM immunoglobulin M
  • subclasses of IgG namely IgGl, IgG2, IgG3, and IgG4.
  • the term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy -terminal portion includes a constant region.
  • the approximate length of a light chain is 211 to 217 amino acids.
  • K kappa
  • X lambda
  • the terms “hypervariable region,” “HVR,” “Complementarity Determining Region,” and “CDR” are used interchangeably.
  • CDR refers to one of three hypervariable regions (Hl, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH 0-sheet framework, or one of three hypervariable regions (LI, L2 or L3) within the non-framework region of the antibody VL P-sheet framework.
  • CDR1 , CDR2 and CDR3 in VH domain are also referred to as HCDR1, HCDR2 and HCDR3, respectively.
  • CDR1, CDR2 and CDR3 in VL domain are also referred to as LCDR1, LCDR2 and LCDR3, respectively. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences.
  • CDR regions are well known to those skilled in the art and have been defined by well- known numbering systems.
  • CDRs Kabat Complementarity Determining Regions
  • Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)).
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Diibel eds., 2d ed. 2010)).
  • the “contact” hypervariable regions are based on an analysis of the available complex crystal structures.
  • Another universal numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp.
  • IMGT immunoglobulins
  • TCR T-cell receptors
  • MHC major histocompatibility complex
  • the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain.
  • location of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified.
  • This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody.
  • An additional numbering system (AHon) has been developed by Honegger and Pliickthun, J. Mol. Biol. 309: 657-70 (2001). Correspondence between the numbering system, including, for example, the
  • CDR complementary determining region
  • individual CDRs e.g., CDR-H1, CDR-H2
  • the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the AbM, IMGT, Kabat, Chothia, or Contact method.
  • the particular amino acid sequence of a CDR is given.
  • CDR regions may also be defined by a combination of various numbering systems, e.g., a combination of Kabat and Chothia numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, the term such as “a CDR1 as set forth in a specific VH” includes any CDR1 as defined by the exemplary CDR numbering systems described above, but is not limited thereby.
  • a variable region e.g., a VH or VL
  • those skilled in the art would understand that CDRs within the region can be defined by different numbering systems or combinations thereof.
  • Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26-35A (Hl), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH.
  • constant region or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor.
  • the term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site.
  • the constant region may contain the CHI, CH2, and CH3 regions of the heavy chain and the CL region of the light chain. Amino acid numbering for constant heavy domain regions typically use the EU numbering system of Kabat.
  • FR refers to those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.
  • Fc domain herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxy 1-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • a “functional Fc region” possesses an “effector function” of a native sequence Fc region. Functional Fc region is typically formed by coming together of two Fc domains (either homodimer or heterodimer) as described herein. Exemplary “effector functions” include Cl q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor), etc.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion).
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide.
  • the variant Fc region herein can possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith.
  • variants when used in relation to an antigen or an antibody may refer to a peptide or polypeptide comprising one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and/or additions as compared to a native or unmodified sequence.
  • a variant of a CD33 antibody may result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native or previously unmodified a CD33 antibody.
  • Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed.
  • Polypeptide variants may be prepared from the corresponding nucleic acid molecules encoding the variants.
  • the CD33 antibody variant at least retains CD33 antibody functional activity, respectively.
  • a CD33 antibody variant binds CD33.
  • the variant is encoded by a single nucleotide polymorphism (SNP) variant of a nucleic acid molecule that encodes anti-CD33 antibody VH or VL regions or subregions, such as one or more CDRs.
  • SNP single nucleotide polymorphism
  • the term “valent” as used herein denotes the presence of a specified number of binding sites in an antigen binding protein.
  • a natural antibody for example or a full length antibody has two binding sites and is bivalent.
  • the terms “monovalent,” “trivalent,” “tetravalent,” “pentavalent” and “hexavalent” denote the presence of one binding site, two binding site, three binding sites, four binding sites, five binding sites, and six binding sites, respectively, in an antibody)
  • the described CD33-specific antibodies or antigen-binding fragments include all isotypes, IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four-chain immunoglobulin structure.
  • the described antibodies or antigen-binding fragments also include the IgY isotype generally found in hen or turkey serum and hen or turkey egg yolk.
  • the CD33 -specific antibodies and antigen-binding fragments may be derived from any species by recombinant means.
  • the antibodies or antigen-binding fragments may be mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof.
  • non-human derived antibodies or antigenbinding fragments may be genetically or structurally altered to be less antigenic upon administration to a human patient.
  • the antibodies or antigen-binding fragments are chimeric.
  • the term “chimeric” refers to an antibody, or antigen-binding fragment thereof, having at least some portion of at least one variable domain derived from the antibody amino acid sequence of a non-human mammal, a rodent, or a reptile, while the remaining portions of the antibody, or antigen-binding fragment thereof, are derived from a human.
  • the antibodies are humanized antibodies.
  • the antibodies or antigen-binding fragments can comprise portions of “humanized” forms of nonhuman (e.g., camelid, murine, non-human primate) antibodies that include sequences from human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as camelid, mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • a nonhuman species e.g., donor antibody
  • one or more FR region residues of the human immunoglobulin sequences are replaced by corresponding nonhuman residues.
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • a humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Humanized antibodies may be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary-determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence.
  • the humanized antibody may include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the antibodies or antigen-binding fragments described herein can occur in a variety of forms, but will include one or more of the antibody CDRs shown in Table 2. Table 2. List of CDR Regions for representative CD33 antibodies.
  • the CD33 -specific antibodies or antigenbinding fragments are human, humanized IgG, or derivatives thereof. While the CD33 -specific antibodies or antigen-binding fragments exemplified herein are human or humanized, the antibodies or antigen-binding fragments exemplified may be chimerized.
  • a CD33 -specific antibody, or an antigen-binding fragment thereof comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 2.
  • a CD33 -specific antibody, or an antigen-binding fragment thereof comprising a heavy chain comprising a CDR1 , a CDR2, and a CDR3 of any one of the antibodies described in Table 2 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 2.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 12, 13, and 14, respectively, and a light chain complementarity determining region 1 (CDR1), a light chain complementarity determining region 2 (CDR2), and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequences of SEQ ID NO: 15, 16, and 17, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same as, or identical to, SEQ ID NO: 42 and a light chain variable domain substantially the same as, or identical to, SEQ ID NO: 43.
  • the CDRs, the heavy chain variable domain, and the light chain variable domain, discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 18, 19, and 14, respectively, and a light chain complementarity determining region 1 (CDR1), a light chain complementarity determining region 2 (CDR2), and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequences of SEQ ID NO: 15, 16, and 17, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same as, or identical to, SEQ ID NO: 42 and a light chain variable domain substantially the same as, or identical to, SEQ ID NO: 43.
  • the CDRs, the heavy chain variable domain, and the light chain variable domain, discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 20, 21, and 14, respectively, and a light chain complementarity determining region 1 (CDR1), a light chain complementarity determining region 2 (CDR2), and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequences of SEQ ID NO: 15, 16, and 17, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same as, or identical to, SEQ ID NO: 42 and a light chain variable domain substantially the same as, or identical to, SEQ ID NO: 43.
  • the CDRs, the heavy chain variable domain, and the light chain variable domain, discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 22, 23, and 24, respectively, and a light chain complementarity determining region 1 (CDR1), a light chain complementarity determining region 2 (CDR2), and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequences of SEQ ID NO: 25, 26, and 17, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same as, or identical to, SEQ ID NO: 42 and a light chain variable domain substantially the same as, or identical to, SEQ ID NO: 43.
  • the CDRs, the heavy chain variable domain, and the light chain variable domain, discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 27, 28, and 29, respectively, and a light chain complementarity determining region 1 (CDR1), a light chain complementarity determining region 2 (CDR2), and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequences of SEQ ID NO: 30, 31, and 32, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same as, or identical to, SEQ ID NO: 44 and a light chain variable domain substantially the same as, or identical to, SEQ ID NO: 45.
  • the CDRs, the heavy chain variable domain, and the light chain variable domain, discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 33, 34, and 29, respectively, and a light chain complementarity determining region 1 (CDR1), a light chain complementarity determining region 2 (CDR2), and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequences of SEQ ID NO: 30, 31, and 32, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same as, or identical to, SEQ ID NO: 44 and a light chain variable domain substantially the same as, or identical to, SEQ ID NO: 45.
  • the CDRs, the heavy chain variable domain, and the light chain variable domain, discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • Also described herein are antibodies or antigen-binding fragments specifically binding membrane-bound CD33 wherein the antigen binding fragment thereof is a single heavy chain variable region (VHH).
  • VHH Single heavy chain variable region
  • dAb fragment refers to an antibody fragment composed of a VH domain (Ward et al., Nature 341 :544 546 (1989)).
  • the antigenbinding fragment of the present disclosure may comprise a VHH.
  • VHHs are well known to the skilled person, see e.g., Hamers-Casterman et al. (1993) Nature 363:446, Roovers et al. (2007) Curr Opin Mol Ther 9:327 and Krah et al. (2016) Immunopharmacol Immunotoxicol 38:21.
  • VHHs comprise a single heavy chain CDR1 , a single heavy chain CDR2 and a single heavy chain CDR3.
  • VHHs may be derived from any species including mouse, human, camel, llama, shark, goat, rabbit, and cow.
  • naturally occurring VHH molecules can be derived from antibodies raised in Camelidae species, for example in camel, dromedary, llama, alpaca and guanaco.
  • a VHH is able to bind selectively to a single specific antigen.
  • VHHs may contain only the variable domain of an immunoglobulin chain, i.e., CDR1, CDR2 and CDR3 and framework regions.
  • the antibodies or antigen-binding fragments comprising a VHH described herein can occur in a variety of forms, but will include one or more of the antibody CDRs shown in Table 3.
  • CD33-specific antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 3.
  • the CD33-specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 46, 47, and 48, respectively.
  • This CD33-specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a single heavy chain variable domain (VHH) substantially the same as, or identical to, SEQ ID NO: 65.
  • the CD33- specific antibodies and antigen-binding fragments comprise a VHH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 65.
  • the CDRs and the VHH discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 49, 50, and 48, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a single heavy chain variable domain (VHH) substantially the same as, or identical to, SEQ ID NO: 65.
  • VHH single heavy chain variable domain
  • the CD33-specific antibodies and antigenbinding fragments comprise a VHH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 65.
  • the CDRs and the VHH discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 51, 52, and 48, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a single heavy chain variable domain (VHH) substantially the same as, or identical to, SEQ ID NO: 65.
  • VHH single heavy chain variable domain
  • the CD33-specific antibodies and antigenbinding fragments comprise a VHH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 65.
  • the CDRs and the VHH discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 53, 54, and 76, respectively.
  • This CD33-specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a single heavy chain variable domain (VHH) substantially the same as, or identical to, SEQ ID NO: 65.
  • VHH single heavy chain variable domain
  • the CD33-specific antibodies and antigenbinding fragments comprise a VHH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 65.
  • the CDRs and the VHH discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 53, 54, and 97, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a single heavy chain variable domain (VHH) substantially the same as, or identical to, SEQ ID NO: 65.
  • VHH single heavy chain variable domain
  • the CD33-specific antibodies and antigenbinding fragments comprise a VHH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:65.
