WO2023049867A1 - Orai1 multispecific antibodies and methods of use - Google Patents

Orai1 multispecific antibodies and methods of use Download PDF

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Publication number
WO2023049867A1
WO2023049867A1 PCT/US2022/076974 US2022076974W WO2023049867A1 WO 2023049867 A1 WO2023049867 A1 WO 2023049867A1 US 2022076974 W US2022076974 W US 2022076974W WO 2023049867 A1 WO2023049867 A1 WO 2023049867A1
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seq
residues
cdr
amino acid
acid sequence
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PCT/US2022/076974
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French (fr)
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Matthew Moyle
Daniel MACAYA
David Presky
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Seeker Biologics, Inc.
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Publication of WO2023049867A1 publication Critical patent/WO2023049867A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C07K16/2818—Immunoglobulins [IGs], 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 CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • 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
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • compositions comprising Orail multispecific antibody agents and methods of their use.
  • Calcium release activated calcium modulator 1 is a transmembrane protein and is the pore forming subunit of calcium release activated calcium (CRAC) channels which mediate store-operated calcium (SOC) influx into cells. Orail is activated by the Ca 2+ sensor, stromal interaction molecule 1 (STIM1), localized to the endoplasmic reticulum (ER) when intracellular Ca 2+ stores are depleted.
  • CRAC calcium release activated calcium
  • SOC stromal interaction molecule 1
  • Orail provides the primary pathway for calcium influx into T cells and plays an important role in T cell activation and immune function.
  • the sustained calcium influx into T cells leads to activation of transcription factors, such as nuclear factor of activated T cells (NF AT) resulting in the transcription of cytokine genes critical for T cell activation.
  • NF AT nuclear factor of activated T cells
  • Orail is a therapeutically relevant target for treating immune disorders, including T cell-mediated disorders, such as autoimmune disease and allergic disease.
  • T cell-mediated disorders such as autoimmune disease and allergic disease.
  • current Orail binders are only weak inhibitors of T cell function. Therefore, there is a need for improved therapeutic agents that can target Orail and inhibit T cell function and/or activity.
  • the present disclosure provides Orail multispecific antibodies and methods of using the Orail multispecific antibodies.
  • the present invention provides a multispecific antibody comprising a first antigen binding domain that binds to Orai 1 ; and a second antigen binding domain that binds to a target on a T cell; wherein the multispecific antibody inhibits T cell activity.
  • the first antigen binding domain of the multispecific antibody competes for binding with, binds a same epitope as, or comprises (a) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2 (b) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (c) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147;
  • the first antigen binding domain of the multispecific antibody comprises (a) residues 27-38 of SEQ ID NO: 5 for CDR-L1, residues 56-65 of SEQ ID NO: 5 for CDR-L2, and residues 105-117 of SEQ ID NO: 5 for CDR- L3; and/or residues 27-38 of SEQ ID NO: 2 for CDR-H1, residues 56-65 of SEQ ID NO: 2 for CDR-H2, and residues 105-117 of SEQ ID NO: 2 for CDR-H3; (b) residues 27-38 of SEQ ID NO: 146 for CDR-L1, residues 56-65 of SEQ ID NO: 146 for CDR-L2, and residues 105-117 of SEQ ID NO: 146 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 140 for CDR-H1, residues 56-65 of SEQ ID NO: 140 for CDR-H2, and residues 105-117 of SEQ ID NO: 140 for CDR
  • the first antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, and residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 2 for CDR-H1, residues 50-65 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 146 for CDR-L1, residues 50-56 of SEQ ID NO: 146 for CDR-L2, and residues 89-97 of SEQ ID NO: 146 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 140 for CDR-H1, residues 50-65 of SEQ ID NO: 140 for CDR-H2, and residues 95-102 of SEQ ID NO: 140 for CDR-H3;
  • the first antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 2 for CDR-H1, residues 52-56 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 146 for CDR-L1, residues 50-56 of SEQ ID NO: 146 for CDR-L2, residues 89-97 of SEQ ID NO: 146 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 140 for CDR-H1, residues 52-56 of SEQ ID NO: 140 for CDR-H2, and residues 95- 102 of SEQ ID NO: 140 for CDR-H3;
  • the first antigen binding domain of the multispecific antibody comprises (a) residues 30-36 of SEQ ID NO: 5 for CDR-L1, residues 46-55 of SEQ ID NO: 5 for CDR-L2, and residues 89-96 of SEQ ID NO: 5 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 2 for CDR-H1, residues 47-58 of SEQ ID NO: 2 for CDR-H2, and residues 93-101 of SEQ ID NO: 2 for CDR-H3; (b) residues 30-36 of SEQ ID NO: 146 for CDR-L1, residues 46-55 of SEQ ID NO: 146 for CDR-L2, and residues 89-96 of SEQ ID NO: 146 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 140 for CDR-H1, residues 47-58 of SEQ ID NO: 140 for CDR-H2, and residues 93-101 of SEQ ID NO: 140 for CDR-H3;
  • the first antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, and residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 2 for CDR-H1, residues 50-58 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 146 for CDR-L1, residues 50-56 of SEQ ID NO: 146 for CDR-L2, and residues 89-97 of SEQ ID NO: 146 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 140 for CDR-H1, residues 50-58 of SEQ ID NO: 140 for CDR-H2, and residues 95-102 of SEQ ID NO: 140 for CDR-H3;
  • the first antigen binding domain of the multispecific antibody comprises (a) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 18, a CDRL2 comprising the amino acid sequence set forth as SEQ ID NO: 19, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 20; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 21, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 22, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 23; (b) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 199, a CDRL2 comprising the amino acid sequence VYN, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 200; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 196, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO:
  • the first antigen binding domain of the multispecific antibody comprises (a) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2; (b) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (c) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or (d) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 151.
  • the first antigen binding domain of the multispecific antibody comprises a light chain comprising an amino acid sequence set forth as SEQ ID NO: 15; and/or a heavy chain comprising an amino acid sequence set forth as SEQ ID NO: 14.
  • the first antigen binding domain of the multispecific antibody comprises a single domain antibody (sdAb).
  • the second antigen binding domain the multispecific antibody binds to a target on activated T cells.
  • the second antigen binding domain of the multispecific antibody comprises a single domain antibody (sdAb).
  • the second antigen binding domain of the multispecific antibody comprises an scFv.
  • the second antigen binding domain of the multispecific antibody binds to programmed cell death protein 1 (PD1).
  • PD1 programmed cell death protein 1
  • the second antigen binding domain of the multispecific antibody competes for binding with, binds a same epitope as, or comprises (a) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 8; (b) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 170; (c) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 171; (d) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 172; or (e) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 173.
  • the second antigen binding domain of the multispecific antibody comprises (a) residues 31-35 of SEQ ID NO: 8 for CDR1, residues 50-65 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3; (b) residues 31-35 of SEQ ID NO: 170 for CDR1, residues 50-65 of SEQ ID NO: 170 for CDR2, and residues 95-102 of SEQ ID NO: 170 for CDR3; (c) residues 31-35 of SEQ ID NO: 171 for CDR1, residues 50-65 of SEQ ID NO:
  • the second antigen binding domain of the multispecific antibody comprises (a) residues 26-32 of SEQ ID NO: 8 for CDR1, residues 52-56 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3; (b) residues 26-32 of SEQ ID NO: 170 for CDR1, residues 52-56 of SEQ ID NO: 170 for CDR2, and residues 95-102 of SEQ ID NO:
  • the second antigen binding domain of the multispecific antibody comprises (a) residues 30-35 of SEQ ID NO: 8 for CDR1, residues 47-58 of SEQ ID NO: 8 for CDR2, and residues 93-101 of SEQ ID NO: 8 for CDR3; (b) residues 30-35 of SEQ ID NO: 170 for CDR1, residues 47-58 of SEQ ID NO: 170 for CDR2, and residues 93-101 of SEQ ID NO:
  • the second antigen binding domain of the multispecific antibody comprises (a) residues 26-35 of SEQ ID NO: 8 for CDR1, residues 50-58 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3; (b) residues 26-35 of SEQ ID NO: 170 for CDR1, residues 50-58 of SEQ ID NO: 170 for CDR2, and residues 95-102 of SEQ ID NO: 170 for CDR3; (c) residues 26-35 of SEQ ID NO: 171 for CDR1, residues 50-58 of SEQ ID NO: 171 for CDR2, and residues 95-102 of SEQ ID NO: 171 for CDR3; (d) residues 26-35 of SEQ ID NO: 172 for CDR1, residues 50-58 of SEQ ID NO: 172 for CDR2, and residues 95-102 of SEQ ID NO: 172 for CDR3; or (e) residues 26-35 of SEQ ID NO: 173 for
  • the second antigen binding domain of the multispecific antibody comprises (a) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 24, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 26; (b) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 314, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 315, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 316; (c) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 322, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 323, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 324; (d) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 330, a CDR2 comprising the amino acid sequence set forth as S
  • the second antigen binding domain of the multispecific antibody comprises the amino acid sequence set forth as SEQ ID NO: 8, 170. 171, 172, or 173.
  • the second antigen binding domain of the multispecific antibody competes for binding with, binds a same epitope as, or comprises (a) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 145 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 143; (b) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 154 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 153; (c) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 158 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO
  • the second antigen binding domain of the multispecific antibody comprises (a) residues 27-38 of SEQ ID NO: 145 for CDR-L1, residues 56-65 of SEQ ID NO: 145 for CDR-L2, and residues 105-117 of SEQ ID NO: 145 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 143 for CDR-H1, residues 56-65 of SEQ ID NO: 143 for CDR-H2, and residues 105-117 of SEQ ID NO: 143 for CDR-H3; (b) residues 27-38 of SEQ ID NO: 154 for CDR-L1, residues 56-65 of SEQ ID NO: 154 for CDR-L2, and residues 105-117 of SEQ ID NO: 154 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 153 for CDR-H1, residues 56-65 of SEQ ID NO: 153 for CDR-H2, and residues 105-117 of SEQ ID NO
  • the second antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 145 for CDR-L1, residues 50-56 of SEQ ID NO: 145 for CDR-L2, and residues 89-97 of SEQ ID NO: 145 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 143 for CDR-H1, residues 50-65 of SEQ ID NO: 143 for CDR-H2, and residues 95-102 of SEQ ID NO: 143 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 154 for CDR-L1, residues 50-56 of SEQ ID NO: 154 for CDR-L2, and residues 89-97 of SEQ ID NO: 154 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 153 for CDR-H1, residues 50-65 of SEQ ID NO: 153 for CDR-H2, and residues 95-102 of SEQ ID NO:
  • the second antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 145 for CDR-L1, residues 50-56 of SEQ ID NO: 145 for CDR-L2, residues 89-97 of SEQ ID NO: 145 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 143 for CDR-H1, residues 52-56 of SEQ ID NO: 143 for CDR-H2, and residues 95-102 of SEQ ID NO: 143 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 154 for CDR-L1, residues SO- 56 of SEQ ID NO: 154 for CDR-L2, residues 89-97 of SEQ ID NO: 154 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 153 for CDR-H1, residues 52-56 of SEQ ID NO: 153 for CDR- H2, and residues 95-102 of SEQ ID NO: 153
  • the second antigen binding domain of the multispecific antibody comprises (a) residues 30-36 of SEQ ID NO: 145 for CDR-L1, residues 46-55 of SEQ ID NO: 145 for CDR-L2, and residues 89-96 of SEQ ID NO: 145 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 143 for CDR-H1, residues 47-58 of SEQ ID NO: 143 for CDR-H2, and residues 93-101 of SEQ ID NO: 143 for CDR-H3; (b) residues 30-36 of SEQ ID NO: 154 for CDR-L1, residues 46-55 of SEQ ID NO: 154 for CDR-L2, and residues 89-96 of SEQ ID NO: 154 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 153 for CDR-H1, residues 47-58 of SEQ ID NO: 153 for CDR-H2, and residues 93-101 of SEQ ID NO:
  • the second antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 145 for CDR-L1, residues 50-56 of SEQ ID NO: 145 for CDR-L2, and residues 89-97 of SEQ ID NO: 145 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 143 for CDR-H1, residues 50-58 of SEQ ID NO: 143 for CDR-H2, and residues 95-102 of SEQ ID NO: 143 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 154 for CDR-L1, residues 50-56 of SEQ ID NO: 154 for CDR-L2, and residues 89-97 of SEQ ID NO: 154 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 153 for CDR-H1, residues 50-58 of SEQ ID NO: 153 for CDR-H2, and residues 95-102 of SEQ ID NO:
  • the second antigen binding domain of the multispecific antibody comprises (a) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 211, a CDRL2 comprising the amino acid sequence AAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 212; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 208, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 209, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 210; (b) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 246, a CDRL2 comprising the amino acid sequence DAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 247; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 243, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO:
  • the second antigen binding domain of the multispecific antibody comprises (a) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 145; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 143; (b) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 154; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 153; (c) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 158; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 157; (d) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 160; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 159; (e) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 162; and/or a heavy chain variable region
  • the second antigen binding domain competes for binding with, binds a same epitope as, or comprises an antigen binding domain having three CDRs of a variable region sequence comprising (a) an amino acid sequence set forth as SEQ ID NO: 35; (b) an amino acid sequence set forth as SEQ ID NO: 36; (c) an amino acid sequence set forth as SEQ ID NO: 37; (d) an amino acid sequence set forth as SEQ ID NO: 38; (e) an amino acid sequence set forth as SEQ ID NO: 39; (f) an amino acid sequence set forth as SEQ ID NO: 40; (g) an amino acid sequence set forth as SEQ ID NO: 41; (h) an amino acid sequence set forth as SEQ ID NO: 42; (i) an amino acid sequence set forth as SEQ ID NO: 43; (j) an amino acid sequence set forth as SEQ ID NO: 44; (k) an amino acid sequence set forth as SEQ ID NO: 45; (1) an amino acid sequence set forth as SEQ ID NO: 46; (m) an amino acid sequence set forth
  • the second antigen binding domain comprises (a) residues 27-38 of SEQ ID NO: 35 for CDR1, residues 56-65 of SEQ ID NO: 35 for CDR2, and residues 105-117 of SEQ ID NO: 35 for CDR3; (b) residues 27-38 of SEQ ID NO: 36 for CDR1, residues 56-65 of SEQ ID NO: 36 for CDR2, and residues 105-117 of SEQ ID NO: 36 for CDR3; (c) residues 27- 38 of SEQ ID NO: 37 for CDR1, residues 56-65 of SEQ ID NO: 37 for CDR2, and residues 105- 117 of SEQ ID NO: 37 for CDR3; (d) residues 27-38 of SEQ ID NO: 38 for CDR1, residues 56- 65 of SEQ ID NO: 38 for CDR2, and residues 105-117 of SEQ ID NO: 38 for CDR3 (e) residues 27-38 of SEQ ID NO: 39 for CDR1, residues 56-65 of SEQ ID NO: 35
  • the second antigen binding domain comprises (a) residues 31-35 of SEQ ID NO: 35 for CDR1, residues 50-65 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 31-35 of SEQ ID NO: 36 for CDR1, residues 50-65 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 31-35 of SEQ ID NO: 37 for CDR1, residues 50-65 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 31-35 of SEQ ID NO: 38 for CDR1, residues 50-65 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 (e) residues 31-35 of SEQ ID NO: 39 for CDR1, residues 50-65 of SEQ ID NO: 39 for CDR1, residue
  • the second antigen binding domain comprises (a) residues 26-32 of SEQ ID NO: 35 for CDR1, residues 52-56 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 26-32 of SEQ ID NO: 36 for CDR1, residues 52-56 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 26-32 of SEQ ID NO: 37 for CDR1, residues 52-56 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 26-32 of SEQ ID NO: 38 for CDR1, residues 52-56 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3; (e) residues 26-32 of SEQ ID NO: 39 for CDR1, residues 52-56 of SEQ ID NO: 39 for CDR1,
  • the second antigen binding domain comprises (a) residues 30-35 of SEQ ID NO: 35 for CDR1, residues 47-58 of SEQ ID NO: 35 for CDR2, and residues 93-101 of SEQ ID NO: 35 for CDR3; (b) residues 30-35 of SEQ ID NO: 36 for CDR1, residues 47-58 of SEQ ID NO: 36 for CDR2, and residues 93-101 of SEQ ID NO: 36 for CDR3; (c) residues 30-35 of SEQ ID NO: 37 for CDR1, residues 47-58 of SEQ ID NO: 37 for CDR2, and residues 93-101 of SEQ ID NO: 37 for CDR3; (d) residues 30-35 of SEQ ID NO: 38 for CDR1, residues 47-58 of SEQ ID NO: 38 for CDR2, and residues 93-101 of SEQ ID NO: 38 for CDR3 (e) residues 30-35 of SEQ ID NO: 39 for CDR1, residues 47-58 of SEQ ID NO: 39 for CDR2, residues
  • the second antigen binding domain comprises (a) residues 26-35 of SEQ ID NO: 35 for CDR1, residues 50-58 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 26-35 of SEQ ID NO: 36 for CDR1, residues 50-58 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 26-35 of SEQ ID NO: 37 for CDR1, residues 50-58 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 26-35 of SEQ ID NO: 38 for CDR1, residues 50-58 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 (e) residues 26-35 of SEQ ID NO: 39 for CDR1, residues 50-58 of SEQ ID NO: 39 for CDR1, residue
  • the second antigen binding domain comprises (a) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 56, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 57, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 58; (b) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 59, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 60, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 61; (c) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 62, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 63, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 64; (d) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 65, a CDR2 comprising the amino acid sequence set forth
  • the second antigen binding domain comprises the amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
  • the first antigen binding domain and the second antigen binding domain the multispecific antibody are linked by a linker.
  • the linker is a peptide linker.
  • the peptide linker comprises a glycine/serine linker.
  • the linker comprises the amino acid sequence set forth as SEQ ID NO: 11, 144, or 174.
  • the multispecific antibody further comprises a half-life extender.
  • the half-life extender binds to human serum albumin, binds directly to FcRn, or binds indirectly to FcRn.
  • inhibiting T cell activity by the multispecific antibody comprises inhibiting cytokine release.
  • the cytokine is interleukin-2 (IL-2), interferon gamma (IFNy), and/or tumor necrosis factor alpha (TNFa).
  • IL-2 interleukin-2
  • IFNy interferon gamma
  • TNFa tumor necrosis factor alpha
  • inhibiting T cell activity by the multispecific antibody comprises inhibiting nuclear factor of activated T-cells (NF AT).
  • NF AT nuclear factor of activated T-cells
  • the multispecific antibody is a bispecific antibody.
  • the multispecific antibody is a trispecific antibody.
  • the present invention provides a nucleic acid encoding any of the multispecific antibodies provided herein.
  • the present invention provides a vector comprising the nucleic acid encoding any of the multispecific antibodies provided herein.
  • the present invention provides a host cell comprising the nucleic acid encoding any of the multispecific antibodies provided herein or a vector comprising the nucleic acid.
  • composition comprising any of the multispecific antibodies provided herein is provided.
  • the present invention provides a method of inhibiting T cell activity comprising contacting a T cell with an effective amount of any of the multispecific antibodies provided herein.
  • a method of treating or preventing an immune disorder in a subject comprising administering to the subject an effective amount of any of the multispecific antibodies provided herein or the pharmaceutical composition provided herein.
  • the immune disorder is a T cell mediated inflammatory disease.
  • the immune disorder is selected from the group consisting of a T cell mediated autoimmune disease, allergic disease, idiopathic thrombocytopenic purpura, warm autoimmune hemolytic anemia, autoimmune glomerulonephritis, autoimmune thyroid disease, systemic sclerosis, immune related adverse events (irAEs), transplant rejection, graft versus host disease (GVHD), rheumatoid arthritis, multiple sclerosis, type-1 diabetes, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, asthma, eosinophilic disease, autoimmune central nervous system (CNS) inflammation, psoriatic arthritis, atopic dermatitis, alopecia aerata, vitiligo, IgA nephropathy, lupus nephritis, chronic spontaneous urticarial, pyoderma gangrenosum, focal segmental glomerular sclerosis, membranous nephropathy,
  • the present invention provides a kit comprising any of the multispecific antibodies or pharmaceutical compositions provided herein.
  • a kit comprising any of the multispecific antibodies or pharmaceutical compositions provided herein.
  • single domain antibodies which bind to suppressor of tumorigenicity 2 (ST2).
  • the present invention provides a single domain antibody which competes for binding with, binds a same epitope as, or comprises an antigen binding domain having three complementarity determining regions (CDRs) of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 35; an amino acid sequence set forth as SEQ ID NO: 36; an amino acid sequence set forth as SEQ ID NO: 37; an amino acid sequence set forth as SEQ ID NO: 38; an amino acid sequence set forth as SEQ ID NO: 39; an amino acid sequence set forth as SEQ ID NO: 40; an amino acid sequence set forth as SEQ ID NO: 41; an amino acid sequence set forth as SEQ ID NO: 42; an amino acid sequence set forth as SEQ ID NO: 43; an amino acid sequence set forth as SEQ ID NO: 44; an amino acid sequence set forth as SEQ ID NO: 45; an amino acid sequence set forth as SEQ ID NO: 46; an amino acid sequence set forth as SEQ ID NO: 47; an amino acid sequence set forth as SEQ ID NO: 48;
  • the single domain antibody comprises (a) residues 27-38 of SEQ ID NO: 35 for CDR1, residues 56-65 of SEQ ID NO: 35 for CDR2, and residues 105-117 of SEQ ID NO: 35 for CDR3; (b) residues 27-38 of SEQ ID NO: 36 for CDR1, residues 56-65 of SEQ ID NO: 36 for CDR2, and residues 105-117 of SEQ ID NO: 36 for CDR3; (c) residues 27- 38 of SEQ ID NO: 37 for CDR1, residues 56-65 of SEQ ID NO: 37 for CDR2, and residues 105- 117 of SEQ ID NO: 37 for CDR3; (d) residues 27-38 of SEQ ID NO: 38 for CDR1, residues 56- 65 of SEQ ID NO: 38 for CDR2, and residues 105-117 of SEQ ID NO: 38 for CDR3 (e) residues 27-38 of SEQ ID NO: 39 for CDR1, residues 56-65 of SEQ ID NO:
  • the single domain antibody comprises (a) residues 31-35 of
  • residues 31-35 of SEQ ID NO: 38 for CDR1, residues 50-65 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 residues 31-35 of SEQ ID NO: 39 for CDR1, residues 50-65 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3;
  • the single domain antibody comprises (a) residues 26-32 of
  • the single domain antibody comprises (a) residues 30-35 of
  • the single domain antibody comprises (a) residues 26-35 of SEQ ID NO: 35 for CDR1, residues 50-58 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 26-35 of SEQ ID NO: 36 for CDR1, residues 50-58 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 26-35 of SEQ ID NO: 37 for CDR1, residues 50-58 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 26-35 of SEQ ID NO: 38 for CDR1, residues 50-58 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 (e) residues 26-35 of SEQ ID NO: 39 for CDR1, residues 50-58 of SEQ ID NO: 39 for CDR2,
  • the single domain antibody comprises (a) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 56, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 57, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 58; (b) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 59, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 60, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 61; (c) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 62, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 63, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 64; (d) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 65, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO
  • the single domain antibody comprises a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
  • the present invention provides a nucleic acid encoding any of the heavy chain variable regions of an ST2 antibody provided herein. In another aspect, the present invention provides a vector comprising a nucleic acid encoding any of the heavy chain variable regions of an ST2 antibody provided herein. In one aspect, the present invention provides a host cell comprising a nucleic acid encoding any of the ST2 single domain antibodies provided herein. In another aspect, the present invention provides a host cell comprising a vector comprising a nucleic acid encoding any of the ST2 single domain antibodies provided herein.
  • composition comprising any of the ST2 single domain antibodies provided herein is provided.
  • FIG. 1 illustrates an exemplary anti-Orail IgG monoclonal antibody (mAb) and an exemplary anti-Orail multispecific antibody (CTAB PR071) comprising an Orail IgG mAb first antigen binding domain and a second antigen binding domain comprising a sdAb (single domain antibody) targeting a T cell antigen.
  • mAb monoclonal antibody
  • CTAB PR071 exemplary anti-Orail multispecific antibody
  • FIG. 2 depicts the percent inhibition of IL-2 release as measured in an IL-2 ELISA.
  • Human PBMCs were incubated with various concentrations of anti-Orai-1 mAB (2C1.1 human IgG2), Anti-Orail/Anti-PDl VHH multispecific antibody (CTAB PR071), anti-Orail mAB and anti-PDl VHH (equimolar), anti-PDl VHH, or cyclosporine A for 1 hour followed by activation with human anti-CD3/CD28 dynabeads for 24 hours.
  • IL-2 was measured by ELISA in the supernatants.
  • FIG. 3 shows the annotated sequences for exemplary Orail/PDl multispecific antibody constructs.
  • the Orail antibody variable regions are shown in yellow
  • the PD1 scFv antibody variable regions are shown in blue
  • the PD1 VHH variable regions are shown in green
  • the linkers are shown in gray.
  • the underlined text indicates the CDR regions as defined by IMGT
  • the red text indicates the CDR regions as defined by Kabat
  • the italic text indicates the CDR regions as defined by Chothia.
  • FIG. 4 depicts the percent inhibition of NF AT as measured in a Jurkat screening assay.
  • Jurkat T cells containing an NF AT luciferase reporter were cultured with various concentrations of an anti -Orail /anti-PDl VHH multispecific antibody (CTAB PR071; anti-Orail IgG/ anti-PDl VHH) or the anti-Orail mAB alone (BB PR001). Luminescence was measured after 48 hours.
  • CTAB PR071 anti-Orail IgG/ anti-PDl VHH multispecific antibody
  • BB PR001 anti-Orail mAB alone
  • FIG. 5 depicts the percent inhibition of NF AT as measured in a Jurkat screening assay.
  • Jurkat T cells containing an NF AT luciferase reporter were cultured with various dilutions of supernatant containing an anti -Orail /anti-PDl VHH multispecific antibody (CTABs), or anti- Orail antibody and PD1 VHH (control).
  • CTABs anti-Orail /anti-PDl VHH multispecific antibody
  • PD1 VHH control
  • Luminescence was measured after 48 hours.
  • Orail is expressed on T cells and is a critical component of a Ca 2+ channel that is required for differentiation and function of T cells.
  • the present inventors have discovered that multispecific antibodies comprising an Orail binding domain and a second antigen binding domain that binds to a target on a T cell exhibit enhanced T cell inhibitory activity compared to the parent anti-Orail antibody.
  • the disclosed multispecific antibodies are both highly specific to Orail expressing targets and cell selective based upon the specificity of the second antigen binding domain and have an improved therapeutic index.
  • the Orail multispecific antibodies provided herein are useful for inhibiting T cell activity and/or function and for treating immune disorders, including, T cell- mediated inflammatory diseases, such as autoimmune disease and allergic disease.
  • Orail multispecific antibodies inhibit T cell activity with a similar efficacy to the standard of care cyclosporine which is a part of the standard of care for a variety of T cell-mediated inflammatory diseases and works downstream of the Orail Ca 2+ channel, supporting the therapeutic use of the Orail multispecific antibodies of the invention.
  • compositions comprising multispecific antibodies comprising at least a first antigen binding domain that binds to Orail and a second binding domain that binds to a target antigen.
  • multispecific antibodies are also called “Orail multispecific antibodies” herein.
  • methods of using the Orai 1 multispecific antibodies to target cell activity and/or function can be a reduction in the release or production of any of various cytokines, such as interleukin-2 (IL-2), as described elsewhere herein.
  • IL-2 interleukin-2
  • antigen binding domain is meant a protein or polypeptide sequence that can bind to a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, specific antigenic determinant, epitope, antigen, or protein.
  • the antigen binding domains used to assemble the Orail multispecific antibody domains of the instant invention can be prepared using any known technique for antibody engineering or antibody selection.
  • the antigen binding domains may be synthesized using known antigen binding domain sequences.
  • the antigen binding domains are selected by screening a library for functional properties.
  • the antigen binding domains may be selected from a stabilized synthetic antibody library that was prepared by (a) generating a primary library comprising a plurality of antibodies, wherein each of the antibodies comprises a diverse CDR1, a diverse CDR2, and a neutral CDR3; (b) selecting a subset of the antibodies having improved developability features; and (c) generating the stabilized synthetic antibody library by replacing the neutral CDR3 of each of the antibodies selected in (b) with a diverse CDR3.
  • neutral CDR is meant a CDR3 that is representative of human CDR3 diversity (see e.g, PCT International Publication No.
  • WO 2019/099454 incorporated by reference herein in its entirety
  • a neutral CDR3 that is representative of a target scaffold for antibody generation i.e., a target scaffold for generation of each of the Orail binding domain and the target binding domain.
  • a neutral CDR3 that is representative of a target scaffold includes an amino acid composition that reflects a pattern observed in a plurality of unique CDR3 sequences for a target scaffold.
  • a neutral CDR3 that is representative of a target scaffold may be determined by (i) identifying a plurality of unique CDR3 sequences for a target scaffold, for example by observing unique CDR3 sequences in antibodies comprising the target scaffold; (ii) selecting multiple subsets of the plurality of unique CDR3 sequences, wherein each subset comprises CDR3 sequences of a same length, and wherein subsets are selected for multiple CDR3 lengths, (iii) performing clustering analysis of CDR3 sequences in randomly selected subsets, (iv) selecting multiple clusters for each CDR3 length, and (v) selecting a representative CDR3 sequence from each cluster.
  • Representative target scaffolds include, for example, either in unmutated or mutated configuration, human heavy chain scaffolds, such as those from the human IGHV germline gene family.
  • the target scaffold is from the human IGHV-3 germline gene family.
  • Representative developability features is based on binding of antibody fragments to Protein A or to Protein L, or based on protein unfolding using treatments selected from high temperature, acid treatment, basic treatment, protease sensitivity, stability in serum, denaturation reagents, urea, and high salt concentration. Increased developability may also include deselection or removal of antibodies characterized as having decreased developability using methods selected from nonspecific absorption, binding to HSP90, and hydrophobic interactions.
  • target a molecule of interest that is expressed by a cell.
  • the target is a non-cytoplasmic target, for example, an extracellular or endosomal target.
  • the target antigen can be any molecule expressed by a cell and the cell can be any cell type of interest.
  • the target is a molecule that is expressed by a particular cell type. In such aspects, the target may not be expressed or has very low expression on other cell types.
  • the cell of interest is a mammalian cell, and preferably a primate cell, and even more preferably a human cell.
  • the cell of interest is an immune cell.
  • the cell of interest is a T cell.
  • the cell of interest is a pancreatic cell (e.g., a pancreatic acinar cell), an epithelia cell (e.g., a lung or intestinal epithelial cell), or a blood cell (e.g., a white blood cell such as a mast cell).
  • a pancreatic cell e.g., a pancreatic acinar cell
  • an epithelia cell e.g., a lung or intestinal epithelial cell
  • a blood cell e.g., a white blood cell such as a mast cell.
  • a “multispecific antibody” or “multispecific binding agent” is one that binds to more than one antigen or epitope. In some cases, the multispecific antibody binds to two, three, four, five or more target antigens or epitopes.
  • the target antigens or epitopes can be on the same cell or on separate cells.
  • the target antigens can be two or more separate and unique antigens or can be different epitopes of the same antigen.
  • multispecific polypeptide “multispecific antibody,” “multispecific binding agent,” and “multispecific antibody construct” are used interchangeably herein.
  • the multispecific antibodies disclosed herein provide cell selective and disease selective targeting, and as such, a “multispecific antibody” as used herein includes a “cell targeted biologic” or “CTAB”.
  • the antibodies useful in the invention are bispecific antibodies.
  • a bispecific antibody or antigen binding protein is an antibody having two different antigen binding sites.
  • Bispecific antibodies are a species of multispecific antibody and may be produced by a variety of techniques known in the art including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148: 1547-1553.
  • a non-limiting example of the synthesis of a bispecific antibody is provided in Example 1 herein.
  • the two binding sites of a bispecific antibody will bind to two different epitopes, Orail and a target antigen.
  • the antibodies useful in the invention are trispecific antibodies.
  • a trispecific antibody or antigen binding protein is an antibody having three different antigen binding sites. In the context of the present invention, two of the binding sites of a trispecific antibody will bind to Orail and a target antigen, and the third antigen binding site can bind to any antigen. In some aspects, the third antigen binding site is a half-life extender.
  • multispecific antibodies of the invention comprise additional sequences including signal peptides, linkers, half-life extenders, and/or tag sequences, such as a His-tag.
  • the first antigen binding domain of the disclosed multispecific antibodies binds to Orail and comprises any of the various antigen binding domains provided herein, such as an antibody, including an antibody variable region, or an sdAb.
  • the first antigen binding domain binds to the Orai with relatively low affinity.
  • the affinity is a measure for the binding strength between an antigenic determinant, i.e. the target, and an antigen-binding domain, i.e., an antigen binding domain of any of the multispecific antibodies provided herein.
  • the affinity can be represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding domain (KD or KD): the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding domain (alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/KD).
  • Affinity can be determined by any manner known in the art depending on the specific antigen of interest.
  • an antigen-binding domain (such as, any of the antigen binding domains of the multispecific antibodies, antibodies, or sdAbs provided herein) are said to bind to their target antigen with a high affinity when the dissociation constant (KD) is 10 -9 to IO -12 moles/liter or less, and preferably 10“ 10 to IO -12 moles/liter or less and more preferably 10 -11 to IO -12 moles/liter (i.e. with an association constant (KA) of 10 9 to 10 12 liter/moles or more, and preferably 10 10 to 10 12 liter/moles or more and more preferably 10 u to 10 12 liter/moles).
  • KD dissociation constant
  • KD dissociation constant
  • KA association constant
  • an antigen-binding domain (such as, any of the antigen binding domains of the multispecific antibodies, antibodies, or sdAbs provided herein) are said to bind to their target antigen with a low affinity when the dissociation constant (KD) is 10 -6 to IO -9 moles/liter or more, and preferably 10 -6 to 10 -8 moles/liter or more and more preferably 10 -6 to IO -7 moles/liter (i.e. with an association constant (KA) of 10 6 to 10 9 liter/moles or more, and preferably 10 6 to 10 8 liter/moles or more and more preferably 10 6 to 10 7 liter/moles).
  • KD dissociation constant
  • KD dissociation constant
  • KA association constant
  • Orai Calcium Release-Activated Calcium Modulator 1 used interchangeably with the term “Orail” refers to the well-known gene and polypeptide encoded by that gene, also known in the art as IMD9, TAM2, ORAT1, CRACM1, TMEM142A, and the ORAI calcium release-activated calcium protein 1.
  • the Orail gene is located in the chromosomal region 12q24.31 (base pairs 121,626,550- 121,642,040 on chromosome 12) and consists of 2 exons. Orail transcripts are differentially expressed throughout the body, including in immune cells (e.g., T cells, such as CD4 T cells and CD8 T cells), muscle tissues, and the gastrointestinal tract.
  • immune cells e.g., T cells, such as CD4 T cells and CD8 T cells
  • Exemplary nucleotide sequences and amino acid sequences of Orail can be found, for example, at GenBank Accession No. NM_032790.3 and UniProt Accession No. Q96D31 for Homo sapiens Orail; GenBank Accession No. NM_175423.3 and UniProt Accession No. Q8BWG9 for Mus musculus Orail; and GenBank Accession No. NM 001013982.1 and UniProt Accession No. Q5M848 for Rattus norvegicus Orai 1. Additional examples of Orai 1 sequences are readily available using, e.g., GenBank, UniProt, and OMIM web site.
  • Orail is a pore-forming subunit of a plasma membrane Ca 2+ release-activated Ca 2+ (CRAC) channel, which mediates store-operated calcium (SOC) influx into cells.
  • CRAC plasma membrane Ca 2+ release-activated Ca 2+
  • SOC store-operated calcium
  • Orail is activated by the Ca 2+ sensor, stromal interaction molecule 1 (STIM1), localized to the endoplasmic reticulum (ER) when intracellular Ca 2+ stores are depleted.
  • STIM1 stromal interaction molecule 1
  • Orail provides the primary pathway for calcium influx into T cells and plays a role in T cell activation and immune function.
  • SCID hereditary severe combined immune deficiency
  • Orail deficient mice exhibited severely attenuated cell- mediated immune responses in vivo that depend on TH1, TH2 and TH I 7 cell function, and absence of contact hypersensitivity responses with tolerance of skin allografts.
  • NF AT nuclear factor of activated T cells
  • cytokines such as IL-2, IL-4, IL- 17, IFN-y, and TNF-a that are crucial for T cell activation.
  • Orail binders are only weak or partial inhibitors of T cell function (see e.g., Lin et al., 2013, J. Pharmacol. Exp. Ther., 345:225; Gaida et al., 2015, J. Immunotoxicol ., 12: 164) such that they are not useful as stand-alone therapeutic molecules.
  • the degree of immunosuppression provided by these Orail antibodies is inferior to cyclosporine, a therapeutically relevant comparator which is a part of the standard of care for a variety of T cell- mediated inflammatory diseases and works downstream of the Orail Ca 2+ channel.