  • the CDRs and the VHH discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 55, 56, and 57, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a single heavy chain variable domain (VHH) substantially the same as, or identical to, SEQ ID NO: 66.
  • VHH single heavy chain variable domain
  • the CD33-specific antibodies and antigenbinding fragments comprise a VHH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 66.
  • the CDRs and the VHH discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 58, 59, and 57, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a single heavy chain variable domain (VHH) substantially the same as, or identical to, SEQ ID NO: 66.
  • VHH single heavy chain variable domain
  • the CD33-specific antibodies and antigenbinding fragments comprise a VHH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 66.
  • the CDRs and the VHH discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 60, 61, and 57, respectively.
  • This CD33 -specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a single heavy chain variable domain (VHH) substantially the same as, or identical to, SEQ ID NO: 66.
  • VHH single heavy chain variable domain
  • the CD33-specific antibodies and antigenbinding fragments comprise a VHH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 66.
  • the CDRs and the VHH discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the CD33 -specific antibodies and antigen-binding fragments comprise a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 62, 63, and 64, respectively.
  • This CD33-specific antibody or antigen-binding fragment may comprise human framework sequences.
  • the CD33-specific antibodies and antigen-binding fragments comprise a single heavy chain variable domain (VHH) substantially the same as, or identical to, SEQ ID NO: 66.
  • VHH single heavy chain variable domain
  • the CD33-specific antibodies and antigenbinding fragments comprise a VHH having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 66.
  • the CDRs and the VHH discussed in this paragraph are suitable for inclusion in multispecific constructs in which one arm is an anti-CD33 arm.
  • the antibodies or antigen-binding fragments are IgG, or derivatives thereof, e.g., IgGl, IgG2, IgG3, and IgG4 isotypes. In some embodiments wherein the antibody is of IgGl isotype, the antibody comprises an IgGl Fc region or IgGFc domain.
  • the CD33 antibodies or antigen-binding fragments thereof bind the C2 domain of CD33.
  • the CD33 antibodies or antigen-binding fragments thereof bind the V domain of CD33.
  • the CD33 antibodies or antigen-binding fragments thereof bind mCD33. [0139] In certain embodiments, the CD33 antibodies or antigen-binding fragments thereof do not significantly bind sCD33.
  • T cells are the most abundant (-75% of blood lymphocytes) and potent immune killer cells.
  • the role of effector T cells in the anti-cancer immune response is strongly supported by in vitro studies and the observation that a high infiltration of CD8+ T cells in several types of cancers correlates with a favorable clinical prognostic.
  • BiTE Bispecific T Cell Engager
  • T cell mediated responses are extremely potent, severe side effects can arise by inducing cytokine storm or directing T cells towards healthy tissues that express low levels of target antigen.
  • Most CD3 bispecific proteins currently in clinical trials are targeting receptors where expression is confined to the hematopoietic lineage (CD 19, CD20, CD 123, etc.), or highly specific cancer antigens, such as CEA, PSMA, and MHCI-gplOO.
  • cancer antigens such as CEA, PSMA, and MHCI-gplOO.
  • CD3-directed T cell redirection with currently available technologies has not shown much efficacy in solid cancers due to various reasons (e.g., recruiting all types of CD3+ T cells, including immature, CD4+, Tregs, pan CD8 (no CTL), exhausted T cells, etc., that could lead to inefficient cancer removal; premature T cell activation that could result in a narrow therapeutic index; suboptimal T cell activation; T cell exhaustion or activation induced death of T cells; induction of cytokine release syndrome that could limit optimal dosing level; inhibition of cancer cell apoptosis; less activation of anti-cancer adaptive immune response; limited ability to combine with other immunotherapies, etc.).
  • reasons e.g., recruiting all types of CD3+ T cells, including immature, CD4+, Tregs, pan CD8 (no CTL), exhausted T cells, etc., that could lead to inefficient cancer removal; premature T cell activation that could result in a narrow therapeutic index; suboptimal T cell activation;
  • CD3+ T cells can be indiscriminately stimulated including various immunoregulatory and immunosuppressive T cells, which are described as playing an active role in immune evasion, and 2) Pan T cell activation that can result in severe side effects can arise by inducing cytokine storm.
  • CD3-redirection limitations alternative strategies to re-direct T cells to cancer cells must be sought.
  • One approach would be to select re-direction of only cytotoxic cells (a subset) that are capable of lysing cancer cells rather than indiscriminately stimulating and recruiting pan-T cells.
  • T cells Another way to recruit T cells is to target specific subset of T cells.
  • yb T cells have provided a great interest in the cancer immunotherapy field.
  • These unconventional T cells well known for their innate immunity, represent only a minor proportion of the peripheral CD3+ T cells (1%— 5%), but constitute a major subset (20%-50%) of T cells in epithelial tissues.
  • Circulating y6 T cells mainly express heterodimers of Vy9 (TRGV9) and V82 (TRDV2) chains whereas tissue y8 T cells preferentially express V81 chains associated with different Vy chains.
  • y8 T cells are endowed with potent anti-cancer functions (high cytotoxicity and interferon y secretion). Moreover, y8 T cells are capable of phagocytosis, a function previously exclusive to innate myeloid lineage cells, and behave as efficient antigen-presenting cells for aP T cells and induce adaptive immune response. y8 T cells have been shown to infiltrate cancers, but the clinical relevance of their presence is still debated. Up to now, all the research efforts have been focused on Vy9V82 T cells, and mainly aimed at activating y5 T cells in vivo or ex vivo for adoptive transfer. Although clinical studies are not yet abundant, preliminary data highlight the importance of considering the y5 T cells subset in T cell-based immunotherapy.
  • V62 refers to a polypeptide capable of forming a T cell receptor together with a Vy9 chain when expressed on the surface of y5 T cells.
  • 62-expressing y5 T cells are among the first T cells to develop in the human fetus and are the predominant y5 T cell subset in healthy adult peripheral blood cells.
  • the term “52” includes any 62 variant, isoform, and species homolog, which is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding the polypeptide.
  • the 62 is a human 62.
  • CD33/ V62 multispecific antibodies or antigen-binding fragments thereof comprising a CD33 antibody or an antigen-binding fragment thereof and a V6 2 antibody or antigen-binding fragment thereof, wherein the CD33 antibody or the antigenbinding fragment thereof, specifically binds to mCD33, and wherein the V62 antibody, or the antigen-binding fragment thereof, specifically binds to the V82 chain of the human Vy9V82 T cell receptor.
  • multispecific antibody refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope.
  • first and second epitopes do not overlap or do not substantially overlap.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain.
  • a multispecific antibody is a bispecific antibody molecule.
  • bispecific antibody refers to a multispecific antibody that binds no more than two epitopes or two antigens.
  • a bispecific antibody is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope (e.g., an epitope on a CD33 antigen) and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope (e.g., an epitope on a V82 antigen).
  • the first and second epitopes are on different antigens, e.g, the different proteins (or different subunits of a multimeric protein).
  • a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a single heavy chain variable domain sequence which has binding specificity for a second epitope.
  • a bispecific antibody comprises a single heavy chain variable domain sequence which has binding specificity for a first epitope and a single heavy chain variable domain sequence which has binding specificity for a second epitope.
  • a bispecific antibody comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope.
  • a bispecific antibody comprises a scFv, or fragment thereof, having binding specificity for a first epitope, and a scFv, or fragment thereof, having binding specificity for a second epitope.
  • the first epitope is located on CD33 and the second epitope is located on V82 chain of the human Vy9V82 T cell receptor.
  • the bispecific antibody may comprise a third, fourth, or fifth immunoglobulin variable domain that binds one or other of the first and second epitope (e.g.
  • the first epitope may have two immunoglobulin variable domains that bind the first epitope and two immunoglobulin variable domains that bind the second epitope, or it may have two immunoglobulin variable domains that bind the first epitope and one immunoglobulin variable domain that bind the second epitope).
  • the CD33 antibody or antigen-binding fragment thereof is an CD33 antibody or antigen-binding fragment thereof described herein and the V62 antibody or antigen-binding fragment thereof comprises a heavy chain HCDR1, HCDR2, and HCDR3, having the amino acid sequences of (1) SEQ ID NOs:77, 78, and 79, respectively, (2) SEQ ID NOs: 80, 81, 82, respectively, (3) SEQ ID NOs: 82, 83, and 79, respectively, or (4) SEQ ID NOs: 84, 85, and 86, respectively.
  • the multispecific antibody of the present disclosure is a diabody, a cross-body, or a multispecific antibody obtained via a controlled Fab arm exchange as those described in the present disclosure.
  • the multispecific antibodies include IgG-like molecules with complementary CH3 domains to force heterodimerisation; recombinant IgG-like dual targeting molecules, wherein the multiple sides of the molecule each contain the binding fragment or part of the binding fragment of at least two different antibodies; IgG fusion molecules, wherein full length IgG antibodies are fused to an extra Fab fragment or parts of Fab fragment; Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant-domains, Fc-regions or parts thereof; Fab fusion molecules, wherein different Fab- fragments are fused together; ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.
  • IgG fusion molecules wherein full length IgG antibodies are fused to an extra Fab fragment
  • IgG-like molecules with complementary CH3 domains molecules include the Triomab/Quadroma (Trion Pharma/Fresenius Biotech), the Knobs-into-Holes (Genentech), CrossMAbs (Roche) and the electrostatically-matched (Amgen), the LUZ-Y (Genentech), the Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), the Biclonic (Merus) and the DuoBody (Genmab A/S).
  • recombinant IgG-like dual targeting molecules include Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX/Pfizer).
  • DT Dual Targeting
  • Genentech Two-in-one Antibody
  • Cross-linked Mabs Karmanos Cancer Center
  • mAb2 F-Star
  • CovX-body CovX/Pfizer
  • IgG fusion molecules include Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (InnClone/Eli Lilly), Ts2Ab (Medlmmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen pout) and TvAb (Roche).
  • DVD Dual Variable Domain
  • IgG-like Bispecific InnClone/Eli Lilly
  • Ts2Ab Medlmmune/AZ
  • BsAb Zymogenetics
  • HERCULES Biogen personal
  • TvAb Roche
  • Fc fusion molecules include to ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics) and Dual(ScFv).sub.2-Fab (National Research Center for Antibody Medicine— China).
  • Fab fusion bispecific antibodies include F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).
  • ScFv-, diabody- based and domain antibodies include but are not limited to Bispecific T Cell Engager (BiTE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain antibodies.
  • BiTE Bispecific T Cell Engager
  • Tiandab Tandem Diabody
  • DART Dual Affinity Retargeting Technology
  • AIT TCR-like Antibodies
  • AIT ReceptorLogics
  • Human Serum Albumin ScFv Fusion Merrimack
  • COMBODY Epigen Biotech
  • Multispecific antibodies disclosed herein may be generated for example using binding arm exchange (or half molecule exchange) between antibodies by introducing substitutions at the heavy chain CH3 interface in each half molecule to favor multimer formation of antibody half molecules having distinct specificity either in vitro in cell-free environment or using co- expression.
  • the Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociation-association of CH3 domains.
  • the heavy-chain disulfide bonds in the hinge regions of the parent mono specific antibodies are reduced.
  • the resulting free cysteines of one of the parent monospecific antibodies form an inter heavy-chain disulfide bond with cysteine residues of a second parent mono specific antibody molecule and simultaneously CH3 domains of the parent antibodies release and reform by dissociation-association.