  • a parent anti-Orail binding domain is a binding domain in monospecific form, that is, not otherwise designed to bind to targets other than Orail.
  • the parent anti-Orail binding domain e.g., an antibody may or may not be functional as a therapeutic agent.
  • a parent Orail binding domain may or may not show T cell inhibition or CRAC channel inhibition as a single agent.
  • the parent Orail antibody has some therapeutic activity as a single agent, such as T cell inhibitory activity and/or CRAC channel inhibitory activity, for example, at least about 50% in routine assays such as any of the assays provided herein, including IL2 reduction and Ca ++ influx, and NF AT inhibition.
  • the parent anti-Orail binding domain may inhibit T cell activity and/or CRAC channel activity by about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less.
  • Orail multispecific antibodies of the present invention demonstrate improved and/or enhanced function as compared to the parent anti-Orail binding domain (e.g., inhibition of CRAC channel activity and/or inhibition of T cell activity).
  • there is at least about a 10% improvement or increase in function or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%, or more improvement or increase in function.
  • Orail multispecific antibody function there is at least about a 2-fold improvement or increase in Orail multispecific antibody function as compared to the parent Orail binding domain, or at least about 5-fold, or at least about 10-fold, or at least about 20-fold, or at least about 25-fold, or at least about 50-fold, or at least about 100-fold, or at least about 200-fold or more improvement or increase in function.
  • Any of the various assays provided elsewhere herein or known in the art can be used to measure the CRAC channel activity and/or the T cell activity.
  • the Orail multispecific design can reveal functionality of a parent Orail antibody that is otherwise considered non-functional.
  • the enhanced activity effect of the Orail multispecific antibodies provided herein may be additive or synergistic in nature as compared to the activity of the parent antigen binding domains alone.
  • Orail multispecific antibodies of the present invention demonstrate improved T cell function (e.g., IL-2 inhibition, NF AT inhibition, and/or Ca ++ influx) as compared to the parent Orail antibody, such that the multispecific antibody achieves a level of efficacy that is comparable to the standard of care cyclosporine.
  • T cell function e.g., IL-2 inhibition, NF AT inhibition, and/or Ca ++ influx
  • the second antigen binding domain of the disclosed Orail multispecific antibodies binds to a cell type-specific antigen or “targeting antigen” and can comprise any of the various antigen binding domains provided herein, such as an antibody, an antibody variable region, or an sdAb.
  • the targeting antigen is expressed on the cell surface of the target cell.
  • the second antigen binding domain binds to the targeting antigen with high affinity.
  • the Orail multispecific antibodies of the invention exploit the avidity of the second antigen binding domain to provide an improved therapeutic index for Orail binders.
  • the second antigen binding domain binds to a targeting antigen on a T cell or on a subset of T cells, including activated T cells, CD4 + T cells (helper T cells or Th cells) including T helper type 1 cells (Thl), T helper type 2 cells (Th2), Th2A, Th9, and Thl7 cells, Th22 cells, CD8 + T cells (cytotoxic or killer T cells) including Tel and Tc2 cells, effector T cells, memory T cells, natural killer T cells (NKT cells), regulatory T cells (Treg), and gamma delta T cells.
  • CD4 + T cells helper T cells or Th cells
  • Thl T helper type 1 cells
  • Th2A T helper type 2 cells
  • Th9 Th9
  • Thl7 cells Th22 cells
  • CD8 + T cells cytotoxic or killer T cells
  • Tel and Tc2 cells including Tel and Tc2 cells, effector T cells, memory T cells, natural killer T cells (NKT cells), regulatory T cells (Treg), and gamm
  • the targeting antigen is a white blood cell (e.g., mast cells), or epithelial cells (e.g., lung epithelial cells and intestinal epithelial cells), or pancreatic cells (e.g., acinar cells).
  • white blood cell e.g., mast cells
  • epithelial cells e.g., lung epithelial cells and intestinal epithelial cells
  • pancreatic cells e.g., acinar cells
  • T cell targeting antigens include, but are not limited to, PD1, CD4, CD3, CTLA4, CXCR4, CD25, ST2, CRTh2, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CLA, IL- 23R, IL-12R, CD52, CD122, CD127, CD27, KLRG-1, ICOS, CD45RA, GITR, 0X40, CD103, LAG3, CD28, CD8, CD2, CD5, CD6, CDl la, CD43, CD132, CD38, CD53, CD69, CD70, CD95, CD45RO, or CD45RB.
  • the targeting antigen is PD1.
  • programmed cell death protein 1 is used interchangeably with the term “PD1” refers to the well- known gene and polypeptide encoded by that gene, also known in the art as PDCD1, PD1, CD279, SLEB2, systemic lupus erythematosus susceptibility 2, HPD-L, HPD-1, ALWB2, and hSLEl.
  • the PD1 gene is located in the chromosomal region 2q37.3 (base pairs 241,848,978- 241,859,811 on chromosome 2) and consists of 6 exons.
  • the PD1 protein is expressed predominantly on immune cells, e.g., T cells.
  • Exemplary nucleotide sequences and amino acid sequences of PD1 can be found, for example, at GenBank AccessionNo. NM_005018.3 and UniProt Accession No. QI 5116 for Homo sapiensVD ,' GenBank AccessionNo. NM_008798.3 and UniProt Accession No. Q02242 for Mus musculus PD1; and GenBank Accession No. NM 001106927.1 and UniProt Accession No. D3ZIN8 for Rattus norvegicus PD1. Additional examples of PD1 sequences are readily available using, e.g., GenBank, UniProt, and OMIM web site.
  • PD1 expression is tightly and dynamically regulated, which makes PD1 an effective targeting antigen in the context of the disclosed Orail multispecific antibodies.
  • PD1 On resting naive T cells, as well as in certain populations of developing thymocytes, PD1 is expressed at low basal levels. Following an initial immune stimulus, PD1 is transiently expressed on multiple immune cell types, including CD4 and CD8 T cells.
  • CD4 and CD8 T cells include CD4 and CD8 T cells.
  • PD1 is down regulated in a time course that is concurrent with or prior to antigen clearance.
  • chronic immune stimulation PD1 remains highly expressed.
  • PD1 functions as an immune inhibitory receptor on the cell surface in activated T cells and regulates T cell functions including those of CD8 T cells.
  • PD1 promotes the differentiation of CD4 T cells into T regulatory cells.
  • Follicular helper T (TFH) cells also constitutively express high PD1 (Bally et al., 2016, J Immunol. 196(6): 2431-37).
  • a second antigen binding domain that binds PD1 may or may not have antagonistic or agonistic activity, but preferably has high binding affinity.
  • the targeting antigen is ST2.
  • ST2 the term, “suppressor of tumorigenicity 2” is used interchangeably with the term “ST2” refers to the well- known gene and polypeptide encoded by that gene, also known in the art as interleukin 1 receptorlike 1, IL1RL1, DER4, FIT-1, IL33R, ST2L, ST2V, Tl, suppression of tumorigenicity 2, EC 3.2.2.6, protein ST2, growth stimulation-expressed, interleukin 1 receptor-related protein, soluble interleukin 1 receptor like 1, and homolog of mouse growth stimulation expressed.
  • the ST2 gene is located in the chromosomal region 2ql2.1 (base pairs 102,311,529- 102,352,367 on chromosome 2) and consists of 13 exons.
  • the PD1 protein is expressed in placenta, kidney, adrenal gland, gall bladder, lung, and on immune cells, e.g., T cells.
  • Exemplary nucleotide sequences and amino acid sequences of ST2 can be found, for example, at NCBI Accession No. NM_016232.5 and UniProt Accession No. Q01638 for Homo sapiens ST2. Additional examples of ST2 sequences are readily available using, e.g., GenBank, UniProt, and OMIM web site. Further information on ST2 is provided, for example, in the NCBI Gene database at http://www.ncbi.nlm.nih.gov/gene/9173. The entire contents of each of the foregoing Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.
  • ST2 plays a role in T cell mediated allergic responses and is differentially expressed in Th2 cells.
  • ST2 is a receptor for IL-33 which drives Th2 cytokine production that has a significant role in the allergic process, including in asthma, atopic dermatitis, and food allergies.
  • a second antigen binding domain that binds ST2 may or may not have antagonistic or agonistic activity, but preferably has high binding affinity.
  • the first and/or second antigen binding domains of the disclosed Orail multispecific antibodies can each comprise a full-length antibody, a monoclonal antibody, a humanized antibody, an IgG antibody, an sdAb (e.g., a nanobody or VHH), an scFv, a variable heavy (VH) domain, a variable light (VL) domain, any antigen binding fragment of an antibody, or any combinations thereof.
  • an sdAb e.g., a nanobody or VHH
  • an scFv e.g., a variable heavy (VH) domain, a variable light (VL) domain, any antigen binding fragment of an antibody, or any combinations thereof.
  • the first antigen binding domain and/or the second antigen binding domain comprises an antibody or fragment thereof, such as a single domain antibody (sdAb).
  • the first and/or the second antigen binding domains comprise antibody-like formats, such as darpins, anticalins, or fibronectin domains.
  • the first antigen binding domain comprises an IgG monoclonal antibody and the second antigen binding domain comprises an sdAb.
  • the Orail binding domain is an antibody. Any Orail antibody can be used in the multispecific antibodies provided herein. Exemplary Orail antibodies include those disclosed in US 2012/0231006, US 2017/0226203, WO 2011/063277, and WO 2013/091903, each of which is herein incorporated by reference in their entirety.
  • Antibodies are immunoglobulin molecules that recognize and bind to a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • a specific target or antigen such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • antibody encompass any type of antibody, including but not limited to monoclonal antibodies, polyclonal antibodies, antigen-binding fragments of intact antibodies (e.g., Fab, Fab’, F(ab’)2, Fd, Fv, Fc, etc.) that retain the ability to specifically bind to a given antigen, bispecific antibodies, multispecific antibodies, heteroconjugate antibodies, fusion proteins having an antibody or antigen-binding fragment thereof, (e.g., a domain antibody), single chain antibodies, (scFv), single domain antibodies (sdAbs, also known as nanobodies and VHH antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that includes an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and
  • antibodies useful in the invention are antibodies that share a same antigen binding region with a reference antibody, for example, antibodies that compete for binding with a reference antibody and/or antibodies that bind to a same epitope as a reference antibody.
  • reference antibody refers to the Orail and target antigen binding domains described herein.
  • the antibodies useful in the invention are human, mouse, rat, rabbit, donkey, or camelid antibodies.
  • antibodies useful in the invention are obtained from camelid species such as dromedary camels, Bactrian camels, wild Bactrian camels, llamas, alpacas, vicunas, and guanacos.
  • camelid species such as dromedary camels, Bactrian camels, wild Bactrian camels, llamas, alpacas, vicunas, and guanacos.
  • camelid species such as dromedary camels, Bactrian camels, wild Bactrian camels, llamas, alpacas, vicunas, and guanacos.
  • CDRs hypervariable regions
  • antibodies useful in the invention are fully human antibodies, humanized antibodies, or chimeric antibodies.
  • Canonical antibodies comprise two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Each light chain is composed of one variable domain (VL) and one constant domain (CL). Light chains of the antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (X), based on the amino acid sequences of their constant domains. Each heavy chain comprises one variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD antibodies, comprises three domains termed CHI, CH2, and CH3 (IgM and IgE have a fourth domain, CH4).
  • variable domains of antibodies show considerable variation in amino acid composition from one antibody to another and are primarily responsible for antigen recognition and binding. Variable regions of each light/heavy chain pair form the antibody binding site such that an intact IgG antibody has two binding sites (i.e., it is bivalent). VH and VL domains comprise three regions of extreme variability, which are termed hypervariable regions, or more commonly, complementarity-determining regions (CDRs), framed and separated by four less variable regions known as framework regions (FRs). Non-covalent association between the VH and the VL region forms the Fv fragment (for "fragment variable") which contains one of the two antigen-binding sites of the antibody.
  • CDRs complementarity-determining regions
  • FRs framework regions
  • the assignment of amino acids to each domain, framework region and CDR may be in accordance with one of the schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5 th Ed.), US Dept, of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; Dubel, Ed.
  • variable region residue numbering is typically as set forth in Chothia or Kabat. Amino acid residues which comprise CDRs as defined by Kabat, Chothia, MacCallum (also known as Contact), Lefranc (also known as IMGT), and AbM as obtained from the Abysis website database (infra.) are set out in Table 1.
  • Variable regions and CDRs in an antibody sequence can be identified according to general rules that have been developed in the art (as set out above, such as, for example, the Kabat numbering system) or by aligning the sequences against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000.
  • Exemplary databases of antibody sequences are described in, and can be accessed through, the “IMGT” (ImMunoGeneTics) website at www.imgt.org; the “Abysis” website at www.bioinf.org.uk/abs (maintained by A.C. Martin in the Department of Biochemistry & Molecular Biology University College London, London, England) and the VBASE2 website at www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671 -D674 (2005). Unless otherwise indicated, all CDRs set forth herein are derived as per one of the definitions in Table 1.
  • epitope refers to the portion of a molecule that is bound by an antigen binding protein (for example, an antibody, an antigen binding domain, or an sdAb).
  • an antigen binding protein for example, an antibody, an antigen binding domain, or an sdAb.
  • the term includes any determinant capable of specifically binding to an antigen binding protein, such as an antibody an antigen binding domain, or an sdAb.
  • An epitope can be contiguous or non-contiguous (e.g., in a single-chain polypeptide, amino acid residues that are not contiguous to one another in the polypeptide sequence but that within the context of the molecule are bound by the antigen binding protein).
  • epitopes may be mimetic in that they comprise a three-dimensional structure that is similar to an epitope used to generate the antigen binding protein, yet comprise none or only some of the amino acid residues found in that epitope used to generate the antigen binding protein. Most often, epitopes reside on proteins, but in some instances may reside on other kinds of molecules, such as nucleic acids. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.
  • antigen binding domains e.g., antibodies
  • competition when used in the context of antigen binding domains (e.g., antibodies) that compete for the same epitope means competition between antigen binding domains is determined by an assay in which the antigen binding domain (e.g., antibody or fragment thereof) under test prevents or inhibits specific binding of a reference antigen binding domain (e.g., a ligand, or a reference antibody) to a common antigen (e.g., Orail or an epitope thereof).
  • a reference antigen binding domain e.g., a ligand, or a reference antibody
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid phase direct biotin-avidin EIA see, e.g., Kirkland et al., 1986, J. Immunol.
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotinavidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82).
  • such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antigen binding domain and a labeled reference antigen binding domain.
  • Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding domain.
  • the test antigen binding domain is present in excess.
  • Antigen binding domains identified by competition assay include antigen binding domains binding to the same epitope as the reference antigen binding domains and antigen binding domains binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding domain for steric hindrance to occur.
  • a competing antigen binding domain when present in excess, it will inhibit specific binding of a reference antigen binding domain to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.
  • an Orail multispecific antibody of the invention comprises a single domain antibody (sdAb), also known as a VHH molecule or nanobody.
  • sdAb single domain antibody
  • single domain antibody or “sdAb” or “single domain antibody fragment” are used interchangeably herein to refer to antigen binding domains or fragments, such as VHH domains or VH or VL domains that have a single antigen binding domain or single variable antibody domain.
  • the sdAbs can be light chain variable domain sequences (e.g., a VL-sequence), or heavy chain variable domain sequences (e.g., a VH-sequence); more specifically, they can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. Accordingly, the sdAbs can be domain antibodies, or immunoglobulin sequences that are suitable for use as domain antibodies, single domain antibodies, or immunoglobulin sequences that are suitable for use as single domain antibodies, or immunoglobulin sequences that are suitable for use as sdAbs or nanobodies, including but not limited to VHH sequences.
  • the structure of an sdAb can be considered, but not limited to, to be comprised of four framework regions or “FR's”, which are referred to in the art and herein as “Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”, respectively; which framework regions are interrupted by three complementary determining regions or “CDR's”, which are referred to in the art as “Complementarity Determining Region 1” or “CDR1”; as “Complementarity Determining Region 2” or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3”, respectively.
  • an sdAb comprising a VH domain comprises: FR1 comprising the amino acid residues at positions 1-30, CDR1 comprising the amino acid residues at positions 31-35, FR2 comprising the amino acids at positions 36-49, CDR2 comprising the amino acid residues at positions 50-65, FR3 comprising the amino acid residues at positions 66-94, CDR3 comprising the amino acid residues at positions 95-102, and FR4 comprising the amino acid residues at positions 103-113.
  • ST2 sdAbs single domain antibodies
  • the ST2 sdAbs provided herein can be the second antigen binding domain of the Orail multispecific antibodies.
  • Exemplary ST2 sdAbs comprise an amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
  • the ST2 sdAbs provided herein compete for binding with, bind a same epitope as, or comprise an antigen binding domain having three complementarity determining regions (CDRs) of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
  • CDRs can be defined by any of the various CDR definitions known in the art, for example, those depicted in Table 1.
  • Exemplary CDR sequences for the ST2 sdAbs provided herein are set forth as SEQ ID NOs 56-118 and are listed in Table 11 in Example 4.
  • the sdAbs can be synthesized by any method known in the art. An exemplary method is described elsewhere herein.
  • polypeptides e.g., antigen binding domains, antibodies, multispecific antibodies, sdAbs, or bispecific antibodies
  • a derived humanized antibody VH or VL domain may exhibit a sequence similarity with the source (e.g., murine) or acceptor (e.g., human) VH or VL domain.
  • a “homologous” polypeptide may exhibit 65%, 70%, 75%, 80%, 85%, or 90% sequence identity. In other aspects a “homologous” polypeptide may exhibit 93%, 95% or 98% sequence identity.
  • the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting Examples below.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. ' l (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 can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • Residue positions which are not identical may differ by conservative amino acid substitutions or by non-conservative amino acid substitutions.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain with similar chemical properties (e.g., charge or hydrophobicity).
  • a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution.
  • the polypeptide exhibiting sequence identity will retain the desired function or activity of the polypeptide of the invention (e.g., antibody).
  • the first antigen binding domain of the Orail multispecific antibodies provided herein comprises an Orail antibody, and in certain aspects, the first antigen binding domain is an Orail antibody as disclosed in U.S. Publication Number 2012/0231006, which is incorporated herein by reference in its entirety, or antibodies that bind to a same antigen binding site as, or that compete for binding with, or that bind to a same epitope as an Orail antibody as disclosed in U.S. Publication Number 2012/0231006.