  • the CH3 domains of the Fab arms may be engineered to favor heterodimerization over homodimerization.
  • the resulting product is a bispecific antibody having two Fab arms or half molecules which each bind a distinct epitope, i.e. an epitope on CD33 and an epitope on CD3.
  • Homodimerization refers to an interaction of two heavy chains having identical CH3 amino acid sequences.
  • Homodimer refers to an antibody having two heavy chains with identical CH3 amino acid sequences.
  • Heterodimerization refers to an interaction of two heavy chains having non-identical CH3 amino acid sequences.
  • Heterodimer as used herein refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.
  • the "knob-into-hole” strategy may be used to generate full length multispecific antibodies. Briefly, selected amino acids forming the interface of the CH3 domains in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into a heavy chain of an antibody specifically binding a first antigen and an amino acid with a large side chain (knob) is introduced into a heavy chain of an antibody specifically binding a second antigen.
  • a heterodimer is formed as a result of the preferential interaction of the heavy chain with a "hole” with the heavy chain with a "knob".
  • Exemplary CH3 substitution pairs forming a knob and a hole are (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): T366Y/F405A, T366W/ F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V. Numbering here is EU numbering.
  • heterodimerization may be promoted by the following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): L351 Y_F405 AY407V/T394W, T366I K392M_T394W/F405 A_Y407V, T366L K392M_T394W/F405 A_Y407V, L351 Y_Y407A/T366 A K409F, L351Y_Y407A/T366V K409F Y407A/T366A_K409F, or T350V_L351Y_F405A Y407V/T350V_T366L_K392L_T394W as described in U.S. Pat. Publ. No. US2012/0149876 or U.S. Pat. Publ. No. US2013/0195849. Numbering here is
  • multispecific antibodies provided herein may be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of at least two mono specific homodimeric antibodies and forming the multispecific heterodimeric antibody from at least two parent monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in Inti. Pat. Publ. No. W02011/131746.
  • the first antibody e.g., CD33 antibody
  • the second antibody e.g., V62 antibody
  • the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange.
  • the incubation conditions may optimally be restored to non-reducing conditions.
  • Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), L-cysteine and betamercaptoethanol, preferably a reducing agent selected from the group consisting of: 2- mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine.
  • the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, comprising the amino sequences of: a. SEQ ID NOs: 12, 13, 14, 15, 16, and 17, respectively; b. SEQ ID NOs: 18, 19, 14, 15, 16, and 17, respectively; c.
  • the CD33 antibody or antigen-binding fragment thereof comprise a VH comprising an amino acid sequence substantially the same as, or identical to, SEQ ID NO: 42 or 44 and a VL comprising an amino acid sequence substantially the same as, or identical to, SEQ ID NO: 43 or 45; and the V62 antibody or antigen-binding fragment thereof comprises a VHH comprising an amino acid sequence substantially the same as, or identical to, SEQ ID NO: 87.
  • the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino sequences of: a. SEQ ID NOs: 46, 47, and 48, respectively; b. SEQ ID NOs: 49, 50, and 48, respectively; c. SEQ ID NOs: 51, 52, and 48, respectively; d. SEQ ID NOs: 53, 54, and 76, respectively; e. SEQ ID NOs: 53, 54, and 111, respectively; f. SEQ ID NOs: 55, 56, and 57, respectively; g. SEQ ID NOs: 58, 59, and 57, respectively; h.
  • the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, comprising the amino sequences of: SEQ ID NOs: 46, 47, and 48, respectively; and the V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: SEQ ID NOs: 77, 78, and 79, respectively.
  • the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, comprising the amino sequences of: SEQ ID NOs: 49, 50, and 48, respectively; and the V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: SEQ ID NOs: 80, 81, and 79, respectively.
  • the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, comprising the amino sequences of: SEQ ID NOs: 51, 52, and 48, respectively; and the V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: SEQ ID NOs: 82, 83, and 79, respectively.
  • the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, comprising the amino sequences of: SEQ ID NOs: 53, 54, and 76, respectively; and the V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: SEQ ID NOs: 84, 85, and 86, respectively.
  • the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, comprising the amino sequences of: SEQ ID NOs: 53, 54, and 111, respectively; and the V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: SEQ ID NOs: 84, 85, and 86, respectively.
  • the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, comprising the amino sequences of: SEQ ID NOs: 55, 56, and 57, respectively; and the V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: SEQ ID NOs: 77, 78, and 79, respectively.
  • the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, comprising the amino sequences of: SEQ ID NOs: 58, 59, and 57, respectively; and the V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: SEQ ID NOs: 80, 81, and 79, respectively.
  • the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, comprising the amino sequences of: SEQ ID NOs: 60, 61, and 57, respectively; and the V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: SEQ ID NOs: 82, 83, and 79, respectively.
  • the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, comprising the amino sequences of: SEQ ID NOs: 62, 63, and 64, respectively; and the V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: SEQ ID NOs: 84, 85, and 86, respectively.
  • the CD33 antibody or antigen-binding fragment thereof comprise a VHH comprising an amino acid sequence substantially the same as, or identical to, SEQ ID NO: 65 or 66; and the V62 antibody or antigen-binding fragment thereof comprises a VHH comprising an amino acid sequence substantially the same as, or identical to, SEQ ID NO: 87.
  • the CD33/ V62 multispecific antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:42, and a VL comprising the amino acid sequence of SEQ ID NO: 43; and a VHH comprising the amino acid sequence of SEQ ID NO: 87.
  • the CD33/ V62 multispecific antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:44, and a VL comprising the amino acid sequence of SEQ ID NO: 45; and a VHH comprising the amino acid sequence of SEQ ID NO: 87.
  • the CD33/ V62 multispecific antibody or antigen-binding fragment thereof comprises a VHH comprising the amino acid sequence of SEQ ID NO: 65; and a VHH comprising the amino acid sequence of SEQ ID NO: 87.
  • the CD33/ V62 multispecific antibody or antigen-binding fragment thereof comprises a VHH comprising the amino acid sequence of SEQ ID NO:66; and a VHH comprising the amino acid sequence of SEQ ID NO: 87.
  • the CD33/ V62 multispecific antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:67; and a heavy chain comprising the amino acid sequence of SEQ ID NO:70, and a light chain comprising the amino acid sequence of SEQ ID NO:71.
  • the CD33/ V62 multispecific antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 67; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 72, and a light chain comprising the amino acid sequence of SEQ ID NO:73
  • the CD33/ V62 multispecific antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:67; and a heavy chain comprising the amino acid sequence of SEQ ID NO:68.
  • the CD33/ V62 multispecific antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:74; and a heavy chain comprising the amino acid sequence of SEQ ID NO:75.
  • the CD33/ V62 multispecific antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:67; and a heavy chain comprising the amino acid sequence of SEQ ID NO:68.
  • the CD33/ V62 multispecific antibodies or antigen-binding fragments thereof bind the C2 domain of CD33.
  • the CD33/ V62 multispecific antibodies or antigen-binding fragments thereof bind the V domain of CD33.
  • the CD33/ V62 multispecific antibodies or antigen-binding fragments thereof bind the mCD33.
  • the CD33/ V62 multispecific antibodies or antigen-binding fragments thereof do not or do not substantially bind sCD33.
  • the CD33/V62 multispecific antibody comprises:
  • SEQ ID NOs: 53, 54, and 111 respectively; and a V62 antibody or the antigen-binding fragment thereof comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: f. SEQ ID NOs: 77, 78, and 79, respectively; g. SEQ ID NOs: 80, 81, and 79, respectively; h. SEQ ID NOs: 82, 83, and 79, respectively; or i. SEQ ID NOs: 84, 85, and 86, respectively; and/or
  • (C) a heavy chain comprising the amino acid sequence of SEQ ID NO: 67 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 68.
  • Variants of the CD33 antibodies and/or the CD33/ V62 multispecific antibodies are intended to be encompassed by the present disclosure. Variations may be a mutation/substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the original antibody or polypeptide. Sites of interest for substitutional mutagenesis include the CDRs and FRs.
  • Amino acid mutations can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid mutations. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids.
  • the mutation, deletion, or insertion includes fewer than 25 amino acid modifications, fewer than 20 amino acid modifications, fewer than 15 amino acid modifications, fewer than 10 amino acid modifications, fewer than 5 amino acid modifications, fewer than 4 amino acid modifications, fewer than 3 amino acid modifications, or fewer than 2 amino acid modifications relative to the original molecule.
  • an amino acid sequence substantially the same as includes molecules having the amino acid sequence (original molecule) with fewer than 25 amino acid modifications, fewer than 20 amino acid modifications, fewer than 15 amino acid modifications, fewer than 10 amino acid modifications, fewer than 5 amino acid modifications, fewer than 4 amino acid modifications, fewer than 3 amino acid modifications, or fewer than 2 amino acid modifications relative to the original molecule.
  • a molecule e.g. polypeptide
  • an amino acid sequence substantially the same as” a reference amino acid sequence has 5, 4, 3, 2 or 1 amino acid substitutions relative to the reference amino acid sequence.
  • the mutation/substitution is a conservative amino acid mutation/substitution made at one or more predicted non-essential amino acid residues. The variation allowed may be determined by systematically making insertions, deletions, or mutations/substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the parental antibodies.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Antibodies generated by conservative amino acid substitutions are included in the present disclosure.
  • a conservative amino acid substitution an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
  • families of amino acid residues having side chains with similar charges have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • substitution mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed and the activity of the protein can be determined.
  • Conservative (e.g., within an amino acid group with similar properties and/or side chains) substitutions may be made, so as to maintain or not significantly change the properties.
  • Amino acids may be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His(H).
  • Naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, for example, with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and to prevent aberrant crosslinking.
  • Non-conservative mutations will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
  • a parent antibody e.g., a humanized or human antibody.
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
  • Alterations may be made in CDRs, e.g, to improve antibody affinity. Such alterations may be made in CDR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant antibody or fragment thereof being tested for binding affinity.
  • CDR “hotspots” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)
  • SDRs a-CDRs
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. More detailed description regarding affinity maturation is provided in the section below.
  • substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • binding affinity may be made in CDRs.
  • each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells, Science, 244:1081-1085 (1989).
  • a residue or group of target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution.
  • Variants may be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • the variations can be made using methods known in the art such as oligonucleotide- mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
  • Site-directed mutagenesis see, e.g., Carter, Biochem J. 237: 1-7 (1986); and Zoller etal., Nucl. Acids Res. 10:6487-500 (1982)
  • cassette mutagenesis see, e.g., Wells et al., Gene 34:315-23 (1985)
  • other known techniques can be performed on the cloned DNA to produce the antibody variant DNA.
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody, or the antigen-binding fragment thereof is an IgG.
  • the antibody, or antigen-binding fragment thereof is an IgGl, IgG2, IgG3, or IgG4 isotype.
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody, or antigenbinding fragment thereof is an IgGl isotype, or comprises a human IgGl Fc domain.
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody, or antigen-binding fragment thereof comprises knob-into-hole (KiH) substitution in the human IgGl Fc domain.
  • One human IgGl Fc domain comprises an T366W substitution (the “knob” Fc domain), while the other human IgGl Fc domain comprises one or more mutations selected from T366S, L368A, and Y407V per the EU numbering (the “hole” Fc domain).
  • one human IgGl Fc domain comprises an T366W substitution (the “knob” Fc domain), while the other human IgGl Fc domain comprises the triple mutations T366S/L368A/Y407V per the EU numbering (the “hole” Fc domain).
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody, or antigen-binding fragment thereof comprises one or more mutations selected from L234A, L235A, and D265S, per the EU numbering, in the human IgGl Fc domain.
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody, or antigen-binding fragment thereof comprises the triple mutations L234A/L235A/D265S in the Fc domain or Fc region.
  • these mutations, known as AAS mutations, in the human IgGl Fc domain are known to limit or abolish antibody interactions with Fc receptors.
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody, or antigen-binding fragment thereof comprises mutations H435R and/or Y436F in the human IgGl Fc domain. In some embodiments, these substitutions in the Fc region are known to disrupt protein A binding of monomeric and homodimerized of “hole” human IgGl Fc domains.
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody, or antigen-binding fragment thereof comprises the triple mutation M252Y/S254T/T256E in the human IgGl Fc domain.
  • these mutations known as YTE mutations, in the human IgGl Fc domain have been shown to increase the serum half-life of antibodies compared to wild-type versions of the same antibody.
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody, or antigen-binding fragment thereof comprises one or more mutations selected from L234A, L235A, and D265S, per the EU numbering, in the human IgGl Fc domain.
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody, or antigen-binding fragment thereof comprises H435R and Y436F substitution, per the EU numbering, in at least one Fc domain.
  • these mutations known as RF mutations, facilitate purification of the desired multispecific molecule.
  • Another species with approximately 25% of the total expression product carries no mutations in their heavy chains and thus, will strongly bind to Protein-A.
  • the main species being about 50% is the desired bispecific comprising the RF double mutation in just one chain, which displays medium affinity to the resin.
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody, or antigen-binding fragment thereof comprises the knob and hole mutations, the AAS mutations and the YTE mutations.
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody, or antigen-binding fragment thereof further comprises the RF mutation in at least one Fc domain.
  • sCD33 levels in a sample may be determined using immunoblots (e.g., Western blots), mass spectrometry, flow cytometry, an immunoassay (e.g., a SIMOA assay from Quanterix, see, e.g., the website: www.quanterix.com/simoa-technology/), a proximity extension assay (e.g., an assay from Olink, see, e.g., the website: www.olink.com/our- platform/our-pea- technology), an electrochemiluminescence-based assay (e.g., an assay from Meso Scale Diagnostics, see, e.g., the website: www.mesoscale.com/en/technical_resources
  • sCD33 levels in a sample are determined using a mass spectrometry-based method.
  • sCD33 levels in a sample may be determined using a Quantitative Liquid Chromatography Multiple-Reaction Monitoring Mass Spectrometry (LCMRM/MS) assay.
  • LCMRM/MS Quantitative Liquid Chromatography Multiple-Reaction Monitoring Mass Spectrometry
  • Determination of sCD33 levels using a Quantitative Liquid Chromatography Multiple-Reaction Monitoring Mass Spectrometry (LCMRM/MS) assay may comprise one or more, or all, of the following steps, as described in Example 5 herein: (a) preparation of calibration standards (e.g., calibration standards may be prepared at levels, e.g., eight levels, covering the assay range by spiking [i.e., adding] CD33 protein, e.g., recombinant CD33 protein, in surrogate matrix, e.g., surrogate matrix for the sample treated with the CD33 antibody and/or the CD33/ V62 multispecific antibody [e.g., a surrogate matrix for cerebrospinal fluid, whole blood, serum or plasma, such as a suitable buffer solution]); (b) preparation of quality control (QC) samples (e.g., QC samples may be prepared at levels, e.g., three levels, covering the assay range by spiking CD33 protein, e.g
  • samples from one or more individuals treated with anti-CD33 antibody e.g., cerebrospinal fluid samples, or blood samples such as a whole blood, serum or plasma samples;
  • the CD33 antibody and/or the CD33/ V62 multispecific antibody or the antigen binding fragment thereof does not bind to sCD33 (or to its surrogate, rCD33).
  • the meaning of the expression “does not bind to sCD33” encompasses absence of significant binding to sCD33 (or to its surrogate, rCD33). Absence of significant binding to sCD33, (e.g. “does not significantly bind sCD33” as used herein) is understood in accordance with its ordinary meaning in the field, and can be assessed by any means, which the skilled person finds appropriate.
  • sCD33 as a free form in human serum is not significantly decreased, or not decreased by more than 50% (e.g., not decreased by more than 40%) [or is decreased by less than 50% (e.g., is decreased by less than 40%)] once placed in contact with 0.2 nM of the CD33 antibody and/or the CD33/ V 6 2 multispecific antibody or the antigen-binding fragment thereof.
  • the antibody and/or the CD33/ V 6 2 multispecific antibody or antigenbinding fragment thereof when placed in contact with a biological sample containing sCD33 (such as a serum sample of an AML human patient), or in a biological sample, which has been spiked with 10 ng/ml of rCD33, the antibody and/or the CD33/ V 6 2 multispecific antibody or antigenbinding fragment thereof (e.g., at a concentration of 0.2 nM) does not significantly decrease the level of free form of sCD33, or at least does not decrease it by more than 50% (e.g., does not decrease it by more than 40%)
  • the T-cell cytotoxicity EC50 value of the antibody and/or the CD33/ V 6 2 multispecific antibody or antigen-binding fragment thereof on tumor cells such as the THP-1 cell line is not significantly impacted by the presence of 10 ng/mL of rCD33 (i.e., the same or substantially the same EC50 value in the absence or in the presence of 10 ng/mL rCD33; cf. T cell cytotoxicity assay of example 9).
  • the CD33 antibody and/or the CD33/ V 6 2 multispecific antibody or antigen-binding fragment thereof has an affinity for sCD33 or rCD33 lower than an antibody that is not mCD33 -specific (i.e., which binds sCD33 or rCD33).
  • the CD33 antibody and/or antibody and/or the CD33/ V 6 2 multispecific antibody or antigen-binding fragment thereof has greater affinity for binding mCD33 than sCD33.
  • the CD33 antibody and/or the CD33/ V62 multispecific antibodies described herein include humanized antibodies.
  • Humanized antibodies such as the humanized antibodies disclosed herein can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400; International publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos.
  • antibodies provided herein can be humanized antibodies that bind to CD33 or CD33 and V62, including human CD33 and human V62.
  • Various methods for humanizing non-human antibodies are known in the art.
  • a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • Humanization may be performed, for example, following the method of Jones et al., Nature 321 :522-25 (1986); Riechmann et al., Nature 332:323-27 (1988); and Verhoeyen et al., Science 239: 1534-36 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody.
  • the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the CDRs of the parent non-human antibody are grafted onto a human antibody framework.
  • CDR grafting in which the amino acid sequences of the CDRs of the parent non-human antibody are grafted onto a human antibody framework.
  • variable domains to be used in making the humanized antibodies can be important to reduce antigenicity.
  • sequence of the variable domain of a non-human antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence that is closest to that of the non-human antibody may be selected as the human framework for the humanized antibody (Sims etal., J. Immunol. 151:2296-308 (1993); and Chothia etal., J. Mol. Biol. 196:901-17 (1987)).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies (Carter etal., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); and Presta etal., J. Immunol. 151 :2623-32 (1993)).
  • the framework is derived from the consensus sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) and VH subgroup III (VHIII).
  • VL6I VL6 subgroup I
  • VHIII VH subgroup III
  • human germline genes are used as the source of the framework regions.
  • FR homology is irrelevant.
  • the method consists of comparison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., J. Immunol. 169:1119-25 (2002)).
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13:819-24 (2002)), Modeller (Sali and Blundell, J. Mol. Biol.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
  • HSC Human String Content
  • Antibody variants may be isolated from phage, ribosome, and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol. 23: 1105-16 (2005); Dufner etal., Trends Biotechnol. 24:523-29 (2006); Feldhaus et al., Nat. Biotechnol. 21: 163-70 (2003); and Schlapschy et al. , Protein Eng. Des. Sei. 17:847-60 (2004)).
  • residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224:487-99 (1992)), or from the more limited set of target residues identified by Baca et al. J. Biol. Chem. 272:10678-84 (1997).
  • FR shuffling whole FRs are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall’Acqua etal., Methods 36:43- 60 (2005)).
  • a one-step FR shuffling process may be used. Such a process has been shown to be efficient, as the resulting antibodies exhibited improved biochemical and physicochemical properties including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60 (2007)).
  • the “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. This methodology typically results in epitope retention and identification of antibodies from multiple subclasses with distinct human V-segment CDRs.
  • the “human engineering” method involves altering a non-human antibody or antibody fragment by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies.
  • the technique involves classifying amino acid residues of a non-human antibody as “low risk,” “moderate risk,” or “high risk” residues. The classification is performed using a global risk/reward calculation that evaluates the predicted benefits of making particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody’s folding.
  • the particular human amino acid residue to be substituted at a given position (e.g., low or moderate risk) of a non-human antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody’s variable regions with the corresponding region of a specific or consensus human antibody sequence.
  • the amino acid residues at low or moderate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment.
  • a composite human antibody can be generated using, for example, Composite Human AntibodyTM technology (Antitope Ltd., Cambridge, United Kingdom).
  • variable region sequences are designed from fragments of multiple human antibody variable region sequences in a manner that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibody.
  • a deimmunized antibody is an antibody in which T-cell epitopes have been removed. Methods for making deimmunized antibodies have been described. See, e.g., Jones etal., Methods Mol Biol. 525:405-23 (2009), xiv, and De Groot et al., Cell. Immunol. 244: 148- 153(2006)).
  • Deimmunized antibodies comprise T-cell epitope-depleted variable regions and human constant regions. Briefly, variable regions of an antibody are cloned and T-cell epitopes are subsequently identified by testing overlapping peptides derived from the variable regions of the antibody in a T cell proliferation assay.
  • T cell epitopes are identified via in silica methods to identify peptide binding to human MHC class II. Mutations are introduced in the variable regions to abrogate binding to human MHC class II. Mutated variable regions are then utilized to generate the deimmunized antibody.
  • the present disclosure relates to synthetic polynucleotides encoding a CD33 antibody or antigen-binding fragment thereof described herein.
  • the present disclosure relates to one or more isolated nucleic acid encoding a CD33 antibody or antigen-binding fragment thereof, or a CD33/ V62 multispecific antibody or antigen-binding fragment thereof described herein.
  • Polynucleotide or “nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • a “polynucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase polynucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • the nucleotide sequence encoding for the anti-CD33 heavy chain of GD33B273 comprises:
  • GD33B273 comprises:
  • the present disclosure relates to one or more vectors comprising one or more synthetic polynucleotide sequences encoding a CD33 antibody, or antigen-binding fragment thereof of, disclosed herein.
  • the present disclosure relates to one or more vectors comprising one or more synthetic polynucleotide sequences encoding a CD33/ V62 multispecific antibody, or antigen-binding fragment thereof of, disclosed herein.
  • vector refers to a substance that is used to carry or include a polynucleotide sequence, including for example, a polynucleotide sequence encoding an antibody or antigen-binding fragment as described herein, in order to introduce a polynucleotide sequence into a host cell.
  • Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media.
  • Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art.