  • Additional exemplary anti-Orai 1 antibodies are also contemplated for use in the multispecific antibodies of the present invention. Non-limiting examples include mAb 10F8 (WO2013/091903) and mAb hR198_HGl/LGl (US 2017/0226203).
  • the first antigen binding domain competes for binding with or binds to a same epitope as an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2.
  • CDRs complementarity determining regions
  • the first antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, and residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 2 for CDR-H1, residues 50-65 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3, wherein the residues are numbered according to Kabat; (b) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 2 for CDR-H1, residues 52-56 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ
  • the first antigen binding domain of an Orail multispecific antibody comprises a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 18, a CDRL2 comprising the amino acid sequence set forth as SEQ ID NO: 19, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 20; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 21, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 22, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 23.
  • the first antigen binding domain of an Orail multispecific antibody comprises a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5, and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2.
  • the first antigen binding domain of an Orail multispecific antibody comprises a light chain comprising an amino acid sequence set forth as SEQ ID NO: 15, and/or a heavy chain comprising an amino acid sequence set forth as SEQ ID NO: 14.
  • the first antigen binding domain competes for binding with or binds to a same epitope as (a) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (b) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or (c) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 151.
  • CDRs complement
  • the first antigen binding domain of the multispecific antibody comprises (a) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (b) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or (c) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 151.
  • the second antigen binding domain can bind to any targeting antigen, and in certain aspects, the targeting antigen is a T cell antigen, for example, PD1.
  • the second antigen binding domain is PD1 antibody, such as a sdAb, including, for example, a PD1 antibody as disclosed in U.S. Patent Number 10,323,090, which is incorporated herein by reference in its entirety, or antibodies that bind to a same antigen binding site as, or that compete for binding with, or that bind to a same epitope as PD1 antibody as disclosed in U.S. Patent Number 10,323,090.
  • the second antigen binding domain is PD1 antibody, such as a scFv or VHH, including, for example, a PD1 antibody as disclosed in US2019/032749, US2019/032749, US 2020/0347135, or US 2019/0322747, each of which is incorporated herein by reference in its entirety, or antibodies that bind to a same antigen binding site as, or that compete for binding with, or that bind to a same epitope as PD1 antibody as disclosed in US2019/032749, US 2020/0347135, and US 2019/0322747.
  • PD1 antibody such as a scFv or VHH
  • the second antigen binding domain is PD1 antibody derived from known antibodies, such as Nivolumab, Pembrolizumab, Sintilimab, Spartalizumab, Cemiplimab, Retifanlimab, Dostarlimab, Ezabenlimab, Cetrelimab, Budigalimab, Sasanlimab, and Vudalimab, or antibodies that bind to a same antigen binding site as, or that compete for binding with, or that bind to a same epitope as any of these antibodies.
  • known antibodies such as Nivolumab, Pembrolizumab, Sintilimab, Spartalizumab, Cemiplimab, Retifanlimab, Dostarlimab, Ezabenlimab, Cetrelimab, Budigalimab, Sasanlimab, and Vudalimab, or antibodies that bind to a same antigen binding site as, or that compete for binding
  • the second antigen binding domain of an Orail multispecific antibody competes for binding with or binds to a same epitope as an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 8.
  • the second antigen binding domain of the multispecific antibody competes for binding with, binds a same epitope as, or comprises (a) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 170; (b) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 171; (c) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 172; or (d) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 173.
  • the second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 31-35 of SEQ ID NO: 8 for CDR1, residues 50-65 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3, wherein the residues are numbered according to Kabat; (b) residues 26-32 of SEQ ID NO: 8 for CDR1, residues 52-56 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3, wherein the residues are numbered according to Chothia; (c) residues 30-35 of SEQ ID NO: 8 for CDR1, residues 47-58 of SEQ ID NO: 8 for CDR2, and residues 93-101 of SEQ ID NO: 8 for CDR3, wherein the residues are numbered according to MacCallum; or (d) residues 26-35 of SEQ ID NO: 8 for CDR1, residues 50-58 of SEQ ID NO: 8 for CDR2, and residues 95-
  • the second antigen binding domain of an Orail multispecific antibody comprises a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 24, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 26.
  • the second antigen binding domain of the multispecific antibody comprises the amino acid sequence set forth as SEQ ID NO: 8.
  • the second antigen binding domain of the multispecific antibody comprises (a) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 314, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 315, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 316; (b) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 322, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 323, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 324; (c) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 330, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 331, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 332; or (d) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 338, a CDR2
  • the second antigen binding domain of the multi specific antibody comprises the amino acid sequence set forth as SEQ ID NO: 8, 170. 171, 172, or 173.
  • the second antigen binding domain is an scFv.
  • the second antigen binding domain of the multispecific antibody competes for binding with, binds a same epitope as, or comprises (a) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 145 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 143; (b) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 154 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 153; (c) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 158 and/or three CDRs of a
  • the second antigen binding domain of the multispecific antibody comprises (a) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 145; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 143; (b) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 154; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 153; (c) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 158; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 157; (d) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 160; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 159; (e) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 162; and/or a heavy chain variable region
  • the second antigen binding domain can bind to ST2 as the T cell targeting antigen.
  • the second antigen binding domain is an ST2 antibody, such as an sdAb, including, for example, the ST2 sdAbs set forth as SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55, or antibodies that bind to a same antigen binding site as, or that compete for binding with, or that bind to a same epitope as the ST2 sdAbs set forth as SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
  • the ST2 second antigen binding domain of an Orail multispecific antibody competes for binding with or binds to a same epitope as an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
  • the ST2 second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 27-38 of SEQ ID NO: 35 for CDR1, residues 56-65 of SEQ ID NO: 35 for CDR2, and residues 105-117 of SEQ ID NO: 35 for CDR3; (b) residues 27-38 of SEQ ID NO: 36 for CDR1, residues 56-65 of SEQ ID NO: 36 for CDR2, and residues 105-117 of SEQ ID NO: 36 for CDR3; (c) residues 27-38 of SEQ ID NO: 37 for CDR1, residues 56-65 of SEQ ID NO: 37 for CDR2, and residues 105-117 of SEQ ID NO: 37 for CDR3; (d) residues 27-38 of SEQ ID NO: 38 for CDR1, residues 56-65 of SEQ ID NO: 38 for CDR2, and residues 105-117 of SEQ ID NO: 38 for CDR3 (e) residues 27-38 of SEQ ID NO: 39 for CDR1, residues
  • the ST2 second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 31-35 of SEQ ID NO: 35 for CDR1, residues 50-65 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 31-35 of SEQ ID NO: 36 for CDR1, residues 50-65 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 31-35 of SEQ ID NO: 37 for CDR1, residues 50-65 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 31-35 of SEQ ID NO: 38 for CDR1, residues 50-65 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 (e) residues 31-35 of SEQ ID NO: 39 for CDR1, residues 50-65 of
  • the ST2 second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 26-32 of SEQ ID NO: 35 for CDR1, residues 52-56 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 26-32 of SEQ ID NO: 36 for CDR1, residues 52-56 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 26-32 of SEQ ID NO: 37 for CDR1, residues 52-56 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 26-32 of SEQ ID NO: 38 for CDR1, residues 52-56 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3; (e) residues 26-32 of SEQ ID NO: 39 for CDR1, residues 52-56
  • the ST2 second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 30-35 of SEQ ID NO: 35 for CDR1, residues 47-58 of SEQ ID NO: 35 for CDR2, and residues 93-101 of SEQ ID NO: 35 for CDR3; (b) residues 30-35 of SEQ ID NO: 36 for CDR1, residues 47-58 of SEQ ID NO: 36 for CDR2, and residues 93-101 of SEQ ID NO: 36 for CDR3; (c) residues 30-35 of SEQ ID NO: 37 for CDR1, residues 47-58 of SEQ ID NO: 37 for CDR2, and residues 93-101 of SEQ ID NO: 37 for CDR3; (d) residues 30-35 of SEQ ID NO: 38 for CDR1, residues 47-58 of SEQ ID NO: 38 for CDR2, and residues 93-101 of SEQ ID NO: 38 for CDR3 (e) residues 30-35 of SEQ ID NO: 39 for CDR1, residues 47-58 of S
  • the ST2 second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 26-35 of SEQ ID NO: 35 for CDR1, residues 50-58 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 26-35 of SEQ ID NO: 36 for CDR1, residues 50-58 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 26-35 of SEQ ID NO: 37 for CDR1, residues 50-58 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 26-35 of SEQ ID NO: 38 for CDR1, residues 50-58 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 (e) residues 26-35 of SEQ ID NO: 39 for CDR1, residues 50-58 of
  • the Orail multispecific antibodies provided herein can comprise any combination of the first and the second antigen binding domains provided herein.
  • An exemplary Orail multispecific antibody of the invention comprises a second antigen binding domain that binds to PD1, for example, a multispecific antibody having the sequence set forth as SEQ ID NO: 17.
  • Additional Orail multispecific antibodies of the invention that comprise a second antigen binding domain that binds to PD1 include those set forth as SEQ ID NOs: 175-195.
  • the design of the Orail multispecific antibodies provided herein can include any of the first and second antigen binding domains as described above and optionally can include other components, such as, a signal peptide, a linker, a half-life extender, and/or a tag.
  • the Orail multispecific antibodies provided herein may comprise one or more linkers to connect the various antigen binding domains. Any suitable linker known in the art can be used to connect the antigen binding domains of the Orail multispecific antibodies. Non-limiting examples of a linker include a peptide linker, a chemical linker, a PEG linker, or a genetic linker. The first antigen binding domain and the second antigen binding domain may be covalently linked.
  • the first antigen binding domain and the second antigen binding domain of the Orail multispecific antibodies provided herein are connected via a peptide linker, i.e., any chain of amino acids.
  • the peptide linker comprises a glycine/ serine linker.
  • a peptide linker can be any length of amino acids, including at least 60 amino acids, at least 55 amino acids, at least 50 amino acids, at least 45 amino acids, at least 40 amino acids, at least 35 amino acids, at least 30 amino acids, at least 25 amino acids, at least 20 amino acids, at least 15 amino acids, at least 10 amino acids, at least 9 amino acids, at least 8 amino acids, at least 7 amino acids, at least 6 amino acids, at least 5 amino acids, at least 4 amino acids, at least 3 amino acids, at least 2 amino acids, or at least 1 amino acid in length.
  • the linker is about 35 amino acids in length.
  • the linker comprises the sequence set forth as SEQ ID NOs: 9, 11, 144, or 174.
  • the Orail multispecific antibodies of the invention comprise a half-life extender.
  • a “half-life extender” refers to any modification that increases the serum half-life of the Orai 1 multispecific antibody as compared to the Orai 1 multispecific antibody without the half-life extender.
  • an Orail multispecific antibody of the invention may be linked (chemically or otherwise) to one or more groups or moieties that extend the half-life (such as PEG), so as to provide Orail multispecific antibodies of the invention with increased halflife.
  • Non-limiting examples of such Orail multispecific antibodies comprising half-life extenders include chemically modified multispecific antibodies (for example, pegylation); Orail multispecific antibodies that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); and Orail multispecific antibodies of the invention which comprise at least one amino acid sequence that is linked to at least one moiety (and in particular at least one amino acid sequence) which increases the half-life of the Orail multispecific antibodies of the invention.
  • the Orail multispecific antibodies of the invention are suitably linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, antibodies, domain antibodies, single domain antibodies (sdAbs), VHH antibodies, or nanobodies that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrin.
  • serum proteins or fragments thereof such as (human) serum albumin or suitable fragments thereof
  • binding units that can bind to serum proteins
  • serum proteins such as, for example, antibodies, domain antibodies, single domain antibodies (sdAbs), VHH antibodies, or nanobodies that can bind to serum proteins
  • serum albumin such as human serum albumin
  • serum immunoglobulins such as IgG, or transferrin.
  • the half-life extender binds to the neonatal Fc receptor (FcRn).
  • the half-life extender can bind to the FcRn directly or indirectly.
  • a multispecific antibody may be directly fused or conjugated to an Fc sequence capable of binding to the FcRn or can bind indirectly via an FcRn binding protein.
  • the half-life extender is an Fc sequence comprising a YTE mutation. Mutations at positions 252, 254, and 256 of human IgGl to a Tyr (Y), Thr (T), and Glu (E), respectively, result in increased serum half-life as described in Dall'acqua WF, et al., Properties of human IgGls engineered for enhanced binding to the neonatal Fc receptor (FcRn), J Biol Chem. (2006) 281 :23514-24, which is incorporated herein in its entirety. See also U.S. Patent Nos.
  • the Orail multi specific antibodies of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding Orail multispecific antibody without the half-life extender.
  • the Orail multispecific antibodies of the invention with increased half-life may have a half-life e.g., in humans that is increased more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding Orail multispecific antibody without the half-life extender.
  • such Orail multispecific antibodies of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more.
  • multispecific antibodies of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).
  • the Orail multispecific antibody constructs of the invention can comprise any first antigen binding domain that bind to Orail, any second antigen binding domain that binds to a targeting antigen, and any linkers and/or half-life extenders provided herein.
  • the Orail multispecific antibody comprises (a) a first antigen binding domain that competes for binding with or binds a same epitope as an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5 and three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2; (b) a linker, for example a linker comprising the sequence set forth as SEQ ID NO: 11; and (c) a second antigen binding domain that competes for binding with or binds a same epitope as an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 8.
  • CDRs complementarity determining regions
  • the Orail the Orail multispecific antibody comprises (a) a first antigen binding domain that competes for binding with or binds a same epitope as (i) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (ii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or (iii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155 and/or three CDRs of a heavy chain variable region comprising an
  • the Orail the Orail multispecific antibody comprises (a) a first antigen binding domain that competes for binding with or binds a same epitope as (i) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (ii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or (iii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155 and/or three CDRs of a heavy chain variable region comprising an
  • the Orail multispecific antibody comprises (a) a first antigen binding domain comprising three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5 and three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2; (b) a linker, for example a linker comprising the sequence set forth as SEQ ID NO: 11; and (c) a second antigen binding domain comprising three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 8.
  • CDRs complementarity determining regions
  • the Orail multispecific antibody comprises (a) a first antigen binding domain comprising a heavy chain sequence comprising the sequence set forth as SEQ ID NO: 12 and a light chain sequence comprising the sequence set forth as SEQ ID NO: 13; (c) a linker comprising the sequence set forth as SEQ ID NO: 11; and (c) a second antigen binding domain comprising the sequence set forth as SEQ ID NO: 16.
  • the Orail multispecific antibody comprises the sequence set forth as SEQ ID NO: 17.
  • the Orail multispecific antibody comprises (a) a heavy chain (HC)- scFv set forth as SEQ ID NO: 175 and a light chain (LC) set forth as SEQ ID NO: 176; (b) a heavy chain (HC)-scFv set forth as SEQ ID NO: 177 and a light chain (LC) set forth as SEQ ID NO: 178; (c) a heavy chain (HC)-scFv set forth as SEQ ID NO: 179 and a light chain (LC) set forth as SEQ ID NO: 180; (d) a heavy chain (HC)-scFv set forth as SEQ ID NO: 181 and a light chain (LC) set forth as SEQ ID NO: 180; (e) a heavy chain (HC)-scFv set forth as SEQ ID NO: 182 and a light chain (LC) set forth as SEQ ID NO: 180; (f) a heavy chain (HC)-scFv set forth as SEQ ID NO: 183 and
  • the Orail multispecific antibody comprises (a) a first antigen binding domain that competes for binding with or binds a same epitope as an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5 and three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2; (b) a linker, for example a linker comprising the sequence set forth as SEQ ID NO: 11; and (c) a second antigen binding domain that competes for binding with or binds a same epitope as an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
  • CDRs complementarity determining regions
  • the present invention provides nucleic acid molecules that encode the Orail multispecific antibodies or the ST2 sdAbs provided herein and vectors and host cells comprising the nucleic acid molecules.
  • the nucleic acid molecules, vectors, and host cells can be used for expressing any of the Orail multispecific antibodies or the ST2 sdAbs provided herein.
  • nucleic acids that that exhibit “sequence identity”, sequence similarity” or “sequence homology” to the nucleic acids that encode the Orail multispecific antibodies or ST2 sdAbs of the invention, wherein the encoded Orail multispecific antibody or the ST2 sdAb exhibits the desired functionality.
  • a “homologous sequence” means a sequence of nucleic acid molecules exhibiting at least about 65%, 70%, 75%, 80%, 85%, or 90% sequence identity. In other aspects, a “homologous sequence” of nucleic acids may exhibit 93%, 95% or 98% sequence identity to the reference nucleic acid.
  • the instant invention also provides vectors comprising nucleic acid molecules that encode the Orail multispecific antibodies or ST2 sdAbs provided herein, which may be operably linked to a promoter (see, e.g., WO 86/05807; WO 89/01036; and U.S.P.N. 5,122,464); and other transcriptional regulatory and processing control elements of the eukaryotic secretory pathway.
  • the invention also provides host cells harboring those vectors and host-expression systems.
  • host-expression system includes any kind of cellular system that can be engineered to generate the nucleic acids, the Orail multispecific antibodies, or the ST2 sdAbs of the invention.
  • host-expression systems include, but are not limited to microorganisms (e.g., E. coli or B.
  • subtilis transformed or transfected with recombinant bacteriophage DNA or plasmid DNA; yeast (e.g., Saccharomyces) transfected with recombinant yeast expression vectors; or mammalian cells (e.g., COS, CHO-S, HEK293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells or viruses (e.g., the adenovirus late promoter).
  • the host cell may be co-transfected with two or more expression vectors, for example, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the host cell may also be engineered to allow the production of an antigen binding molecule with various characteristics (e.g., modified glycoforms or proteins having GnTIII activity).
  • cell lines that stably express the selected antibody, ST2 sdAb, or Orail multispecific antibody may be engineered using standard art recognized techniques and form part of the invention.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter or enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter or enhancer sequences, transcription terminators, polyadenylation sites, etc.
  • a selectable marker e.g., promoter or enhancer sequences, transcription terminators, polyadenylation sites, etc.
  • Any of the selection systems well known in the art may be used, including the glutamine synthetase gene expression system (the GS system) which provides an efficient approach for enhancing expression under selected conditions.
  • the GS system is discussed in whole or part in connection with EP 0 216 846, EP 0 256 055, EP 0 323 997 and EP 0 338 841 and U.S. Patent Nos. 5,591,639 and 5,879,936.
  • Another compatible expression system for the development of stable cell lines is the FreedomTM CHO-S Kit (Life Technologies).
  • an Orail multispecific antibody or ST2 sdAb of the invention may be purified or isolated by methods known in the art in that it is identified and separated and/or recovered from its natural environment and separated from contaminants that would interfere with therapeutic uses for the multispecific antibody.