  • both polynucleotide molecules can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding polynucleotides can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • the introduction of polynucleotide molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced polynucleotide sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • the present disclosure relates to a host cell comprising a synthetic polynucleotide encoding a CD33 antibody and/or a CD33/ V62 multispecific antibody, or an antigen-binding fragment thereof, described herein.
  • the term “host cell” as used herein refers to a particular subject cell that may be transfected with a polynucleotide molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the polynucleotide molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the polynucleotide molecule into the host cell genome.
  • transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • Any host cell known to those skilled in the art in view of the present disclosure can be used for recombinant expression of antibodies or antigen-binding fragments thereof, disclosed herein.
  • Mammalian, fungal, bacterial or plant cells are particularly suitable. Mammalian cells, including human, monkey, rat, mouse, guinea pig are routinely used and suitable. Fungal cells including yeast (Saccharomyces cerevisiae), Pichia pastoris, Neurospora crassa could also be used. Bacterial, such as E. coli can also be used.
  • mammalian cell lines such as CHO, HEK and NS0 are the preferred and most commonly used expression system for recombinant antibody production (e.g., for clinical use) are most conveniently expressed in the often because they easily allow post-translational modifications and can produce antibodies with human-like glycosylation patterns.
  • the host cells are E. coli TGI or BL21 cells (for expression of, e.g., an scFv or Fab antibody), CHO-DG44 or CHO-K1 cells or HEK293 cells or NSO cells (for expression of, e.g., a full-length IgG antibody).
  • the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed.
  • the present disclosure relates to a method of producing a CD33 antibody, or antigen-binding fragment thereof, described herein, comprising culturing a cell comprising one or more polynucleotide sequences (molecules) encoding the CD33 antibody or antigen-binding fragment thereof under conditions to produce a CD33 antibody, or antigenbinding fragment thereof, disclosed herein, and recovering the CD33 antibody, or antigenbinding fragment thereof, from the cell or cell culture (e.g., from the supernatant).
  • the present disclosure relates to a method of producing a CD33/ V62 multispecific antibody, or antigen-binding fragment thereof, disclosed herein, comprising culturing a cell comprising one or more polynucleotide sequences (molecules) encoding the bispecific antibody or, antigen-binding fragment thereof, under conditions to produce a CD33/ V 62 multispecific antibody, or antigen-binding fragment thereof, disclosed herein, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant).
  • Expressed antibodies or antigen-binding fragments thereof can be harvested from the cells and purified according to conventional techniques known in the art and as described herein.
  • the present disclosure relates to a pharmaceutical composition comprising a CD33 antibody, or antigen-binding fragment thereof, described herein and a pharmaceutically acceptable carrier.
  • the present disclosure relates to a pharmaceutical composition comprising a CD33/ V62 multispecific antibody, or antigen-binding fragment thereof, described herein and a pharmaceutically acceptable carrier.
  • pharmaceutical composition as used herein means a product comprising an antibody described herein together with a pharmaceutically acceptable carrier. Antibodies of the present disclosure and compositions comprising them are also useful in the manufacture of a medicament for therapeutic applications mentioned herein.
  • the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application.
  • the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effectiveness of a composition according to the present disclosure or the biological activity of a composition according to the present disclosure. According to particular embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in an antibody pharmaceutical composition can be used.
  • the present disclosure relates to a method of producing a pharmaceutical composition comprising a CD33 antibody or antigen-binding fragment thereof, described herein, comprising combining a CD33 antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
  • the present disclosure relates to a method of producing a pharmaceutical composition comprising a CD33/ V62 multispecific antibody, or antigen-binding fragment thereof, described herein, comprising combining a CD33/ V62 multispecific antibody or antigenbinding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
  • the present disclosure relates to a method of targeting CD33 on a cancer cell surface in a subject, the method comprising administering to the subject a CD33 antibody or antigen binding fragment thereof that specifically binds CD33 or a CD33/ V62 multispecific antibody or antigen binding fragment thereof or a pharmaceutical composition comprising either, as described herein.
  • the CD33 antibody, or antigen binding fragment thereof, or CD33/ V62 antibody, or antigen binding fragments thereof or a pharmaceutical composition comprising either are administered to the subject (e.g. human) in need thereof in a therapeutically effective amount.
  • the functional activity of antibodies and antigen-binding fragments thereof that bind CD33 can be characterized by methods known in the art and as described herein.
  • Methods for characterizing antibodies and antigen-binding fragments thereof that bind CD33 include, but are not limited to, affinity and specificity assays including Biacore, ELISA, and OctetRed analysis; binding assays to detect the binding of antibodies to CD33 on cancer cells by FACS.
  • the methods for characterizing antibodies and antigen-binding fragments thereof that bind CD33 include those described below.
  • the present disclosure relates to a method of treating a hematological cancer in a subject in need thereof, comprising administering to the subject a CD33 antibody, a CD33/ V62 multispecific antibody or antigen binding fragment thereof that specifically binds CD33 or a pharmaceutical composition of the present disclosure.
  • the hematologic cancer can, for example, be a leukemia, a lymphoma, and a myeloma.
  • the hematologic cancer can be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low or high risk), acute lymphocytic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • ALL acute lymphocytic leukemia
  • ALL diffuse large B-cell lymphoma
  • CML chronic myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic cell neoplasm
  • the present disclosure relates to a CD33 antibody, a CD33/ V6 2 multispecific antibody or antigen binding fragment thereof or a pharmaceutical composition comprising either, as described herein, for use in therapy.
  • the present disclosure relates to a CD33 antibody, a CD33/ V6 2 multispecific antibody or antigen binding fragment thereof or a pharmaceutical composition comprising either, as described herein, for use in the treatment of a hematological cancer.
  • the present disclosure relates to use of a CD33 antibody, a CD33/ V62 multispecific antibody or antigen binding fragment thereof or a pharmaceutical composition comprising either, as described herein, in the manufacture of a medicament for treating a hematological cancer.
  • the hematologic cancer can, for example, be a leukemia, a lymphoma, and a myeloma.
  • the hematologic cancer can be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS, low or high risk), acute lymphocytic leukemia (ALL, including all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • ALL acute lymphocytic leukemia
  • ALL diffuse large B-cell lymphoma
  • CML chronic myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic cell neoplasm
  • the subject with the hematologic cancer e.g. AML or MDS, lacks the single nucleotide polymorphism (SNP) rsl2459419 in CD33 gene.
  • SNP single nucleotide polymorphism
  • the subject with the hematologic cancer e.g. AML or MDS
  • SNP single nucleotide polymorphism
  • the pharmaceutical composition comprises a therapeutically effective amount of a CD33 antibody, a CD33/ V62 multispecific antibody or antigen-binding fragment(s) thereof.
  • therapeutically effective amount refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject.
  • a therapeutically effective amount means an amount of the CD33 antibody, a CD33/ V62 multispecific, or antigen-binding fragment thereof, that modulates an immune response in a subject in need thereof.
  • a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the
  • OS overall survival
  • PFS progression free survival
  • the therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • compositions described herein are formulated to be suitable for the intended route of administration to a subject.
  • the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.
  • the terms “treat,” “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to a cancer, which is not necessarily discernible in the subject, but can be discernible in the subject.
  • the terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as a tumor or more preferably a cancer.
  • “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.
  • the invention is generally disclosed herein using affirmative language to describe the numerous embodiments.
  • the invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis.
  • the invention is generally not expressed herein in terms of what the invention does not include, aspects that are not expressly included in the invention are nevertheless disclosed herein.
  • Embodiment 1 is an antibody or an antigen-binding fragment specifically binding to membrane-bound human CD 33 (mCD33).
  • Embodiment 2 is the antibody or the antigen-binding fragment thereof of embodiment 1 comprising: a. a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 12, 13, and 14, respectively; and a light chain complementarity determining region 1 (CDR1), a light chain complementarity determining region 2 (CDR2), and a light chain complementarity determining region 3 (CDR3) comprising the amino acid sequences of SEQ ID NO: 15, 16, and 17, respectively; b.
  • CDR1 heavy chain complementarity determining region 1
  • CDR2 heavy chain complementarity determining region 2
  • CDR3 heavy chain complementarity determining region 3
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 18, 19, and 14, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 15, 16, and 17, respectively; c. a heavy chain CDR1 , a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 20, 21, and 14, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 15, 16, and 17, respectively; or d.
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 22, 23, and 24, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 25, 26, and 17, respectively.
  • Embodiment 3 is the antibody or antigen-binding fragment thereof of embodiment 1 comprising: a. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 27, 28, and 29, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 30, 31, and 32, respectively; b.
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 33, 34, and 29, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 30, 31, and 32, respectively; c. a heavy chain CDR1 , a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 35, 36, and 29, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 30, 31, and 32, respectively; or d.
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 37, 38, and 39, respectively; and a light chain CDR1 , a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences of SEQ ID NO: 40, 41, and 32, respectively.
  • Embodiment 4 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 3, comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 42 or 44.
  • VH heavy chain variable region
  • Embodiment 5 is the antibody or the antigen-binding fragment thereof of embodiment 1 to 4, comprising a VH comprising SEQ ID NO: 42 or 44.
  • Embodiment 6 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 5, comprising a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43 or 45.
  • VL light chain variable region
  • Embodiment 7 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 6, comprising a VL comprising SEQ ID NO: 43 or 45.
  • Embodiment 8 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 7, comprising a VH comprising SEQ ID NO: 42, and a VL comprising SEQ ID NO: 43.
  • Embodiment 9 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 8, comprising a VH comprising SEQ ID NO: 44, and a VL comprising SEQ ID NO: 45.
  • Embodiment 10 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises an immunoglobulin (IgG) Fc domain.
  • IgG immunoglobulin
  • Embodiment 11 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein the IgGFc domain is a human IgGl Fc domain.
  • Embodiment 12 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 11, wherein the human IgGl Fc domain comprises one or more mutations selected from T366S, L368A, T366W and Y407V per the EU numbering system.
  • Embodiment 13 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 12, wherein the human IgGl Fc domain further comprises one or more mutations selected from L234A, L235A, and D265S per the EU numbering system.
  • Embodiment 14 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 13, wherein the human IgGl Fc domain further comprises mutations H435R and/or Y436F per the EU numbering system.
  • Embodiment 15 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 14, wherein the human IgGl Fc domain further comprises the triple mutation M252Y/S254T/T256E per the EU numbering system.
  • Embodiment 16 is the antibody or the antigen-binding fragment thereof of embodiment 1 , wherein the antibody is a heavy chain only antibody.
  • Embodiment 17 is the antibody or the antigen-binding fragment thereof of embodiment 1 , wherein the antigen-binding fragment thereof is a single heavy chain variable region (VHH).
  • Embodiment 18 is the antibody or the antigen-binding fragment thereof of embodiment 16 or embodiment 17 comprising: a. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 46, 47, and 48, respectively; b.
  • a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 49, 50, and 48, respectively; c. a heavy chain CDR1 , a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 51, 52, and 48, respectively; d. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 53, 54, and 76, respectively; or e. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 53, 54, and 111, respectively.
  • Embodiment 19 is the antibody or the antigen-binding fragment thereof of embodiment 16 or embodiment 17 comprising: a. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 55, 56, and 57, respectively; b. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 58, 59, and 57, respectively; c. a heavy chain CDR1 , a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 60, 61, and 57, respectively; or d. a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences of SEQ ID NO: 62, 63, and 64, respectively.