  • isolated preparations may be purified using various art-recognized techniques, such as, for example, ion exchange and size exclusion chromatography, dialysis, diafiltration, and affinity chromatography, particularly Protein A or Protein G affinity chromatography. Compatible methods are discussed more fully in the Examples below.
  • the Orail multispecific antibodies of the present invention are designed to specifically target T cells and inhibit the activity and/or function of T cells, either in vitro and in vivo.
  • the Orail first antigen binding domain and the second antigen binding domain of the disclosed multispecific antibodies both bind to targets on T cells.
  • the first antigen binding domain is specific for a functional target on T cells (i.e., Orail) and binding of the first antigen binding domain to Orail on the T cell inhibits or reduces T cell activity.
  • the second antigen binding domain functions as a targeting component and directs the Orail multispecific antibody to a T cell by binding to a target molecule on the T cell.
  • inhibitor as used herein, is used interchangeably with “reduce,” “decrease,” and other similar terms, and includes any level of inhibition of T cell activity.
  • Inhibiting or reducing T cell activity can be any decrease in any T cell activity, including, for example, a decrease of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95% or greater from T cell activity observed in the absence of the Orail multispecific antibody.
  • Inhibiting or reducing T cell activity can also be an inhibition of any T cell activity by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 50-fold, about 100-fold or more from T cell activity observed in the absence of the Orail multispecific antibody.
  • any T cell activity can be inhibited by the Orail multispecific antibodies disclosed herein, including, but not limited to, cytokine release, such as IL-2, IFN-y, or TNF-a, T cell proliferation, up-regulation/expression of T cell activation markers (e.g., IL2RA/CD25), nuclear translocation and/or activity of NF AT, or intracellular calcium influx as described in, for example, U.S. Patent No. 10,981,995; U.S. Application Nos. 2017/0226203, 2012/0231006; Zappasodi R. et al., 2020, Methods EnzymoL 631 :43-59; Maguire, O. et al., 2013 Cytometry A.
  • cytokine release such as IL-2, IFN-y, or TNF-a
  • T cell proliferation up-regulation/expression of T cell activation markers (e.g., IL2RA/CD25), nuclear translocation and/or activity of NF AT, or intracellular
  • the inhibition of T cell activity may be specific to particular subsets of T cells as described herein.
  • the Orail multispecific constructs inhibit Th2 helper cell activity.
  • T cell function and/or activity may be assessed by a variety of methods known in the art, and exemplary assays are provided herein in Examples 2 and 3.
  • T cell proliferation is assessed by measuring incorporation of a radioactive tracer (e.g., [ 3 H]thymidine) or a fluorescent dye (e.g., bromodeoxyuridine) into T cells, which reflects the total amount of DNA synthesized in the T cell population.
  • a radioactive tracer e.g., [ 3 H]thymidine
  • a fluorescent dye e.g., bromodeoxyuridine
  • T cell proliferation is assessed by flow cytometry.
  • T cells are loaded with a dye such as fluorescent 5,6-carboxyfluorescein succinimidyl ester (CFSE), cell trace violet (CTV), and violet proliferation dye 450 (VPD-450), followed by incubation with ligands such as anti-CD3/anti-CD28 antibodies and assessment of cell divisions using a flow cytometer.
  • CFSE fluorescent 5,6-carboxyfluorescein succinimidyl ester
  • CTV cell trace violet
  • VPD-450 violet proliferation dye 450
  • a ligand or ionophore e.g., anti-CD3 antibody, anti-CD28 antibody, glucocorticoid-induced TNFR-related protein (GITR) ligand, thapsigargin, phorbol myristate acetate (PMA), ionomycin
  • a ligand or ionophore e.g., anti-CD3 antibody, anti-CD28 antibody, glucocorticoid-induced TNFR-related protein (GITR) ligand, thapsigargin, phorbol myristate acetate (PMA), ionomycin
  • effector cytokines e.g., FFNy, TNFa, IL-2, IL-4, IL-10, IL-17
  • cytokine secretion/release is measured by assays including, for example, enzyme-linked immunosolvent assay (ELISA), enzyme-linked immunospot (ELISPOT), or intracellular cytokine staining
  • T cell function and/or activity is evaluated by analyzing subcellular localization and/or activity of NF AT.
  • T cell function and/or activity is evaluated by assessing calcium influx.
  • Assays for assessing calcium influx are known in the art and include utilizing a calcium-binding dye (e.g., Fura-2) in combination with a fluorescent imaging plate reader (FLIPR) assay.
  • FLIPR fluorescent imaging plate reader
  • the Orail multispecific antibodies of the present invention may be used for treating or preventing diseases or disorders that can benefit from Orail inhibition, i.e., Orail -associated diseases and disorders.
  • the disclosed Orail multispecific antibodies are particularly useful for treating subjects with immune disorders including T cell mediated inflammatory disease, such as autoimmune disease and allergic disease.
  • the Orail multispecific antibodies can be administered alone or in combination with additional antiinflammatory or other agents.
  • treating refers to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more signs or symptoms associated with and Orail -associated disease or disorder, for example, an immune disease or disorder such as a T cell-mediated inflammatory disease, autoimmune disease and allergic disease. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. "Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.
  • an “effective amount” is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remediate the damage that results from or is associated with an Orail -associated disease or disorder.
  • the effective amount is a therapeutically effective amount or a prophylactically effective amount.
  • a “therapeutically effective amount” is an amount sufficient to remedy a disease state (e.g., transplant rejection or autoimmune disease) or symptom(s), particularly a state or symptom(s) associated with the disease state, or otherwise prevent, hinder, retard or reverse the progression of the disease state or any other undesirable symptom associated with the disease in any way whatsoever (i.e., that provides “therapeutic efficacy”).
  • a “prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of symptoms associated with a disease state, or reducing the likelihood of the onset (or reoccurrence) of symptoms.
  • the full therapeutic or prophylactic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses.
  • a therapeutically or prophylactically effective amount may be administered in one or more administrations.
  • the therapeutically or prophylactically effective amount may vary depending on the Orail multispecific antibody, how the Orail multispecific antibody is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated. Determination of an effective amount is within the skill of a trained clinician.
  • the Orail multispecific antibodies disclosed herein may be used to treat or prevent any of the various immune disorders or diseases as are recognized in the art.
  • the Orail multispecific antibodies are used to treat immune disorders or diseases.
  • the immune disorder is associated with T cells, including, for example, autoimmune disease and allergic disease.
  • the immune disease is a T cell-mediated inflammatory disease.
  • the T cell-mediated inflammatory disease is a chronic disease.
  • Non-limiting examples of immune disorders or diseases include, T cell-mediated chronic inflammatory diseases, autoimmune diseases, allergic diseases, rheumatoid arthritis, multiple sclerosis, type-1 diabetes, systemic lupus erythematosus (SLE), vitiligo, autoimmune thyroid disease (e.g., Graves’ disease, Hashimoto’s thyroiditis), pernicious anemia, alopecia areata, immune (idiopathic) thrombocytopenic purpura, celiac disease, atopic dermatitis (AD), eosinophilic esophagitis, nasal polyps, chronic rhinosinusitis with nasal polyps (CRSwNP), psoriasis, chronic plaque psoriasis (PsO), psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis), asthma, allergic rhinitis, e
  • a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g. , a monkey, and a chimpanzee), or a non-primate (such as a rat, or a mouse).
  • a primate such as a human, a non-human primate, e.g. , a monkey, and a chimpanzee
  • a non-primate such as a rat, or a mouse.
  • the subject is a human.
  • the human subject is being treated or assessed for an immune disorder or disease, or is at risk for developing an immune disorder or disease, that would benefit from reduction in T cell activity.
  • an Orail multispecific antibody disclosed herein is administered in combination with one or more additional therapies known to be effective in treating immune disorders (e.g., a T cell-mediated inflammatory disease, such as an autoimmune disease, or an allergic disease, or a symptom of such a disorder).
  • the Orail multispecific antibody may be administered before, after, or concurrent with the one or more additional therapies.
  • the Orail multispecific antibody is administered before the one or more additional therapies.
  • the Orail multispecific antibody is administered after the one or more additional therapies.
  • the Orail multispecific antibody is administered concurrent with the one or more additional therapies.
  • the Orail multispecific antibody is administered in conjunction with the one or more additional therapies.
  • the one or more additional therapies may be an additional therapeutic agent(s).
  • the Orail multispecific antibody and the additional therapeutic agent(s) can be administered in combination in the same composition or the additional therapeutic agent(s) can be administered as part of a separate composition.
  • the one or more additional therapies is a non-antibody therapeutic agent that is effective to treat the disorder or symptoms of the disorder. In some aspects, the one or more additional therapies is an antibody therapeutic agent that is effective to treat the disorder or symptoms of the disorder.
  • the Orail multispecific antibodies provided herein may, in certain aspects provide an enhanced effect (e.g., additive or synergistic in nature) that potentiates the mode of action of another administered therapeutic agent.
  • an enhanced effect e.g., additive or synergistic in nature
  • at least additive effects are generally desirable, any increased effect above one of the single therapies is beneficial.
  • the invention does not require the combined treatment to exhibit synergistic effects. However, those skilled in the art will appreciate that with certain selected combinations that comprise preferred aspects, synergism may be observed.
  • the one or more additional therapies may be a compound that also inhibits Orail.
  • Such compounds include, but are not limited to, CM4620, DS-2741a monoclonal antibody, CM6325, VV2003, SYL116011, SPLUNC peptide, PRCL-02, RP4010, RP3128, CM2489, CM3457, GSK- 7975A, RO2959, 10F8 monoclonal antibody.
  • the additional therapeutic agent(s) is an immunosuppressive agent, an immunomodulatory agent, an anti-inflammatory agent, or a biologic.
  • additional agents include, but are not limited to, corticosteroids (e.g., prednisone, prednisolone, methylprednisolone, budesonide, beclomethasone), calcineurin inhibitors [e.g., cyclosporine, also known as cyclosporin A (Sandimmune, Neoral, Gengraf), tacrolimus (Protopic, Prograf, FK506), pimecrolimus (Elidel)], methotrexate (Trexall, Otrexup, Xatmep, others), azathioprine (Azasan, Imuran), mercaptopurine (Purinethol, Purixan), leflunomide (Arava), hydroxychloroquine (Plaquenil), 5-aminosalicylates [e.g., s, s
  • the additional therapeutic agent(s) is a blocker of T cell costimulatory signals, such as a blocker of CD28, CTLA-4, CD80, and CD86, or a blocker of interactions between the receptor (e.g., CD28, CTLA-4) and the ligands (e.g., CD80, CD86).
  • the additional therapeutic agent is CTLA-4-IgG.
  • the additional therapeutic agent is an antibiotic, such as ciprofloxacin and metronidazole. In some aspects, the additional therapeutic agent is intestinal microbiome.
  • the additional therapeutic agent(s) aims to replenish cellular components or functions depleted or affected by the disease, the disorder, or the pathological process.
  • the additional therapeutic agent is thyroid hormone (to treat autoimmune thyroid disease), insulin (to treat type-1 diabetes), or vitamin B12 (to treat pernicious anemia).
  • the additional therapeutic agent aims to relieve symptoms or signs.
  • additional agents include, but are not limited to: pain relievers (see NSAIDs above); nutritional supplements [e.g., fish oil, dehydroepiandrosterone (DHEA)]; ointment, wet dressings, vitamin D analogs [e.g., calcipotriene, calcitriol (Vectical)], coal tar, anthralin (for diseases, disorders, and pathological processes involving skin conditions, e.g., atopic dermatitis, chronic plaque psoriasis); anti-diarrheals [e.g., psyllium powder (Metamucil), methylcellulose (Citrucel), loperamide (Imodium A-D)] (for diseases, disorders, and pathological processes involving bowel conditions, e.g., Crohn’s disease); proton pump inhibitor (for diseases, disorders, and pathological processes involving acid reflux, e.g.,
  • the one or more additional therapies is one or more of regulatory T cell (Treg) augmentation therapy, extracorporeal photopheresis, plasmapheresis, physical therapy, occupational therapy, acupuncture, psychological therapy, nutrition/dietary therapy, phototherapy (for skin conditions), renal replacement therapy (e.g., dialysis, for nephritis and renal failure), or surgery.
  • Surgery to treat diseases involving arthritis includes synovectomy, tendon repair, j oint fusion, and total j oint replacement.
  • Surgery for inflammatory bowel disease includes partial or total bowel resection.
  • Surgery for eosinophilic esophagitis includes dilatation of esophagus.
  • Surgery for asthma includes bronchial thermoplasty.
  • the second therapy may comprise transplant, e.g., kidney transplant (for renal failure, e.g., lupus nephritis), or pancreatic or islet cell transplant (for type-1 diabetes).
  • compositions or medicaments comprising the Orail multispecific antibodies or the ST2 sdAbs are also provided herein.
  • the disclosed Orail multispecific antibodies or the ST2 sdAbs of the invention may be formulated as pharmaceutical compositions as desired using art-recognized techniques.
  • the pharmaceutical compositions of the invention may be administered neat or with a minimum of additional components while others may optionally be formulated to contain suitable pharmaceutically acceptable carriers e.g., vehicles, adjuvants, and diluents) comprising excipients and auxiliaries that are well known in the art, including for example, pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, radioprotectants and the like.
  • Certain non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • Certain nonlimiting exemplary radioprotectants include ascorbic acid, gentisic acid, ethanol, and combinations thereof.
  • the compounds and compositions of the invention may be administered to a subject by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation.
  • routes including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation.
  • compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms suitable for the particular mode of administration, including, for example, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.
  • the particular dosage regimen for administering Orail multispecific antibodies of the invention z.e., dose, timing and repetition, will depend on the particular subject and that subject’s medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.). Frequency of administration may be determined and adjusted over the course of therapy.
  • a therapeutically effective dose is a dose sufficient to provide a clinical benefit to the subject, including for example, a dose sufficient to reduce swelling and/or redness, reduce skin rashes, hives and/or itching, reduce wheezing and/or shortness of breath, reduce sneezing and/or runny/stuffy nose, reduce anaphylaxis, decrease fatigue, reduce fever, reduce abdominal pain or digestive issues, decrease muscle aches, and/or reduce swelling in glands. Dosage administered may be adjusted or attenuated to manage potential side effects and/or toxicity.
  • kits that include a suitable container containing a Orail multispecific antibody or ST2 sdAb of the invention or a pharmaceutical composition of a Orail multispecific antibody or ST2 sdAb provided herein.
  • kits include one or more Orail multispecific antibodies or ST2 sdAbs and instructions for use, e.g., instructions for administering a therapeutically or prophylactically effective amount of a Orail multispecific antibody.
  • the Orail multispecific antibody or ST2 sdAb may be in a vial or a pre-filled syringe.
  • the kits may optionally further comprise means for administering the Orai 1 multispecific antibody or ST2 sdAb (e.g., an injection device, such as a pre-filled syringe or an intrathecal pump) or means for measuring the inhibition of T cell activity (e.g, means for measuring the inhibition of cytokine release).
  • Such means for measuring the inhibition of T cells may comprise a means for obtaining a sample from a subject, such as, e.g., a blood or plasma sample.
  • the kits of the invention may optionally further comprise means for determining the therapeutically effective amount.
  • the individual components of the pharmaceutical composition may be provided in one container.
  • the kit may be packaged in a number of different configurations such as one or more containers in a single box.
  • the different components can be combined, e.g., according to instructions provided with the kit.
  • the components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition.
  • the kit can also include a delivery device.
  • an element means one element or more than one element, e.g., a plurality of elements.
  • sample includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject.
  • biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like.
  • An anti-Orail monoclonal human IgG2 antibody with a lambda light chain (mAb 2C1.1; US 2012/0231006) was used as the Orail/first antigen binding domain in the synthesis of Orail multispecific antibodies as described herein.
  • an Orail multispecific antibody was prepared comprising a second antigen binding domain that is an anti-PDl VHH (sdAb) as disclosed in U.S. Patent Number 10,323,090.
  • This exemplary multispecific antibody is also referred to herein as “CTAB PR071” or “PR071”.
  • the expression constructs for the anti-Orai 1 antibody and the anti-PDl single chain VHH variants were cloned into a high expression mammalian vector and the DNA sequences of the gene insert were confirmed. Each DNA construct was scaled up for transfection and the DNA sequence was confirmed after DNA scale-up. A 0.1 Liter transient production was completed in CHO cells (TunaCHOTM extended 14-day process) for each.
  • the anti-Orai 1 antibody variants were purified by Protein A chromatography, and a final yield range of 6.93 mg to 30.95 mg was obtained.
  • the anti-PDl single chain VHH variants were purified by IMAC, and a final yield range of 0.72 mg to 30.34 mg was obtained. CE-SDS analysis was performed.
  • CTABs Additional exemplary anti-Orail/PDl multispecific antibodies
  • Tables 6 and 7 provide the components, including variable regions, linkers, and constant region sequences for the multispecific antibodies.
  • the Orail antibody components were from Orail antibodies disclosed in US 2012/0231006, W02013/091903, and US 2017/0226203.
  • the PD1 portion of the exemplary multispecific antibody constructs comprises a VHH or an scFv binding domain sequence.
  • the PD1 sequences are as disclosed in U.S.
  • Table 8 illustrates the sequences of the full-length constructs, including the Orail multispecific antibody constructs
  • Table 9 provides exemplary CDR sequences of the Orail/PDl multispecific antibody constructs.
  • Suspension CHO cells were seeded in a shake flask and expanded using a serum-free and chemically defined medium. On the day of transfection, the expanded cells were seeded into a new vessel with fresh medium. After transfection, the cells were maintained as a batch-fed culture until the end of the production run.
  • Clarified conditioned media from the production run was loaded onto a Protein A column pre-equilibrated with binding buffer. Washing buffer was passed through the column until the OD280 value returned to baseline. The protein was eluted with a low pH buffer, fractions were collected, buffered, and the OD280 value of each fraction was recorded. Fractions containing the protein of interest were pooled and filtered through a 0.2 pm membrane filter. The protein concentration was calculated from the OD280 value and the calculated extinction coefficient. IMAC (Immobilized Metal Affinity Chromatography) Purification of His Tagged Protein
  • Clarified and buffered conditioned media from the production run was loaded onto an IMAC column preequilibrated with binding buffer. Washing buffer containing 40 mM imidazole was passed through the column until the OD280 value returned to baseline. The protein was eluted with a linear gradient of increasing imidazole concentration up to 0.5 M. The eluate was collected in fractions, and the OD280 value of each fraction was recorded. Denaturing capillary electrophoresis (CE-SDS, LabChip GXII, Perkin Elmer) of each fraction was performed and analyzed. Fractions containing the protein of interest were pooled and dialyzed into buffer. The protein was filtered through a 0.2 pm membrane filter and the protein concentration was calculated using the OD280 value and the calculated extinction coefficient.