  • Embodiment 22 is the antibody or the antigen-binding fragment thereof of any one of embodiments 16 to 21, particularly embodiment 19, comprising a VHH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 66.
  • Embodiment 23 is the antibody or the antigen-binding fragment thereof of embodiment 22, comprising a VHH comprising SEQ ID NO: 66.
  • Embodiment 24 is the antibody or the antigen-binding fragment thereof of any one of embodiments 16 to 23, wherein the antibody or antigen-binding fragment thereof comprises an immunoglobulin (IgG) Fc domain.
  • IgG immunoglobulin
  • Embodiment 25 is the antibody or the antigen-binding fragment thereof of any one of embodiments 16 to 24, wherein the IgG Fc domain is a human IgGl Fc domain.
  • Embodiment 26 is the antibody or the antigen-binding fragment thereof of any one of embodiments 16 to 25, wherein the human IgGl Fc domain comprises one or more mutations selected from T366S, L368A, T366W and Y407V per the EU numbering system.
  • Embodiment 27 is the antibody or the antigen-binding fragment thereof of any one of embodiments 16 to 26, wherein the human IgGl Fc domain further comprises one or more mutations selected from L234A, L235A, and D265S per the EU numbering system.
  • Embodiment 28 is the antibody or the antigen-binding fragment thereof of any one of embodiments 16 to 27, wherein the human IgGl Fc domain further comprises mutations H435R and/or Y436F per the EU numbering system.
  • Embodiment 29 is the antibody or the antigen-binding fragment thereof of any one of embodiments 16 to 28, wherein the human IgGl Fc domain further comprises the triple mutation M252Y/S254T/T256E per the EU numbering system.
  • Embodiment 30 is the antibody or the antigen-binding fragment of any one of embodiments 1 to 29, wherein the antibody or antigen-binding fragment binds the C2 domain of human CD33.
  • Embodiment 31 is the antibody or the antigen-binding fragment of any one of embodiments 1 to 30, wherein the antibody or the antigen-binding fragment thereof does not bind to soluble human CD33 (sCD33).
  • Embodiment 32 is the antibody or antigen-binding fragment thereof of any one of embodiments 1 to 31 , wherein the antibody or the antigen-binding fragment thereof is chimeric.
  • Embodiment 33 is the antibody or the antigen-binding fragment thereof of any one of embodiments 1 to 32, wherein the antibody or the antigen-binding fragment thereof is human or humanized.
  • Embodiment 34 is a synthetic polynucleotide encoding the antibody or the antigenbinding fragment thereof of any one of embodiments 1 to 33.
  • Embodiment 35 is a vector comprising the synthetic polynucleotide of embodiment 34.
  • Embodiment 36 is a host cell comprising the vector of embodiment 35.
  • Embodiment 37 is a pharmaceutical composition, comprising the antibody or the antigenbinding fragment thereof of any one of embodiments 1 to 33 and a pharmaceutically acceptable carrier.
  • Embodiment 38 is a method of treating a hematological cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of embodiment 36.
  • Embodiment 39 is the method of embodiment 38, wherein the hematological cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • CML chronic myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic cell neoplasm
  • Embodiment 40 is a method of producing the antibody or the antigen-binding fragment thereof of any one of embodiments 1-33, comprising culturing a cell comprising a polynucleotide encoding the antibody or the antigen-binding fragment thereof under conditions to produce the antibody or the antigen-binding fragment, and recovering the antibody or the antigen-binding fragment thereof from the cell or culture.
  • Embodiment 41 is a method of producing a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof of any one of embodiments 1-33, comprising combining the antibody or the antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
  • Embodiment 42 is a CD33/V62 multispecific antibody comprising a CD33 antibody or an antigen-binding fragment thereof and a V62 antibody or an antigen-binding fragment thereof, wherein the CD33 antibody or the antigen-binding fragment thereof specifically binds to mCD33, and wherein the V62 antibody or the antigen-binding fragment thereof specifically binds to the V82 chain of the human Vy9V82 T cell receptor.
  • Embodiment 43 the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of embodiment 42, wherein the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, comprising the amino sequences of: a. SEQ ID NOs: 12, 13, 14, 15, 16, and 17, respectively; b. SEQ ID NOs: 18, 19, 14, 15, 16, and 17, respectively; c. SEQ ID NOs: 20, 21, 14, 15, 16, and 17, respectively; or d.
  • V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: e. SEQ ID NOs: 77, 78, and 79, respectively; f. SEQ ID NOs: 80, 81, and 79, respectively; g. SEQ ID NOs: 82, 83, and 79, respectively; or h. SEQ ID NOs: 84, 85, and 86, respectively.
  • Embodiment 44 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of embodiment 42, wherein the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, and a light chain CDR3, comprising the amino sequences of: a. SEQ ID NOs: 27, 28, 29, 30, 31, and 32, respectively; b. SEQ ID NOs: 33, 34, 29, 30, 31, and 32, respectively; c. SEQ ID NOs: 35, 36, 29, 30, 31, and 32, respectively; or d.
  • V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: e. SEQ ID NOs: 77, 78, and 79, respectively; f. SEQ ID NOs: 80, 81, and 79, respectively; g. SEQ ID NOs: 82, 83, and 79, respectively; or h. SEQ ID NOs: 84, 85, and 86, respectively.
  • Embodiment 45 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of embodiment 42, wherein the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino sequences of: a. SEQ ID NOs: 46, 47, and 48, respectively; b. SEQ ID NOs: 49, 50, and 48, respectively; c. SEQ ID NOs: 51, 52, and 48, respectively; d. SEQ ID NOs: 53, 54, and 76, respectively; or e. SEQ ID NOs: 53, 54, and 111, respectively.
  • V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: f. SEQ ID NOs: 77, 78, and 79, respectively; g. SEQ ID NOs: 80, 81, and 79, respectively; h. SEQ ID NOs: 82, 83, and 79, respectively; or i. SEQ ID NOs: 84, 85, and 86, respectively.
  • Embodiment 46 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of embodiment 42, wherein the CD33 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino sequences of: a. SEQ ID NOs: 55, 56, and 57, respectively; b. SEQ ID NOs: 58, 59, and 57, respectively; c. SEQ ID NOs: 60, 61, and 57, respectively; or d.
  • V62 antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, comprising the amino acid sequences of: e. SEQ ID NOs: 77, 78, and 79, respectively; f. SEQ ID NOs: 80, 81, and 79, respectively; g. SEQ ID NOs: 82, 83, and 79, respectively; or h. SEQ ID NOs: 84, 85, and 86, respectively.
  • Embodiment 47 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 44, wherein the CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 42 or 44, and a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO:43 or 45; and the V62 antibody or the antigen-binding fragment thereof comprises a heavy chain only variable region (VHH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 87.
  • VH heavy chain variable region
  • VL light chain variable region
  • VHH heavy chain only variable region
  • Embodiment 48 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of embodiment 47 comprising: a. a VH comprising the amino acid sequence of SEQ ID NO:42, and a VL comprising the amino acid sequence of SEQ ID NO: 43; and a VHH comprising the amino acid sequence of SEQ ID NO: 87; or b. a VH comprising the amino acid sequence of SEQ ID NO:44, and a VL comprising the amino acid sequence of SEQ ID NO: 45; and a VHH comprising the amino acid sequence of SEQ ID NO: 87.
  • Embodiment 49 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42, 45 and 46, wherein the CD33 antibody or the antigen-binding fragment thereof comprises a VHH comprising an amino acid sequence at least 95% identical to SEQ ID NO: 65 or 66; and the V62 antibody or the antigen-binding fragment thereof comprises a VHH comprising an amino acid sequence at least 95% identical to SEQ ID NO: 87.
  • Embodiment 50 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of embodiment 49 comprising: a. a VHH comprising the amino acid sequence of SEQ ID NO:65; and a VHH comprising the amino acid sequence of SEQ ID NO: 87; or b. a VHH comprising the amino acid sequence of SEQ ID NO: 66; and a VHH comprising the amino acid sequence of SEQ ID NO: 87.
  • Embodiment 51 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42, 44, 47 and 48, comprising: a. a heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: a. a heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: a. a heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: a heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID
  • Embodiment 51b is the CD33/V52 multispecific antibody or the antigen-binding fragment thereof of embodiment 51 , comprising: a. a heavy chain comprising the amino acid sequence of SEQ ID NO:67; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 70, and a light chain comprising the amino acid sequence of SEQ ID NO:71; b. a heavy chain comprising the amino acid sequence of SEQ ID NO: 67; and a heavy chain comprising the amino acid sequence of SEQ ID NO:72, and a light chain comprising the amino acid sequence of SEQ ID NO:73; c.
  • a heavy chain comprising the amino acid sequence of SEQ ID NO: 67; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 68; d. a heavy chain comprising the amino acid sequence of SEQ ID NO: 74; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 75 or e. a heavy chain comprising the amino acid sequence of SEQ ID NO:91 ; and a heavy chain comprising the amino acid sequence of SEQ ID NO: 92.
  • Embodiment 52 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 51, wherein the multispecific antibody is a bispecific antibody.
  • Embodiment 53 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 52, wherein the CD33/V62 multispecific antibody or the antigen-binding fragment thereof comprises an immunoglobulin (IgG) Fc domain.
  • IgG immunoglobulin
  • Embodiment 54 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 53, wherein the IgG Fc domain is a human IgGl Fc domain.
  • Embodiment 55 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 54, wherein the human IgGl Fc domain comprises one or more mutations selected from T366S, L368A, T366W and Y407V per the EU numbering system.
  • Embodiment 56 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 55, wherein the human IgGl Fc domain further comprises one or more mutations selected from L234A, L235A, and D265S per the EU numbering system.
  • Embodiment 57 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 56, wherein the human IgGl Fc domain further comprises mutations H435R and/or Y436F per the EU numbering system.
  • Embodiment 58 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 57, wherein the human IgGl Fc domain further comprises the triple mutation M252Y/S254T/T256E per the EU numbering system.
  • Embodiment 59 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 58, wherein CD33/V62 multispecific antibody or the antigen-binding fragment thereof binds the C2 domain of human CD33.
  • Embodiment 60 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 59, wherein the CD33/V62 multispecific antibody or the antigen-binding fragment thereof does not bind to sCD33.
  • Embodiment 61 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 60, wherein the CD33/V62 multispecific antibody or the antigen-binding fragment thereof is chimeric.
  • Embodiment 62 is the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 61, wherein the CD33/V62 multispecific antibody or the antigen-binding fragment thereof is human or humanized.
  • Embodiment 63 is a synthetic polynucleotide encoding the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 62.
  • Embodiment 64 is a vector comprising the synthetic polynucleotide of embodiment 63.
  • Embodiment 65 is a host cell comprising the vector of embodiment 64.
  • Embodiment 66 is a pharmaceutical composition, comprising the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42 to 62 and a pharmaceutically acceptable carrier.
  • Embodiment 67 is a method of treating a hematological cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of embodiment 66.
  • Embodiment 68 is the method of embodiment 67, wherein the hematological cancer is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • CML chronic myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic cell neoplasm
  • Embodiment 69 is a method of producing the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42-62, comprising culturing a cell comprising a polynucleotide encoding the CD33/V62 multispecific antibody or the antigenbinding fragment thereof under conditions to produce the CD33/V62 multispecific antibody or the antigen-binding fragment thereof, and recovering the CD33/V62 multispecific antibody or the antigen-binding fragment thereof from the cell or culture.