  • CE-SDS Denaturing capillary electrophoresis
  • CE-SDS Capillary Electrophoresis using Sodium Dodecyl Sulfate
  • CE-SDS analysis of the antibody protein was performed using a LabChip GXII (Perkin Elmer).
  • Intact mass analysis by mass spectrometry was performed (Xevo G2-XS Qtof, Waters). The observed molecular weights for the IgG were within the expected range.
  • Endotoxin in a sample of the purified product was quantified using the chromogenic Limulus Amebocyte Lysate method (Endosafe-MCS, Charles River). The sample was run in duplicate. Endotoxin measurements confirmed all samples met the ⁇ 1 EU/mg requirement.
  • SEC size exclusion chromatography
  • CE-SDS Capillary Electrophoresis- Sodium Dodecyl Sulfate
  • SE-UPLC Size Exclusion-Ultra High Pressure Liquid Chromatography
  • Table 4 Exemplary CDR Sequences for Orail and Target Antigen Binding Domains
  • Table 5 Additional Exemplary Orail Antibody Sequences
  • the Orail/PDl multispecific antibodies provided herein were tested in an IL-2 cytokine release assay to determine the effect on T cell activity.
  • Human PBMCs were seeded in 24-well tissue culture plates at a density of 10 6 cells/ml in a volume of 900 pL. The PBMCs were treated for 1 hour at 37°C with various concentrations of Anti-Orai-1 mAB (2C1.1 human IgG2), Anti- Orail/Anti-PDl VHH multispecific antibody, anti-Orail antibody and anti-PDl VHH (equimolar), anti-PDl VHH, or cyclosporine A.
  • Anti-Orai-1 mAB (2C1.1 human IgG2)
  • Anti- Orail/Anti-PDl VHH multispecific antibody anti-Orail antibody and anti-PDl VHH (equimolar)
  • anti-PDl VHH or cyclosporine A.
  • FIG. 2 depicts the percent inhibition of IL-2 release with the various treatments.
  • the graph in FIG. 2 shows that the Orail/PDl VHH multispecific antibody inhibited IL-2 release.
  • the dose-response curve in FIG. 2 demonstrates that the anti -Orail/PDl VHH multispecific antibody exhibited enhanced inhibition of IL-2 release as compared to the parent anti-Orail antibody.
  • the level of IL-2 cytokine suppression achieved with the anti- Orail/PDl VHH multispecific antibody was equivalent to therapeutically relevant levels of the immunosuppressant agent cyclosporine.
  • PD1 -expressing Jurkat T cells containing a nuclear factor of activated T-cells (NFAT) luciferase reporter were utilized to assess activity of Cell Targeting Biologies (CTABs).
  • Reporter cells were cultured in assay media consisting of RPMI (Gibco, 72400-047) supplemented with 10% FBS (Gibco, J1211).
  • Supernatants from CHO cells secreting CTAB proteins targeting PD1 and Orail were diluted 1 : 10, 1 :20, or 1 : 50 in assay media and 50uL of each was dispensed in wells of a white, flat-bottom 96 well plate (Thermo, 136102).
  • Reporter cells were next thawed and diluted in assay media according to manufacturer’s instructions and 50uL of cell suspension was added to each well. Cells and test supernatants were next mixed by pipette and preincubated for 1 hour at 37C at 5% CO2. After incubation, cells were mixed by pipetting and 80uL was transferred to a lug/mL anti-CD3 (Biolegend, 300465) coated 96 well plate for stimulation. The coated stimulation plates were white, flat-bottomed plates coated overnight with lug/mL anti-CD3 diluted in PBS (Gibco, 14190-144). Plates were washed three times with PBS and aspirated immediately prior to the addition of reporter cells.
  • FIG. 4 depicts the percent inhibition of NF AT at various nM concentrations of the anti- Orail/PDl VHH multispecific antibody (CTAB PR071; anti-Orail IgG/anti-PDl VHH).
  • CTAB PR071 anti-Orail IgG/anti-PDl VHH.
  • the dose-response curve in FIG. 4 demonstrates that the anti-Orail/PDl VHH multispecific antibody exhibited enhanced inhibition of NF AT as compared to the parent anti-Orail antibody (BB PR001; anti-Orail IgG).
  • FIG. 5 depicts the percent inhibition of NF AT at various dilutions of various anti- Orail/PDl multispecific antibodies (CTABs). The results show that the Orail/PDl multispecific antibodies exhibited enhanced inhibition of NF AT as compared to the Orail and PD1 individual antibody components.
  • CTABs anti- Orail/PDl multispecific antibodies
  • ST2 single domain (VHH) antigen binding domains were prepared as described in methods provided elsewhere herein.
  • Exemplary sequences of ST2 VHH antibodies are depicted as SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 in Table 10.
  • Exemplary CDR sequences for the ST2 VHH antibodies are illustrated in Table 11.
  • Orail multispecific antibodies comprising any of the various ST2 VHH antigen binding domains provided herein can be synthesized as described in Example 1.
  • ST2-expressing T cells are generated essentially as described in Calise et al. (2021, J. Allergy Clin. Immunol. 148(3):867-875), which is incorporated herein by reference in its entirety.
  • human PBMCs are obtained from subjects with allergy to peanut, alder pollen, grass pollen, or dust mite.
  • the PBMCs are stimulated with allergen plus or minus recombinant human IL-33 for 18 hours.
  • the cells are enriched for CD154 expressing cells using anti-CD154 antibody and magnetic beads. ST2 expression is monitored by flow cytometry.
  • the ST2-expressing T cells are treated with any of the anti-Orail/anti-ST2 multispecific antibodies provided herein and a cytokine release assay is performed as described in Example 2.

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Abstract

The disclosure relates to multispecific antibody compositions that bind to Orai1 and a second target, and methods of using such multispecific antibody compositions, for example, to inhibit T cell activity and/or treat or prevent immune disorders or diseases.

Description

ORAI1 MULTISPECIFIC ANTIBODIES AND METHODS OF USE
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Application No.
63/248,399, filed on September 24, 2021, and U.S. Provisional Application No. 63/316,742, filed on March 4, 2022, the entire contents of each of which are hereby incorporated herein by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on September 23, 2022, is named F113316_1020WO_SL.xml and is 322,269 bytes in size.
FIELD OF THE INVENTION
The disclosure generally relates to compositions comprising Orail multispecific antibody agents and methods of their use.
BACKGROUND OF THE INVENTION
Calcium release activated calcium modulator 1 (Orail) is a transmembrane protein and is the pore forming subunit of calcium release activated calcium (CRAC) channels which mediate store-operated calcium (SOC) influx into cells. Orail is activated by the Ca2+ sensor, stromal interaction molecule 1 (STIM1), localized to the endoplasmic reticulum (ER) when intracellular Ca2+ stores are depleted.
Orail provides the primary pathway for calcium influx into T cells and plays an important role in T cell activation and immune function. The sustained calcium influx into T cells leads to activation of transcription factors, such as nuclear factor of activated T cells (NF AT) resulting in the transcription of cytokine genes critical for T cell activation.
Accordingly, Orail is a therapeutically relevant target for treating immune disorders, including T cell-mediated disorders, such as autoimmune disease and allergic disease. However, current Orail binders are only weak inhibitors of T cell function. Therefore, there is a need for improved therapeutic agents that can target Orail and inhibit T cell function and/or activity. BRIEF SUMMARY OF THE INVENTION
The present disclosure provides Orail multispecific antibodies and methods of using the Orail multispecific antibodies.
In one aspect, the present invention provides a multispecific antibody comprising a first antigen binding domain that binds to Orai 1 ; and a second antigen binding domain that binds to a target on a T cell; wherein the multispecific antibody inhibits T cell activity.
In one embodiment, the first antigen binding domain of the multispecific antibody competes for binding with, binds a same epitope as, or comprises (a) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2 (b) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (c) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or (d) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 151. In one embodiment, the first antigen binding domain of the multispecific antibody comprises (a) residues 27-38 of SEQ ID NO: 5 for CDR-L1, residues 56-65 of SEQ ID NO: 5 for CDR-L2, and residues 105-117 of SEQ ID NO: 5 for CDR- L3; and/or residues 27-38 of SEQ ID NO: 2 for CDR-H1, residues 56-65 of SEQ ID NO: 2 for CDR-H2, and residues 105-117 of SEQ ID NO: 2 for CDR-H3; (b) residues 27-38 of SEQ ID NO: 146 for CDR-L1, residues 56-65 of SEQ ID NO: 146 for CDR-L2, and residues 105-117 of SEQ ID NO: 146 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 140 for CDR-H1, residues 56-65 of SEQ ID NO: 140 for CDR-H2, and residues 105-117 of SEQ ID NO: 140 for CDR-H3; (c) residues 27-38 of SEQ ID NO: 149 for CDR-L1, residues 56-65 of SEQ ID NO: 149 for CDR-L2, and residues 105-117 of SEQ ID NO: 149 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 147 for CDR-H1, residues 56-65 of SEQ ID NO: 147 for CDR-H2, and residues 105-117 of SEQ ID NO: 147 for CDR-H3; or (d) residues 27-38 of SEQ ID NO: 155 for CDR-L1, residues 56-65 of SEQ ID NO: 155 for CDR-L2, and residues 105-117 of SEQ ID NO: 155 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 151 for CDR-H1, residues 56-65 of SEQ ID NO: 151 for CDR- H2, and residues 105-117 of SEQ ID NO: 151 for CDR-H3; wherein the residues are numbered according to Lefranc (IMGT).
In one embodiment, the first antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, and residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 2 for CDR-H1, residues 50-65 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 146 for CDR-L1, residues 50-56 of SEQ ID NO: 146 for CDR-L2, and residues 89-97 of SEQ ID NO: 146 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 140 for CDR-H1, residues 50-65 of SEQ ID NO: 140 for CDR-H2, and residues 95-102 of SEQ ID NO: 140 for CDR-H3; (c) residues 24-34 of SEQ ID NO: 149 for CDR-L1, residues 50-56 of SEQ ID NO: 149 for CDR-L2, and residues 89-97 of SEQ ID NO: 149 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 147 for CDR-H1, residues 50-65 of SEQ ID NO: 147 for CDR-H2, and residues 95-102 of SEQ ID NO: 147 for CDR-H3; or (d) residues 24- 34 of SEQ ID NO: 155 for CDR-L1, residues 50-56 of SEQ ID NO: 155 for CDR-L2, and residues 89-97 of SEQ ID NO: 155 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 151 for CDR-H1, residues 50-65 of SEQ ID NO: 151 for CDR-H2, and residues 95-102 of SEQ ID NO: 151 for CDR-H3; wherein the residues are numbered according to Kabat.
In one embodiment, the first antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 2 for CDR-H1, residues 52-56 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 146 for CDR-L1, residues 50-56 of SEQ ID NO: 146 for CDR-L2, residues 89-97 of SEQ ID NO: 146 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 140 for CDR-H1, residues 52-56 of SEQ ID NO: 140 for CDR-H2, and residues 95- 102 of SEQ ID NO: 140 for CDR-H3; (c) residues 24-34 of SEQ ID NO: 149 for CDR-L1, residues 50-56 of SEQ ID NO: 149 for CDR-L2, residues 89-97 of SEQ ID NO: 149 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 147 for CDR-H1, residues 52-56 of SEQ ID NO: 147 for CDR- H2, and residues 95-102 of SEQ ID NO: 147 for CDR-H3; or (d) residues 24-34 of SEQ ID NO: 155 for CDR-L1, residues 50-56 of SEQ ID NO: 155 for CDR-L2, residues 89-97 of SEQ ID NO: 155 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 151 for CDR-H1, residues 52-56 of SEQ ID NO: 151 for CDR-H2, and residues 95-102 of SEQ ID NO: 151 for CDR-H3; wherein the residues are numbered according to Chothia.
In one embodiment, the first antigen binding domain of the multispecific antibody comprises (a) residues 30-36 of SEQ ID NO: 5 for CDR-L1, residues 46-55 of SEQ ID NO: 5 for CDR-L2, and residues 89-96 of SEQ ID NO: 5 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 2 for CDR-H1, residues 47-58 of SEQ ID NO: 2 for CDR-H2, and residues 93-101 of SEQ ID NO: 2 for CDR-H3; (b) residues 30-36 of SEQ ID NO: 146 for CDR-L1, residues 46-55 of SEQ ID NO: 146 for CDR-L2, and residues 89-96 of SEQ ID NO: 146 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 140 for CDR-H1, residues 47-58 of SEQ ID NO: 140 for CDR-H2, and residues 93-101 of SEQ ID NO: 140 for CDR-H3; (c) residues 30-36 of SEQ ID NO: 149 for CDR-L1, residues 46-55 of SEQ ID NO: 149 for CDR-L2, and residues 89-96 of SEQ ID NO: 149 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 147 for CDR-H1, residues 47-58 of SEQ ID NO: 147 for CDR-H2, and residues 93-101 of SEQ ID NO: 147 for CDR-H3; or (d) residues SOSO of SEQ ID NO: 155 for CDR-L1, residues 46-55 of SEQ ID NO: 155 for CDR-L2, and residues 89-96 of SEQ ID NO: 155 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 151 for CDR-H1, residues 47-58 of SEQ ID NO: 151 for CDR-H2, and residues 93-101 of SEQ ID NO: 151 for CDR-H3; wherein the residues are numbered according to MacCallum.
In one embodiment, the first antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, and residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 2 for CDR-H1, residues 50-58 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 146 for CDR-L1, residues 50-56 of SEQ ID NO: 146 for CDR-L2, and residues 89-97 of SEQ ID NO: 146 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 140 for CDR-H1, residues 50-58 of SEQ ID NO: 140 for CDR-H2, and residues 95-102 of SEQ ID NO: 140 for CDR-H3; (c) residues 24-34 of SEQ ID NO: 149 for CDR-L1, residues 50-56 of SEQ ID NO: 149 for CDR-L2, and residues 89-97 of SEQ ID NO: 149 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 147 for CDR-H1, residues 50-58 of SEQ ID NO: 147 for CDR-H2, and residues 95-102 of SEQ ID NO: 147 for CDR-H3; or (d) residues 24-
Figure imgf000006_0001
89-97 of SEQ ID NO: 155 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 151 for CDR-H1, residues 50-58 of SEQ ID NO: 151 for CDR-H2, and residues 95-102 of SEQ ID NO: 151 for CDR-H3; wherein the residues are numbered according to AbM.
In another embodiment, the first antigen binding domain of the multispecific antibody comprises (a) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 18, a CDRL2 comprising the amino acid sequence set forth as SEQ ID NO: 19, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 20; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 21, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 22, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 23; (b) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 199, a CDRL2 comprising the amino acid sequence VYN, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 200; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 196, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 197, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 198; (c) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 223, a CDRL2 comprising the amino acid sequence STS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 224; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 220, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 221, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 222; or (d) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 235, a CDRL2 comprising the amino acid sequence WAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 236; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 232, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 233, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 234.
In one embodiment, the first antigen binding domain of the multispecific antibody comprises (a) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2; (b) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (c) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or (d) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 151.
In another embodiment, the first antigen binding domain of the multispecific antibody comprises a light chain comprising an amino acid sequence set forth as SEQ ID NO: 15; and/or a heavy chain comprising an amino acid sequence set forth as SEQ ID NO: 14.
In some embodiments, the first antigen binding domain of the multispecific antibody comprises a single domain antibody (sdAb).
In one embodiment, the second antigen binding domain the multispecific antibody binds to a target on activated T cells.
In some embodiments, the second antigen binding domain of the multispecific antibody comprises a single domain antibody (sdAb).
In some embodiments, the second antigen binding domain of the multispecific antibody comprises an scFv.
In another embodiment, the second antigen binding domain of the multispecific antibody binds to programmed cell death protein 1 (PD1).
In one embodiment, the second antigen binding domain of the multispecific antibody competes for binding with, binds a same epitope as, or comprises (a) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 8; (b) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 170; (c) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 171; (d) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 172; or (e) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 173.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) residues 31-35 of SEQ ID NO: 8 for CDR1, residues 50-65 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3; (b) residues 31-35 of SEQ ID NO: 170 for CDR1, residues 50-65 of SEQ ID NO: 170 for CDR2, and residues 95-102 of SEQ ID NO: 170 for CDR3; (c) residues 31-35 of SEQ ID NO: 171 for CDR1, residues 50-65 of SEQ ID NO:
171 for CDR2, and residues 95-102 of SEQ ID NO: 171 for CDR3; (d) residues 31-35 of SEQ ID NO: 172 for CDR1, residues 50-65 of SEQ ID NO: 172 for CDR2, and residues 95-102 of SEQ ID NO: 172 for CDR3; or (e) residues 31-35 of SEQ ID NO: 173 for CDR1, residues 50-65 of SEQ ID NO: 173 for CDR2, and residues 95-102 of SEQ ID NO: 173 for CDR3; wherein the residues are numbered according to Kabat.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) residues 26-32 of SEQ ID NO: 8 for CDR1, residues 52-56 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3; (b) residues 26-32 of SEQ ID NO: 170 for CDR1, residues 52-56 of SEQ ID NO: 170 for CDR2, and residues 95-102 of SEQ ID NO:
170 for CDR3; (c) residues 26-32 of SEQ ID NO: 171 for CDR1, residues 52-56 of SEQ ID NO:
171 for CDR2, and residues 95-102 of SEQ ID NO: 171 for CDR3; (d) residues 26-32 of SEQ ID NO: 172 for CDR1, residues 52-56 of SEQ ID NO: 172 for CDR2, and residues 95-102 of SEQ ID NO: 172 for CDR3; or (e) residues 26-32 of SEQ ID NO: 173 for CDR1, residues 52-56 of SEQ ID NO: 173 for CDR2, and residues 95-102 of SEQ ID NO: 173 for CDR3; wherein the residues are numbered according to Chothia.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) residues 30-35 of SEQ ID NO: 8 for CDR1, residues 47-58 of SEQ ID NO: 8 for CDR2, and residues 93-101 of SEQ ID NO: 8 for CDR3; (b) residues 30-35 of SEQ ID NO: 170 for CDR1, residues 47-58 of SEQ ID NO: 170 for CDR2, and residues 93-101 of SEQ ID NO:
170 for CDR3; (c) residues 30-35 of SEQ ID NO: 171 for CDR1, residues 47-58 of SEQ ID NO:
171 for CDR2, and residues 93-101 of SEQ ID NO: 171 for CDR3; (d) residues 30-35 of SEQ ID NO: 172 for CDR1, residues 47-58 of SEQ ID NO: 172 for CDR2, and residues 93-101 of SEQ ID NO: 172 for CDR3; or (e) residues 30-35 of SEQ ID NO: 173 for CDR1, residues 47-58 of SEQ ID NO: 173 for CDR2, and residues 93-101 of SEQ ID NO: 173 for CDR3; wherein the residues are numbered according to MacCallum.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) residues 26-35 of SEQ ID NO: 8 for CDR1, residues 50-58 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3; (b) residues 26-35 of SEQ ID NO: 170 for CDR1, residues 50-58 of SEQ ID NO: 170 for CDR2, and residues 95-102 of SEQ ID NO: 170 for CDR3; (c) residues 26-35 of SEQ ID NO: 171 for CDR1, residues 50-58 of SEQ ID NO: 171 for CDR2, and residues 95-102 of SEQ ID NO: 171 for CDR3; (d) residues 26-35 of SEQ ID NO: 172 for CDR1, residues 50-58 of SEQ ID NO: 172 for CDR2, and residues 95-102 of SEQ ID NO: 172 for CDR3; or (e) residues 26-35 of SEQ ID NO: 173 for CDR1, residues 50-58 of SEQ ID NO: 173 for CDR2, and residues 95-102 of SEQ ID NO: 173 for CDR3; wherein the residues are numbered according to AbM.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 24, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 26; (b) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 314, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 315, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 316; (c) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 322, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 323, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 324; (d) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 330, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 331, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 332; or (e) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 338, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 339, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 340.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises the amino acid sequence set forth as SEQ ID NO: 8, 170. 171, 172, or 173.