  • Embodiment 70 is a method of producing a pharmaceutical composition comprising the CD33/V62 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42-62, comprising combining the CD33/V62 multispecific antibody or the antigenbinding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.
  • Embodiment 71 is the CD33/V52 multispecific antibody or the antigen-binding fragment thereof of any one of embodiments 42-62 or the pharmaceutical composition of embodiment 66 for use in treating a hematological cancer in a subject in need thereof, such as a hematological cancer selected from acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML) or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • CML chronic myeloid leukemia
  • BPDCN blastic plasmacytoid dendritic cell neoplasm
  • Expression constructs encoding the human CD33 extracellular domain (ECD) or its subdomains were designed based on the sequence of myeloid cell surface antigen CD33 (Uniprot accession # P20138) and its domain annotation with either 6X His-tag sequence (SEQ ID NO: 246) or as a fusion protein to a C34S variant of human serum albumin (HSA) with a 6X His-tag sequence at the C-terminus.
  • Similar expression constructs encoding CD33 (ECD) or its subdomains from cynomolgus monkey (Macaca fascicularis) were designed based on NCBI Accession # XP_005590138.1. The amino acid sequences of the generated antigens are shown in Table 5.
  • the human and cyno CD33 full-length ECD or sub-domain expression constructs were transfected into Expi293 cells (HEK293 derived), using Expifectamine as per the manufacturer's guidelines. Following incubation for 5 days at 37 °C with 8% C02 on an orbital shaker, cells expressing the target protein were separated via centrifugation. The soluble CD33 proteins bearing his-tags were then purified from the culture media using immobilized metal-ion affinity chromatography (IMAC) facilitated by Ni NTA Sepharose 6 Fast Flow resin (GE Healthcare).
  • IMAC immobilized metal-ion affinity chromatography
  • the production process of anti-CD33 antibodies involved the use of Ablexis transgenic mice technologies. Initially, AlivaMab mice engineered to yield human/mouse immunoglobulins were immunized with recombinant human CD33 protein. Lymphocytes were obtained from secondary lymphoid organs and either fused with FO mouse myeloma cells for hybridoma generation or individually sorted via FACS. Hybridoma supernatants underwent screening using MSD electrochemiluminescence to detect binding to human CD33 ECD-overexpressing HEK cells. Positive samples were further evaluated by FACS for confirmation of binding to the overexpressed CD33 ECD, and confirmed cell binders underwent light chain isotyping through ELISA.
  • the single cell sorting supernatants were screened for binding to recombinant human CD33 protein via MSD electrochemiluminescence. Hits exhibiting the desired binding profile were selected and sequenced for further analysis.
  • the process of V region cloning ensued, involving cDNA synthesis from RNA using the Smarter cDNA synthesis kit, followed by amplification of VH and VL fragments via PCR. Subsequently, the VH and VL genes were cloned directionally into Lonza mother vectors (VH and VL) using the In-Fusion® HD Cloning Kit. Confirmation of complete V-gene fragments was done through Sanger sequencing of mini- prepped DNAs.
  • the subsequent step involved the expression of anti-CD33 antibodies in ExpiCHO-STM cells through transient transfection using purified plasmid DNA encoding the proteins.
  • the cells were maintained in suspension using ExpiCHOTM expression medium under specific environmental conditions.
  • Transfection was conducted using the ExpiF ectamineTM CHO transfection kit, and culture supernatants were harvested after seven days.
  • Protein purification was carried out by loading the filtered cell culture supernatant onto a MabSelect Sure Protein A column, followed by washing and elution steps. Eluted protein fractions were neutralized and pooled, followed by filtration. The quality of the purified protein was assessed using analytical size exclusion HPLC via an Agilent HPLC system.
  • the amino acid sequences of the CDRs and the heavy and light chain variable regions of exemplary antibodies generated through this method are outlined in Table 6 and Table 7, respectively.
  • the polynucleotide sequences are outlined in Table 8.
  • Single heavy chain CD33 antibodies were derived from a VHH phage display campaign.
  • the amino acid sequences of the CDRs and the heavy chain variable regions (VHHs) of exemplary antibodies generated through this method are outlined in Table 9 and Table 10, respectively.
  • the polynucleotide sequences are outlined in Table 11 .
  • Bispecific molecules were developed through the implementation of knob-into-hole mutations, creating heterodimers consisting of a CD33 binder fused to Fc and a V62 binder fused to Fc In these constructs, the CD33 binder is located on the Hole Fc, while the V82 binder is on the Knob Fc.
  • the CD33 VH and human CHI constant region are combined with a hinge on Fc, featuring several mutations: L234A/L235A/D265S_M252Y/S254T/T256E_T366S/L368A/Y407V_H435R/Y436F; and the V82 binder is fused to the hinge and Knob Fc, incorporating the following mutations:
  • the V82 arm may be prepared as described in the Examples section of patent application WO 2015/156673A1 and WO 2023/037333 Al.
  • the V62 binding sequences utilized were derived from WO2023/037333 (see Table 10 and Table 11 below).
  • Table 10 and Table 11 below The art already provides the necessary teachings for the person skilled in the art to prepare the CD33xV82 bispecific antibody of the disclosure, having knowledge of the actual sequences of the two arms, as provided herein.
  • the AAS mutations (L234A/L235A/D265S) are deliberately introduced into the Fc portion of both heavy chains to reduce the Fc receptor binding. Additionally, the YTE mutations (M252Y/S254T/T256E) are incorporated into both heavy chains of to extend the bispecific antibodies’ half-life.
  • the RF mutations are introduced on the hole heavy chain to aid in purification. These RF mutations may be introduced on the hole heavy chain of the CD33 arm, as disclosed hereinabove or as in SEQ ID NO. 34, or on the hole heavy chain of the V82 arm as in SEQ ID NO. 35. Alternatively, these RF mutations maybe introduced on both heavy chains.
  • the bispecific antibody CD33xV82 may be produced by cultivation of recombinant Chinese hamster ovary cells, followed by isolation, chromatographic purification, and formulation.
  • the protein molecules may be co-produced in a CHO cell line by co-transfection of each of the encoding expression plasmids and may be purified using a two-step process involving ProA capture followed by CHI affinity capture.
  • the antibodies may undergo purification via a Mab Select SuRe Protein A column from GE Healthcare.
  • the column may be prepped with PBS at pH 7.2 and then loaded with fermentation supernatant at a flow rate of 2 mL/min. Following loading, the column may undergo a wash with 4 column volumes of PBS, succeeded by elution using 30 mM sodium acetate at pH 3.5.
  • the fractions containing protein peaks, detected by absorbance at 280 nm, may be combined and their pH neutralized to 5.0 using a 1% solution of 3 M sodium acetate at pH 9.0.
  • Variants of the sequences disclosed herein preferably comprise conservative modifications of the disclosed sequences. “Conservative modifications” refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid modifications. Conservative modifications include amino acid substitutions, additions and deletions. Conservative amino acid substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain.
  • amino acids with acidic side chains e.g., aspartic acid, glutamic acid
  • basic side chains e.g., lysine, arginine, histidine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan
  • aromatic side chains e.g., phenylalanine, tryptophan, histidine, tyrosine
  • aliphatic side chains e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine
  • amide e.g., asparagine, glutamine
  • any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; Sasaki et al., (1988) Adv Biophys 35: 1-24).
  • Amino acid substitutions to the antibodies of the disclosure may be made by known methods for example by PCR mutagenesis (U.S. Patent No. 4,683,195).
  • libraries of variants may be generated for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp).
  • NNK random
  • DVK codons which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp).
  • the resulting variants may be tested for their characteristics using assays described herein.
  • the CD33 binder was located on the Hole Fc, while the V62 binder was on the Knob Fc.
  • the CD33 VH and human CHI constant region were combined with a hinge on Fc, featuring several mutations — L234A/L235A/D265S_M252Y/S254T/T256E_T366S/L368A/Y407V_H435R/Y436F. These mutations were deliberately introduced into the Fc portion of both heavy chains to reduce the Fc receptor binding. Additionally, the YTE mutations (M252Y/S254T/T256E) were incorporated into both heavy chains of to extend the bispecific antibodies’ half-life.
  • V62 binder was fused to the hinge and Knob Fc, incorporating the following mutations: C220S_L234A/L235A/D265S_M252Y/S254T/T256E_T366W.
  • the molecules were produced in a CHO cell line by co-transfection of the encoding expression plasmids and purified using a two-step process involving ProA capture followed by CHI affinity capture. Initially, the antibodies underwent purification via a Mab Select SuRe Protein A column from GE Healthcare. The column was prepped with PBS at pH 7.2 and then loaded with fermentation supernatant at a flow rate of 2 mL/min. Following loading, the column underwent a wash with 4 column volumes of PBS, succeeded by elution using 30 mM sodium acetate at pH 3.5.
  • Table 14 presents the heavy and/or light chains for exemplary CD33 X V82 bispecific antibodies.
  • GD33B134 is identical to GD33B273 except the H435R and Y436F substitutions (RF substitutions) are on heavy chain 1 instead of heavy chain 2.
  • the NullxV82 antibody which lacks a CD33 binding arm, and is used for comparison against the bispecific CD33xV82 antibodies disclosed herein, has the following sequences: EXAMPLE 4. BISPECIFIC ANTIBODIES TARGETING CD33 X V82 EXHIBIT BINDING AFFINITY TOWARD Vy9V82 CELLS.
  • Vy9V82 T cells were incubated at 37°C for 60 minutes with varying concentrations (ranging from 300 to 0.00508 nM) of the bispecific antibody GD33B273 or with a variant lacking the CD33 binding arm NullxV82, aka GD33B73.
  • Detection of bound V82 antibody was achieved by incubating with MonoRabTM Anti-Rabbit Camelid-anti-VHH AF647 secondary detection antibody (GenScript, A02019-200) for 30 minutes at 4°C.
  • FACS Lyric (BD) was used to measure samples, and FlowJo software (FlowJo) was employed for data analysis.
  • the nullxV82 antibody GD33B73 also exhibited binding to the Vy9V82 T cells.
  • Soluble CD33 containing serum samples from patients (e.g., AML patients) were mixed with specified concentrations of the test CD33 X V82 bispecific antibody (test molecule).
  • the test molecule's binding to soluble CD33 was determined by capturing soluble CD33 bound to the test molecule using magnetic beads coated with an antimolecule antibody. This process effectively removed the formed complex from the solution.
  • the supernatant serum was subjected to analysis using LC-MS (liquid chromatography-mass spectrometry) to identify soluble CD33 in the serum samples.
  • the LC-MS assay was formulated specifically to quantify the quantity of unbound sCD33 in the sera of AML patients subsequent to the addition of the specified antibody.
  • FIGs. 2A-2D showed limited binding of the various tested CD33xV82 bispecific antibodies to sCD33, demonstrating that these antibodies bind specifically to mCD33.
  • the reference antibody JL5 is non-specific and displayed dosage dependent binding to sCD33.
  • EXAMPLE 6 BISPECIFIC CD33 X V82 ANTIBODIES CAN MEDIATE CYTOTOXICITY AGAINST CD33 -EXPRESSING CELLS
  • THP1 target cells human leukemia monocytic cell line, available from SIGMA ALDRICH or from ATCC
  • CellTrace CFSE proliferation kit Thermo Scientific, C34554
  • bispecific CD33xV82 antibody or a negative control antibody nullxV82, aka GD33B73
  • Pan T cells E
  • E:T 10: 1 ratio
  • EXAMPLE 7 EVALUATING BISPECIFIC CD33 X V82 ANTIBODIES VIA SURFACE PLASMON RESONANCE ANALYSIS
  • a Biacore 8k instrument (Cytiva, cytivalifesciences.com) was utilized for SPR binding experiments by capture of the test antibodies onto a CM4 sensor SPR chip with Goat anti-Fc. CD33 antigen was flowed over at 100, 33, 11, and 3.7 nM in single cycle kinetics mode.
  • Running buffer utilized was HBSP+0.05%BSA+3mM EDTA. All instrument maintenance and liquid supply line priming was performed according to manufacturer operating instructions.
  • EXAMPLE 8 CHARACTERIZATION OF CD33 CELL BINDING SPECIFICITY FOR BISPECIFIC CD33 X V82 ANTIBODIES VIA FLUORESCENT STAINING AND FLOW
  • test molecules were incubated with cells at 37°C for 1 hour, following which the cells were washed via centrifugation and then suspended in staining buffer. Subsequently, a detection secondary antibody was introduced, the samples were incubated for 30 minutes, washed, suspended again, and finally, the samples were analyzed on the iQue instrument to quantify the fluorescent staining.
  • FIGs 4A-4D Results of CD33 cell binding analysis.
  • FIG. 4E depicts the cell binding of GD33B273 to an isogenic THP- 1_C2 cell line that specifically express the IgC2 domain of CD33.
  • EXAMPLE 9 EFFICACY OF CD33 X V82 BISPECIFIC ANTIBODIES IN THE PRESENCE OF sCD33
  • rCD33 human recombinant CD33 (SEQ ID NO: 9) ECD was utilized as a surrogate for sCD33.
  • a T-cell cytotoxicity assay was used where THP-1 cells and pan-T cells were incubated with increasing amounts of rCD33 protein prior to addition of a dose range of the CD33 X V82 bispecific antibody (cf e.g., example 3).
  • EXAMPLE 11 CD33 X V82 BISPECIFIC ANTIBODY MEDIATES SELECTIVE CYTOTOXICITY OF CD33+ CANCER CELLS
  • FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D show how bispecific CD33x82 antibody GD33B273 mediates selective cytotoxicity of CD33+ cancer cells (MOLM-13 and THP-1) and selective activation of V82 T cells when compared to the antibody lacking the CD33 arm (NullxV82, aka GD33B73).
  • T cells from healthy donors were evaluated in T-cell cytotoxicity and activation assays with GD33B273 and NullxV82 antibodies and target cell lines. Cancer cells cytotoxicity was determined for GD33B273 in flow-cytometry based assays at 72 hours with MOLM-13 and THP-1 as target cancer cells (FIG. 8 A and 8C). The assay was conducted at a relative E:T ratio of 0.5: 1.
  • T-cell activation was assessed by evaluating the expression of CD25 on V82 T cells (FIG. 8B and 8D) and was measured after 72 hours.
  • FIG. 8E and 8F show how bispecific CD33x82 antibody GD33B273 mediates selective cytotoxicity of THP-1 C2 cells expressing CD33 IgC2 domain (FIG. 8E) and selective activation of V82 T cells (FIG. 8F) when compared to the antibody lacking the CD33 arm (NullxV82, aka GD33B73).
  • FIG. 8G and 8H show how bispecific CD33x82 antibody GD33B273 does not mediate selective cytotoxicity of OCI-LylO / CD33negative cells, i.e., not expressing CD33 (FIG. 8G), and does not mediate or shows minimal selective activation of V82 T cells (FIG. 8H) when compared to the antibody lacking the CD33 arm (NullxV82, aka GD33B73).
  • OCI-LylO cells are a human-derived cell line and are used as target cancer cells.
  • EXAMPLE 12 EFFICACY OF CD33 X V82 ANTIBODY IN AML BM BLAST MODEL
  • T cells from healthy donors were tested in T-cell cytotoxicity assays with GD33B273 and AML patient-derived bone marrow (BM) cells, performed at a relative E:T ratio of 2:1. Cytotoxicity of CD33+ blast was assessed 24 hours. DN, donor. CD33x82 bispecific antibody GD33B273 was observed to induce potent cytotoxicity of AML BM cells from all tested donors (FIG. 10).
  • EXAMPLE 13 EVALUATING BISPECIFIC CD33 X V82 ANTIBODY TOXICITY ON HEMATOPOIETIC CELLS
  • Naive V82+ T cells were isolated from healthy donor PBMCs and used as effectors at 1 : 1 relative E:T ratio with CD34+ HSPC cells or THP-1 cells as target cells in a CFU assay.
  • HSPC hematopoietic stem and progenitor cells
  • CD cluster of differentiation
  • CFU colony forming unit
  • E:T effector to target
  • PBMC peripheral blood mononuclear cell.
  • GD33B273 showed low risk of hematopoietic toxicity while showing cytotoxicity for the cancer cells THP-1 in a dose response fashion.
  • CD34+ HSPC are stem cells from healthy donors, and the presence of surviving robust colonies (light grey bars) shows that the antibody does not impact them. In contrast, THP-1 cancer cells colonies (black bars) decrease survival in a dose-dependent relation to the antibody concentration.
  • EXAMPLE 14 EFFICACY OF CD33 X V82 ANTIBODIES IN MOLM-13 PREVENTION MODEL
  • the antitumor effect of the CD33 X V82 bispecific antibody GD33B134 was evaluated in a prophylactic MOLM-13 admixture tumor model with expanded pan-T cells or enriched V82 T cells as effector cells in T-cell humanized mice.
  • Female NSG i.e., non-obese diabetic [NOD] severe combined immunodeficiency [scid] gamma or NOD.Cg Prkdc scld Il rg ⁇ '/SzJ mice were used to provide a suitable host for reconstituting a human CD3+ T-cell compartment.
  • mice were inoculated subcutaneously (SC) with an admixture of 1 *10 6 MOLM-13 AML cells and 5*10 6 expanded pan-T cells or 2.5*10 6 enriched V82 T cells in Matrigel on Day 0.
  • T- cell humanized mice were given Fc block antibody (a commercial antibody that is used to block the Fc receptor in immune receptor cells) and intravenous immunoglobulin (IVIg) intraperitoneally (IP) at least 30 minutes prior to bispecific antibody dosing to correct for the low Ig environment in NSG mice.
  • Fc block antibody a commercial antibody that is used to block the Fc receptor in immune receptor cells
  • IVIg intravenous immunoglobulin intraperitoneally
  • NSG mice were randomized into groups of 8 animals and IP treatment was initiated on Day 1 post cell implantation with bispecific antibody GD33B134 at 1, 3, and 10 mg/kg twice weekly for a total of 11 doses. Treatment with bispecific antibody did not affect body weight of the animals.
  • TGI Percent tumor growth inhibition
  • EXAMPLE 15 EFFICACY OF A CD33 X V82 ANTIBODY IN MOLM-13 REGRESSION MODEL
  • the antitumor efficacy of the CD33 X V82 bispecific antibody GD33B134 was evaluated in a disseminated MOLM-13 regression model with enriched V82 T cells as effector cells in humanized mice (female NOG mice).
  • hIL-15 NOG mice were injected intravenously (IV) with 1 *10 6 MOLM-13 AML cells on Day 0.
  • 9.4* 10 6 enriched V82 T cells were injected IV and after 24 hours, T-cell-humanized mice were given Fc block and IVIg at least 30 minutes prior to bispecific antibody dosing.
  • mice were randomized into groups of 8 animals and IP treatment with 1, 3, and 10 mg/kg bispecific antibody was initiated on Day 4 post cell implantation twice weekly for a total of 8 doses.
  • a second injection of enriched V82 T cells (7.9* 10 6 ) was administered IV.
  • Mice were monitored for survival and body weight loss.
  • Statistically significant increased life span (ILS) was observed with bispecific antibody GD33B134 at 1, 3, and 10 mg/kg resulting in 117.9%, 112.8%, and 102.6% ILS, respectively, compared with vehicle-treated (DPBS) animals (FIG. 13 A). Reduction in body weight (reaching -23%) was observed in all treated animals following the injection of V82 T cells.
  • hIL-15 human interleukin-15
  • NOG non-obese diabetic (NOD)/Shi-severe combined immunodeficiency (scid) IL2rgamma(null)
  • DPBS Dulbecco’s phosphate-buffered saline.
  • EXAMPLE 16 A CD33 X VA2 ANTIBODY MEDIATES ANTI-TUMOR ACTIVITY IN DISSEMINATED MOLM-13 IN VIVO MODEL
  • FIG. 14A and FIG. 14B present the efficacy of the bispecific CD33x82 antibody GD33B273 in a MOLM-13 regression mice model.
  • the MOLM-13 cancer cells were labelled with luciferase. This labeled MOLM-13 cancer cells were implanted on Day 0 in the hIL-15 NOG mice. The same mice were injected with the T cells on Day 3 and 16, and dosed with antibody at the indicated doses (0, 0.3, and 3 mg/Kg) on Days 4, 7, 10, 14, 17, 21, and 24 (the dosing period is indicated by the solid bar under the X-axis, i.e. from day 4 to day 24 for both FIGs. 14A and 14B). Results are presented as average tumor radiance (FIG.
  • FIG. 14A shows an increase in average radiance for the untreated mice (0 mg/Kg) when compared to the treated mice with 0.3 and 3 mg/Kg antibody.
  • FIG. 14A also shows 61% and 54.9% of tumor growth inhibition at day 17 for treated mice with antibody at 0.3 and 3 mg/Kg, respectively, when compared to untreated mice (0 mg/Kg).
  • FIG. 14B shows an increase in the survival of the mice treated with 0.3 and 3 mg/Kg of antibody when compared to untreated mice (0 mg/Kg).
  • ILS increased life span
  • TGI tumor growth inhibition.

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Abstract

L'invention concerne des anticorps CD33, et des fragments de liaison à l'antigène de ceux-ci, et des anticorps multispécifiques CD33/Vδ2, ou des fragments de liaison à l'antigène de ceux-ci. L'invention concerne également des polynucléotides codant pour les anticorps, des compositions comprenant les anticorps, des procédés de production des anticorps, et des procédés d'utilisation des anticorps pour traiter ou prévenir des maladies, telles que des cancers hématologiques.
PCT/IB2024/062299 2023-12-08 2024-12-06 ANTICORPS CD33, ANTICORPS MULTISPÉCIFIQUES CD33/Vδ2 ET LEURS UTILISATIONS Pending WO2025120583A2 (fr)

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WO2026033458A1 (fr) * 2024-08-08 2026-02-12 Janssen Biotech, Inc. Doses et compositions pharmaceutiques d'un anticorps bispécifique cd33 x vδ2 pour le traitement du cancer

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WO2026033458A1 (fr) * 2024-08-08 2026-02-12 Janssen Biotech, Inc. Doses et compositions pharmaceutiques d'un anticorps bispécifique cd33 x vδ2 pour le traitement du cancer

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