In one embodiment, the second antigen binding domain of the multispecific antibody competes for binding with, binds a same epitope as, or comprises (a) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 145 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 143; (b) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 154 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 153; (c) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 158 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 157; (d) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 160 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 169; (e) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 162 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 161; (f) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 164 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 163; (g) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 167 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 165; or (h) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 168 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 169.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) residues 27-38 of SEQ ID NO: 145 for CDR-L1, residues 56-65 of SEQ ID NO: 145 for CDR-L2, and residues 105-117 of SEQ ID NO: 145 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 143 for CDR-H1, residues 56-65 of SEQ ID NO: 143 for CDR-H2, and residues 105-117 of SEQ ID NO: 143 for CDR-H3; (b) residues 27-38 of SEQ ID NO: 154 for CDR-L1, residues 56-65 of SEQ ID NO: 154 for CDR-L2, and residues 105-117 of SEQ ID NO: 154 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 153 for CDR-H1, residues 56-65 of SEQ ID NO: 153 for CDR-H2, and residues 105-117 of SEQ ID NO: 153 for CDR-H3; (c) residues 27-38 of SEQ ID NO: 158 for CDR-L1, residues 56-65 of SEQ ID NO: 158 for CDR-L2, and residues 105- 117 of SEQ ID NO: 158 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 157 for CDR-H1, residues 56-65 of SEQ ID NO: 157 for CDR-H2, and residues 105-117 of SEQ ID NO: 157 for CDR-H3; (d) residues 27-38 of SEQ ID NO: 160 for CDR-L1, residues 56-65 of SEQ ID NO: 160 for CDR-L2, and residues 105-117 of SEQ ID NO: 160 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 159 for CDR-H1, residues 56-65 of SEQ ID NO: 159 for CDR-H2, and residues 105- 117 of SEQ ID NO: 159 for CDR-H3; (e) residues 27-38 of SEQ ID NO: 162 for CDR-L1, residues 56-65 of SEQ ID NO: 162 for CDR-L2, and residues 105-117 of SEQ ID NO: 162 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 161 for CDR-H1, residues 56-65 of SEQ ID NO: 161 for CDR-H2, and residues 105-117 of SEQ ID NO: 161 for CDR-H3; (f) residues 27-38 of SEQ ID NO: 164 for CDR-L1, residues 56-65 of SEQ ID NO: 164 for CDR-L2, and residues 105-117 of SEQ ID NO: 164 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 163 for CDR-H1, residues 56-65 of SEQ ID NO: 163 for CDR-H2, and residues 105-117 of SEQ ID NO: 163 for CDR-H3; (g) residues 27-38 of SEQ ID NO: 167 for CDR-L1, residues 56-65 of SEQ ID NO: 167 for CDR- L2, and residues 105-117 of SEQ ID NO: 167 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 165 for CDR-H1, residues 56-65 of SEQ ID NO: 165 for CDR-H2, and residues 105-117 of SEQ ID NO: 165 for CDR-H3; or (h) residues 27-38 of SEQ ID NO: 168 for CDR-L1, residues 56-65 of SEQ ID NO: 168 for CDR-L2, and residues 105-117 of SEQ ID NO: 168 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 169 for CDR-H1, residues 56-65 of SEQ ID NO: 169 for CDR- H2, and residues 105-117 of SEQ ID NO: 169 for CDR-H3; wherein the residues are numbered according to Lefranc (IMGT).
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 145 for CDR-L1, residues 50-56 of SEQ ID NO: 145 for CDR-L2, and residues 89-97 of SEQ ID NO: 145 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 143 for CDR-H1, residues 50-65 of SEQ ID NO: 143 for CDR-H2, and residues 95-102 of SEQ ID NO: 143 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 154 for CDR-L1, residues 50-56 of SEQ ID NO: 154 for CDR-L2, and residues 89-97 of SEQ ID NO: 154 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 153 for CDR-H1, residues 50-65 of SEQ ID NO: 153 for CDR-H2, and residues 95-102 of SEQ ID NO: 153 for CDR-H3; (c) residues 24-34 of SEQ ID NO: 158 for CDR-L1, residues 50-56 of SEQ ID NO: 158 for CDR-L2, and residues 89-97 of SEQ ID NO: 158 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 157 for CDR-H1, residues 50-65 of SEQ ID NO: 157 for CDR-H2, and residues 95-102 of SEQ ID NO: 157 for CDR-H3; or (d) residues 24-34 of SEQ ID NO: 160 for CDR-L1, residues 50-56 of SEQ ID NO: 160 for CDR-L2, and residues 89-97 of SEQ ID NO: 160 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 159 for CDR-H1, residues 50-65 of SEQ ID NO: 159 for CDR-H2, and residues 95-102 of SEQ ID NO: 159 for CDR-H3; (e) residues 24-34 of SEQ ID NO: 162 for CDR-L1, residues 50-56 of SEQ ID NO: 162 for CDR-L2, and residues 89-97 of SEQ ID NO: 162 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 161 for CDR-H1, residues 50-65 of SEQ ID NO: 161 for CDR-H2, and residues 95-102 of SEQ ID NO: 161 for CDR-H3; (f) residues 24-34 of SEQ ID NO: 164 for CDR- Ll, residues 50-56 of SEQ ID NO: 164 for CDR-L2, and residues 89-97 of SEQ ID NO: 164 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 163 for CDR-H1, residues 50-65 of SEQ ID NO: 163 for CDR-H2, and residues 95-102 of SEQ ID NO: 163 for CDR-H3; (g) residues 24-34 of SEQ ID NO: 167 for CDR-L1, residues 50-56 of SEQ ID NO: 167 for CDR-L2, and residues 89- 97 of SEQ ID NO: 167 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 165 for CDR-H1, residues 50-65 of SEQ ID NO: 165 for CDR-H2, and residues 95-102 of SEQ ID NO: 165 for CDR-H3; or (h) residues 24-34 of SEQ ID NO: 168 for CDR-L1, residues 50-56 of SEQ ID NO: 168 for CDR-L2, and residues 89-97 of SEQ ID NO: 168 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 169 for CDR-H1, residues 50-65 of SEQ ID NO: 169 for CDR-H2, and residues 95- 102 of SEQ ID NO: 169 for CDR-H3; wherein the residues are numbered according to Kabat.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 145 for CDR-L1, residues 50-56 of SEQ ID NO: 145 for CDR-L2, residues 89-97 of SEQ ID NO: 145 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 143 for CDR-H1, residues 52-56 of SEQ ID NO: 143 for CDR-H2, and residues 95-102 of SEQ ID NO: 143 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 154 for CDR-L1, residues SO- 56 of SEQ ID NO: 154 for CDR-L2, residues 89-97 of SEQ ID NO: 154 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 153 for CDR-H1, residues 52-56 of SEQ ID NO: 153 for CDR- H2, and residues 95-102 of SEQ ID NO: 153 for CDR-H3; (c) residues 24-34 of SEQ ID NO: 158 for CDR-L1, residues 50-56 of SEQ ID NO: 158 for CDR-L2, residues 89-97 of SEQ ID NO: 158 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 157 for CDR-H1, residues 52-56 of SEQ ID NO: 157 for CDR-H2, and residues 95-102 of SEQ ID NO: 157 for CDR-H3; or (d) residues 24- 34 of SEQ ID NO: 160 for CDR-L1, residues 50-56 of SEQ ID NO: 160 for CDR-L2, residues 89-97 of SEQ ID NO: 160 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 159 for CDR-H1, residues 52-56 of SEQ ID NO: 159 for CDR-H2, and residues 95-102 of SEQ ID NO: 159 for CDR-H3; (e) residues 24-34 of SEQ ID NO: 162 for CDR-L1, residues 50-56 of SEQ ID NO: 162 for CDR-L2, residues 89-97 of SEQ ID NO: 162 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 161 for CDR-H1, residues 52-56 of SEQ ID NO: 161 for CDR-H2, and residues 95-102 of SEQ ID NO: 161 for CDR-H3; (f) residues 24-34 of SEQ ID NO: 164 for CDR-L1, residues 50- 56 of SEQ ID NO: 164 for CDR-L2, residues 89-97 of SEQ ID NO: 164 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 163 for CDR-H1, residues 52-56 of SEQ ID NO: 163 for CDR- H2, and residues 95-102 of SEQ ID NO: 163 for CDR-H3; (g) residues 24-34 of SEQ ID NO: 167 for CDR-L1, residues 50-56 of SEQ ID NO: 167 for CDR-L2, residues 89-97 of SEQ ID NO: 167 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 165 for CDR-H1, residues 52-56 of SEQ ID NO: 165 for CDR-H2, and residues 95-102 of SEQ ID NO: 165 for CDR-H3; or (h) residues 24- 34 of SEQ ID NO: 168 for CDR-L1, residues 50-56 of SEQ ID NO: 168 for CDR-L2, residues 89-97 of SEQ ID NO: 168 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 169 for CDR-H1, residues 52-56 of SEQ ID NO: 169 for CDR-H2, and residues 95-102 of SEQ ID NO: 169 for CDR-H3; wherein the residues are numbered according to Chothia.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) residues 30-36 of SEQ ID NO: 145 for CDR-L1, residues 46-55 of SEQ ID NO: 145 for CDR-L2, and residues 89-96 of SEQ ID NO: 145 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 143 for CDR-H1, residues 47-58 of SEQ ID NO: 143 for CDR-H2, and residues 93-101 of SEQ ID NO: 143 for CDR-H3; (b) residues 30-36 of SEQ ID NO: 154 for CDR-L1, residues 46-55 of SEQ ID NO: 154 for CDR-L2, and residues 89-96 of SEQ ID NO: 154 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 153 for CDR-H1, residues 47-58 of SEQ ID NO: 153 for CDR-H2, and residues 93-101 of SEQ ID NO: 153 for CDR-H3; (c) residues 30-36 of SEQ ID NO: 158 for CDR-L1, residues 46-55 of SEQ ID NO: 158 for CDR-L2, and residues 89-96 of SEQ ID NO: 158 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 157 for CDR-H1, residues 47-58 of SEQ ID NO: 157 for CDR-H2, and residues 93-101 of SEQ ID NO: 157 for CDR-H3; or (d) residues 30-36 of SEQ ID NO: 160 for CDR-L1, residues 46-55 of SEQ ID NO: 160 for CDR-L2, and residues 89-96 of SEQ ID NO: 160 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 159 for CDR-H1, residues 47-58 of SEQ ID NO: 159 for CDR-H2, and residues 93-101 of SEQ ID NO: 159 for CDR-H3; (e) residues 30-36 of SEQ ID NO: 162 for CDR-L1, residues 46-55 of SEQ ID NO: 162 for CDR-L2, and residues 89-96 of SEQ ID NO: 162 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 161 for CDR-H1, residues 47-58 of SEQ ID NO: 161 for CDR-H2, and residues 93-101 of SEQ ID NO: 161 for CDR-H3; (f) residues 30-36 of SEQ ID NO: 164 for CDR- Ll, residues 46-55 of SEQ ID NO: 164 for CDR-L2, and residues 89-96 of SEQ ID NO: 164 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 163 for CDR-H1, residues 47-58 of SEQ ID NO: 163 for CDR-H2, and residues 93-101 of SEQ ID NO: 163 for CDR-H3; (g) residues 30-36 of SEQ ID NO: 167 for CDR-L1, residues 46-55 of SEQ ID NO: 167 for CDR-L2, and residues 89-
96 of SEQ ID NO: 167 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 165 for CDR-H1, residues 47-58 of SEQ ID NO: 165 for CDR-H2, and residues 93-101 of SEQ ID NO: 165 for CDR-H3; or (h) residues 30-36 of SEQ ID NO: 168 for CDR-L1, residues 46-55 of SEQ ID NO: 168 for CDR-L2, and residues 89-96 of SEQ ID NO: 168 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 169 for CDR-H1, residues 47-58 of SEQ ID NO: 169 for CDR-H2, and residues 93- 101 of SEQ ID NO: 169 for CDR-H3; wherein the residues are numbered according to MacCallum.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) residues 24-34 of SEQ ID NO: 145 for CDR-L1, residues 50-56 of SEQ ID NO: 145 for CDR-L2, and residues 89-97 of SEQ ID NO: 145 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 143 for CDR-H1, residues 50-58 of SEQ ID NO: 143 for CDR-H2, and residues 95-102 of SEQ ID NO: 143 for CDR-H3; (b) residues 24-34 of SEQ ID NO: 154 for CDR-L1, residues 50-56 of SEQ ID NO: 154 for CDR-L2, and residues 89-97 of SEQ ID NO: 154 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 153 for CDR-H1, residues 50-58 of SEQ ID NO: 153 for CDR-H2, and residues 95-102 of SEQ ID NO: 153 for CDR-H3; (c) residues 24-34 of SEQ ID NO: 158 for CDR-L1, residues 50-56 of SEQ ID NO: 158 for CDR-L2, and residues 89-97 of SEQ ID NO: 158 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 157 for CDR-H1, residues 50-58 of SEQ ID NO: 157 for CDR-H2, and residues 95-102 of SEQ ID NO: 157 for CDR-H3; or (d) residues 24-34 of SEQ ID NO: 160 for CDR-L1, residues 50-56 of SEQ ID NO: 160 for CDR-L2, and residues 89-97 of SEQ ID NO: 160 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 159 for CDR-H1, residues 50-58 of SEQ ID NO: 159 for CDR-H2, and residues 95-102 of SEQ ID NO: 159 for CDR-H3; (e) residues 24-34 of SEQ ID NO: 162 for CDR-L1, residues 50-56 of SEQ ID NO: 162 for CDR-L2, and residues 89-97 of SEQ ID NO: 162 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 161 for CDR-H1, residues 50-58 of SEQ ID NO: 161 for CDR-H2, and residues 95-102 of SEQ ID NO: 161 for CDR-H3; (f) residues 24-34 of SEQ ID NO: 164 for CDR- Ll, residues 50-56 of SEQ ID NO: 164 for CDR-L2, and residues 89-97 of SEQ ID NO: 164 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 163 for CDR-H1, residues 50-58 of SEQ ID NO: 163 for CDR-H2, and residues 95-102 of SEQ ID NO: 163 for CDR-H3; (g) residues 24-34 of SEQ ID NO: 167 for CDR-L1, residues 50-56 of SEQ ID NO: 167 for CDR-L2, and residues 89-
97 of SEQ ID NO: 167 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 165 for CDR-H1, residues 50-58 of SEQ ID NO: 165 for CDR-H2, and residues 95-102 of SEQ ID NO: 165 for CDR-H3; or (h) residues 24-34 of SEQ ID NO: 168 for CDR-L1, residues 50-56 of SEQ ID NO: 168 for CDR-L2, and residues 89-97 of SEQ ID NO: 168 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 169 for CDR-H1, residues 50-58 of SEQ ID NO: 169 for CDR-H2, and residues 95- 102 of SEQ ID NO: 169 for CDR-H3; wherein the residues are numbered according to AbM.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 211, a CDRL2 comprising the amino acid sequence AAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 212; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 208, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 209, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 210; (b) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 246, a CDRL2 comprising the amino acid sequence DAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 247; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 243, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 244, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 245; (c) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 258, a CDRL2 comprising the amino acid sequence WAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 259; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 255, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 256, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 257; (d) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 270, a CDRL2 comprising the amino acid sequence VAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 271; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 267, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 268, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 269; (e) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 282, a CDRL2 comprising the amino acid sequence DAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 283; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 279, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 280, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 281; (f) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 292, a CDRL2 comprising the amino acid sequence AAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 293; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 243, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 290, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 291; (g) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 302, a CDRL2 comprising the amino acid sequence YAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 303; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 299, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 300, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 301; or (h) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 311, a CDRL2 comprising the amino acid sequence YAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 312; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 299, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 300, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 301.
In one embodiment, the second antigen binding domain of the multispecific antibody comprises (a) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 145; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 143; (b) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 154; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 153; (c) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 158; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 157; (d) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 160; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 159; (e) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 162; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 161; (f) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 164; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 163; (g) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 167; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 165; or (h) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 168; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 169. In another embodiment, the second antigen binding domain binds to suppressor of tumorigenicity 2 (ST2).
In one embodiment, the second antigen binding domain competes for binding with, binds a same epitope as, or comprises an antigen binding domain having three CDRs of a variable region sequence comprising (a) an amino acid sequence set forth as SEQ ID NO: 35; (b) an amino acid sequence set forth as SEQ ID NO: 36; (c) an amino acid sequence set forth as SEQ ID NO: 37; (d) an amino acid sequence set forth as SEQ ID NO: 38; (e) an amino acid sequence set forth as SEQ ID NO: 39; (f) an amino acid sequence set forth as SEQ ID NO: 40; (g) an amino acid sequence set forth as SEQ ID NO: 41; (h) an amino acid sequence set forth as SEQ ID NO: 42; (i) an amino acid sequence set forth as SEQ ID NO: 43; (j) an amino acid sequence set forth as SEQ ID NO: 44; (k) an amino acid sequence set forth as SEQ ID NO: 45; (1) an amino acid sequence set forth as SEQ ID NO: 46; (m) an amino acid sequence set forth as SEQ ID NO: 47; (n) an amino acid sequence set forth as SEQ ID NO: 48; (o) an amino acid sequence set forth as SEQ ID NO: 49; (p) an amino acid sequence set forth as SEQ ID NO: 50; (q) an amino acid sequence set forth as SEQ ID NO: 51; (r) an amino acid sequence set forth as SEQ ID NO: 52; (s) an amino acid sequence set forth as SEQ ID NO: 53; (t) an amino acid sequence set forth as SEQ ID NO: 54; or (u) an amino acid sequence set forth as SEQ ID NO: 55.
In one embodiment, the second antigen binding domain comprises (a) residues 27-38 of SEQ ID NO: 35 for CDR1, residues 56-65 of SEQ ID NO: 35 for CDR2, and residues 105-117 of SEQ ID NO: 35 for CDR3; (b) residues 27-38 of SEQ ID NO: 36 for CDR1, residues 56-65 of SEQ ID NO: 36 for CDR2, and residues 105-117 of SEQ ID NO: 36 for CDR3; (c) residues 27- 38 of SEQ ID NO: 37 for CDR1, residues 56-65 of SEQ ID NO: 37 for CDR2, and residues 105- 117 of SEQ ID NO: 37 for CDR3; (d) residues 27-38 of SEQ ID NO: 38 for CDR1, residues 56- 65 of SEQ ID NO: 38 for CDR2, and residues 105-117 of SEQ ID NO: 38 for CDR3 (e) residues 27-38 of SEQ ID NO: 39 for CDR1, residues 56-65 of SEQ ID NO: 39 for CDR2, and residues 105-117 of SEQ ID NO: 39 for CDR3; (f) residues 27-38 of SEQ ID NO: 40 for CDR1, residues 56-65 of SEQ ID NO: 40 for CDR2, and residues 105-117 of SEQ ID NO: 40 for CDR3; (g) residues 27-38 of SEQ ID NO: 41 for CDR1, residues 56-65 of SEQ ID NO: 41 for CDR2, and residues 105-117 of SEQ ID NO: 41 for CDR3; (h) residues 27-38 of SEQ ID NO: 42 for CDR1, residues 56-65 of SEQ ID NO: 42 for CDR2, and residues 105-117 of SEQ ID NO: 42 for CDR3; (i) residues 27-38 of SEQ ID NO: 43 for CDR1, residues 56-65 of SEQ ID NO: 43 for CDR2, and residues 105-117 of SEQ ID NO: 43 for CDR3; (j) residues 27-38 of SEQ ID NO: 44 for CDR1, residues 56-65 of SEQ ID NO: 44 for CDR2, and residues 105-117 of SEQ ID NO: 44 for CDR3; (k) residues 27-38 of SEQ ID NO: 45 for CDR1, residues 56-65 of SEQ ID NO: 45 for CDR2, and residues 105-117 of SEQ ID NO: 45 for CDR3; (1) residues 27-38 of SEQ ID NO: 46 for CDR1, residues 56-65 of SEQ ID NO: 46 for CDR2, and residues 105-117 of SEQ ID NO: 46 for CDR3; (m) residues 27-38 of SEQ ID NO: 47 for CDR1, residues 56-65 of SEQ ID NO: 47 for CDR2, and residues 105-117 of SEQ ID NO: 47 for CDR3; (n) residues 27-38 of SEQ ID NO: 48 for CDR1, residues 56-65 of SEQ ID NO: 48 for CDR2, and residues 105-117 of SEQ ID NO: 48 for CDR3; (o) residues 27-38 of SEQ ID NO: 49 for CDR1, residues 56-65 of SEQ ID NO: 49 for CDR2, and residues 105-117 of SEQ ID NO: 49 for CDR3; (p) residues 27-38 of SEQ ID NO: 50 for CDR1, residues 56-65 of SEQ ID NO: 50 for CDR2, and residues 105-117 of SEQ ID NO: 50 for CDR3; (q) residues 27-38 of SEQ ID NO: 51 for CDR1, residues 56-65 of SEQ ID NO: 51 for CDR2, and residues 105-117 of SEQ ID NO: 51 for CDR3; (r) residues 27-38 of SEQ ID NO: 52 for CDR1, residues 56-65 of SEQ ID NO: 52 for CDR2, and residues 105-117 of SEQ ID NO: 52 for CDR3; (s) residues 27-38 of SEQ ID NO: 53 for CDR1, residues 56-65 of SEQ ID NO: 53 for CDR2, and residues 105-117 of SEQ ID NO: 53 for CDR3; (t) residues 27-38 of SEQ ID NO: 54 for CDR1, residues 56-65 of SEQ ID NO: 54 for CDR2, and residues 105-117 of SEQ ID NO: 54 for CDR3; or (u) residues 27-38 of SEQ ID NO: 55 for CDR1, residues 56-65 of SEQ ID NO: 55 for CDR2, and residues 105-117 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Lefranc (IMGT).
In one embodiment, the second antigen binding domain comprises (a) residues 31-35 of SEQ ID NO: 35 for CDR1, residues 50-65 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 31-35 of SEQ ID NO: 36 for CDR1, residues 50-65 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 31-35 of SEQ ID NO: 37 for CDR1, residues 50-65 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 31-35 of SEQ ID NO: 38 for CDR1, residues 50-65 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 (e) residues 31-35 of SEQ ID NO: 39 for CDR1, residues 50-65 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3; (f) residues 31-35 of SEQ ID NO: 40 for CDR1, residues 50-65 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3; (g) residues 31-35 of SEQ ID NO: 41 for CDR1, residues 50-65 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3; (h) residues 31-35 of SEQ ID NO: 42 for CDR1, residues 50-65 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3; (i) residues 31-35 of SEQ ID NO: 43 for CDR1, residues 50-65 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3; (j) residues 31-35 of SEQ ID NO: 44 for CDR1, residues 50-65 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3; (k) residues 31-35 of SEQ ID NO: 45 for CDR1, residues 50-65 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3; (1) residues 31-35 of SEQ ID NO: 46 for CDR1, residues 50-65 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3; (m) residues 31-35 of SEQ ID NO: 47 for CDR1, residues 50-65 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3; (n) residues 31-35 of SEQ ID NO: 48 for CDR1, residues 50-65 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3; (o) residues 31-35 of SEQ ID NO: 49 for CDR1, residues 50-65 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3; (p) residues 31-35 of SEQ ID NO: 50 for CDR1, residues 50-65 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3; (q) residues 31-35 of SEQ ID NO: 51 for CDR1, residues 50-65 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3; (r) residues 31-35 of SEQ ID NO: 52 for CDR1, residues 50-65 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3; (s) residues 31-35 of SEQ ID NO: 53 for CDR1, residues 50-65 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3; (t) residues 31-35 of SEQ ID NO: 54 for CDR1, residues 50-65 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or (u) residues 31- 35 of SEQ ID NO: 55 for CDR1, residues 50-65 of SEQ ID NO: 55 for CDR2, and residues 95- 102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Kabat.
In one embodiment, the second antigen binding domain comprises (a) residues 26-32 of SEQ ID NO: 35 for CDR1, residues 52-56 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 26-32 of SEQ ID NO: 36 for CDR1, residues 52-56 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 26-32 of SEQ ID NO: 37 for CDR1, residues 52-56 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 26-32 of SEQ ID NO: 38 for CDR1, residues 52-56 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3; (e) residues 26-32 of SEQ ID NO: 39 for CDR1, residues 52-56 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3; (f) residues 26-32 of SEQ ID NO: 40 for CDR1, residues 52-56 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3; (g) residues 26-32 of SEQ ID NO: 41 for CDR1, residues 52-56 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3; (h) residues 26-32 of SEQ ID NO: 42 for CDR1, residues 52-56 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3; (i) residues 26-32 of SEQ ID NO: 43 for CDR1, residues 52-56 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3; (j) residues 26-32 of SEQ ID NO: 44 for CDR1, residues 52-56 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3; (k) residues 26-32 of SEQ ID NO: 45 for CDR1, residues 52-56 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3; (1) residues 26-32 of SEQ ID NO: 46 for CDR1, residues 52-56 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3; (m) residues 26-32 of SEQ ID NO: 47 for CDR1, residues 52-56 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3; (n) residues 26-32 of SEQ ID NO: 48 for CDR1, residues 52-56 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3; (o) residues 26-32 of SEQ ID NO: 49 for CDR1, residues 52-56 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3; (p) residues 26-32 of SEQ ID NO: 50 for CDR1, residues 52-56 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3; (q) residues 26-32 of SEQ ID NO: 51 for CDR1, residues 52-56 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3; (r) residues 26-32 of SEQ ID NO: 52 for CDR1, residues 52-56 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3; (s) residues 26-32 of SEQ ID NO: 53 for CDR1, residues 52-56 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3; (t) residues 26-32 of SEQ ID NO: 54 for CDR1, residues 52-56 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or (u) residues 26- 32 of SEQ ID NO: 55 for CDR1, residues 52-56 of SEQ ID NO: 55 for CDR2, and residues 95- 102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Chothia.
In one embodiment, the second antigen binding domain comprises (a) residues 30-35 of SEQ ID NO: 35 for CDR1, residues 47-58 of SEQ ID NO: 35 for CDR2, and residues 93-101 of SEQ ID NO: 35 for CDR3; (b) residues 30-35 of SEQ ID NO: 36 for CDR1, residues 47-58 of SEQ ID NO: 36 for CDR2, and residues 93-101 of SEQ ID NO: 36 for CDR3; (c) residues 30-35 of SEQ ID NO: 37 for CDR1, residues 47-58 of SEQ ID NO: 37 for CDR2, and residues 93-101 of SEQ ID NO: 37 for CDR3; (d) residues 30-35 of SEQ ID NO: 38 for CDR1, residues 47-58 of SEQ ID NO: 38 for CDR2, and residues 93-101 of SEQ ID NO: 38 for CDR3 (e) residues 30-35 of SEQ ID NO: 39 for CDR1, residues 47-58 of SEQ ID NO: 39 for CDR2, and residues 93-101 of SEQ ID NO: 39 for CDR3; (f) residues 30-35 of SEQ ID NO: 40 for CDR1, residues 47-58 of SEQ ID NO: 40 for CDR2, and residues 93-101 of SEQ ID NO: 40 for CDR3; (g) residues 30-35 of SEQ ID NO: 41 for CDR1, residues 47-58 of SEQ ID NO: 41 for CDR2, and residues 93-101 of SEQ ID NO: 41 for CDR3; (h) residues 30-35 of SEQ ID NO: 42 for CDR1, residues 47-58 of SEQ ID NO: 42 for CDR2, and residues 93-101 of SEQ ID NO: 42 for CDR3; (i) residues 30-35 of SEQ ID NO: 43 for CDR1, residues 47-58 of SEQ ID NO: 43 for CDR2, and residues 93-101 of SEQ ID NO: 43 for CDR3; (j) residues 30-35 of SEQ ID NO: 44 for CDR1, residues 47-58 of SEQ ID NO: 44 for CDR2, and residues 93-101 of SEQ ID NO: 44 for CDR3; (k) residues 30-35 of SEQ ID NO: 45 for CDR1, residues 47-58 of SEQ ID NO: 45 for CDR2, and residues 93-101 of SEQ ID NO: 45 for CDR3; (1) residues 30-35 of SEQ ID NO: 46 for CDR1, residues 47-58 of SEQ ID NO: 46 for CDR2, and residues 93-101 of SEQ ID NO: 46 for CDR3; (m) residues 30-35 of SEQ ID NO: 47 for CDR1, residues 47-58 of SEQ ID NO: 47 for CDR2, and residues 93-101 of SEQ ID NO: 47 for CDR3; (n) residues 30-35 of SEQ ID NO: 48 for CDR1, residues 47-58 of SEQ ID NO: 48 for CDR2, and residues 93-101 of SEQ ID NO: 48 for CDR3; (o) residues 30-35 of SEQ ID NO: 49 for CDR1, residues 47-58 of SEQ ID NO: 49 for CDR2, and residues 93-101 of SEQ ID NO: 49 for CDR3; (p) residues 30-35 of SEQ ID NO: 50 for CDR1, residues 47-58 of SEQ ID NO: 50 for CDR2, and residues 93-101 of SEQ ID NO: 50 for CDR3; (q) residues 30-35 of SEQ ID NO: 51 for CDR1, residues 47-58 of SEQ ID NO: 51 for CDR2, and residues 93-101 of SEQ ID NO: 51 for CDR3; (r) residues 30-35 of SEQ ID NO: 52 for CDR1, residues 47-58 of SEQ ID NO: 52 for CDR2, and residues 93-101 of SEQ ID NO: 52 for CDR3; (s) residues 30-35 of SEQ ID NO: 53 for CDR1, residues 47-58 of SEQ ID NO: 53 for CDR2, and residues 93-101 of SEQ ID NO: 53 for CDR3; (t) residues 30-35 of SEQ ID NO: 54 for CDR1, residues 47-58 of SEQ ID NO: 54 for CDR2, and residues 93-101 of SEQ ID NO: 54 for CDR3; or (u) residues 30- 35 of SEQ ID NO: 55 for CDR1, residues 47-58 of SEQ ID NO: 55 for CDR2, and residues 93- 101 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to MacCallum.
In one embodiment, the second antigen binding domain comprises (a) residues 26-35 of SEQ ID NO: 35 for CDR1, residues 50-58 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 26-35 of SEQ ID NO: 36 for CDR1, residues 50-58 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 26-35 of SEQ ID NO: 37 for CDR1, residues 50-58 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 26-35 of SEQ ID NO: 38 for CDR1, residues 50-58 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 (e) residues 26-35 of SEQ ID NO: 39 for CDR1, residues 50-58 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3; (f) residues 26-35 of SEQ ID NO: 40 for CDR1, residues 50-58 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3; (g) residues 26-35 of SEQ ID NO: 41 for CDR1, residues 50-58 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3; (h) residues 26-35 of SEQ ID NO: 42 for CDR1, residues 50-58 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3; (i) residues 26-35 of SEQ ID NO: 43 for CDR1, residues 50-58 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3; (j) residues 26-35 of SEQ ID NO: 44 for CDR1, residues 50-58 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3; (k) residues 26-35 of SEQ ID NO: 45 for CDR1, residues 50-58 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3; (1) residues 26-35 of SEQ ID NO: 46 for CDR1, residues 50-58 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3; (m) residues 26-35 of SEQ ID NO: 47 for CDR1, residues 50-58 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3; (n) residues 26-35 of SEQ ID NO: 48 for CDR1, residues 50-58 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3; (o) residues 26-35 of SEQ ID NO: 49 for CDR1, residues 50-58 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3; (p) residues 26-35 of SEQ ID NO: 50 for CDR1, residues 50-58 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3; (q) residues 26-35 of SEQ ID NO: 51 for CDR1, residues 50-58 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3; (r) residues 26-35 of SEQ ID NO: 52 for CDR1, residues 50-58 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3; (s) residues 26-35 of SEQ ID NO: 53 for CDR1, residues 50-58 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3; (t) residues 26-35 of SEQ ID NO: 54 for CDR1, residues 50-58 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or (u) residues 26- 35 of SEQ ID NO: 55 for CDR1, residues 50-58 of SEQ ID NO: 55 for CDR2, and residues 95- 102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to AbM.
In one embodiment, the second antigen binding domain comprises (a) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 56, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 57, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 58; (b) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 59, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 60, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 61; (c) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 62, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 63, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 64; (d) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 65, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 66, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 67; (e) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 68, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 70; (f) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 71, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 72, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 73; (g) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 74, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 75, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 76; (h) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 77, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 78, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 79; (i) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 80, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 81, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 82; (j) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 83, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 84, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 85; (k) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 86, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 87, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 88; (1) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 89, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 91; (m) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 92, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 93, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 94; (n) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 95, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 96, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 97; (o) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 98, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 99, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 100; (p) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 101, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 102, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 103; (q) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 104, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 105, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 106; (r) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 107, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 108, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 109; (s) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 110, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 111, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 112; (t) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 113, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 114, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 115; or (u) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 116, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 117, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 118.
In one embodiment, the second antigen binding domain comprises the amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
In some embodiments, the first antigen binding domain and the second antigen binding domain the multispecific antibody are linked by a linker.
In one embodiment, the linker is a peptide linker. In another embodiment, the peptide linker comprises a glycine/serine linker. In yet another embodiment, the linker comprises the amino acid sequence set forth as SEQ ID NO: 11, 144, or 174.
In some embodiments, the multispecific antibody further comprises a half-life extender. In one embodiment, the half-life extender binds to human serum albumin, binds directly to FcRn, or binds indirectly to FcRn. In one embodiment, inhibiting T cell activity by the multispecific antibody comprises inhibiting cytokine release. In one embodiment, the cytokine is interleukin-2 (IL-2), interferon gamma (IFNy), and/or tumor necrosis factor alpha (TNFa).
In one embodiment, inhibiting T cell activity by the multispecific antibody comprises inhibiting nuclear factor of activated T-cells (NF AT).
In some embodiments, the multispecific antibody is a bispecific antibody.
In some embodiments, the multispecific antibody is a trispecific antibody.
In one aspect, the present invention provides a nucleic acid encoding any of the multispecific antibodies provided herein.
In another aspect, the present invention provides a vector comprising the nucleic acid encoding any of the multispecific antibodies provided herein.
In another aspect, the present invention provides a host cell comprising the nucleic acid encoding any of the multispecific antibodies provided herein or a vector comprising the nucleic acid.
In one aspect, a pharmaceutical composition comprising any of the multispecific antibodies provided herein is provided.
In one aspect, the present invention provides a method of inhibiting T cell activity comprising contacting a T cell with an effective amount of any of the multispecific antibodies provided herein.
In another aspect, a method of treating or preventing an immune disorder in a subject is provided, the method comprising administering to the subject an effective amount of any of the multispecific antibodies provided herein or the pharmaceutical composition provided herein.
In one embodiment, the immune disorder is a T cell mediated inflammatory disease.
In another embodiment, the immune disorder is selected from the group consisting of a T cell mediated autoimmune disease, allergic disease, idiopathic thrombocytopenic purpura, warm autoimmune hemolytic anemia, autoimmune glomerulonephritis, autoimmune thyroid disease, systemic sclerosis, immune related adverse events (irAEs), transplant rejection, graft versus host disease (GVHD), rheumatoid arthritis, multiple sclerosis, type-1 diabetes, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, asthma, eosinophilic disease, autoimmune central nervous system (CNS) inflammation, psoriatic arthritis, atopic dermatitis, alopecia aerata, vitiligo, IgA nephropathy, lupus nephritis, chronic spontaneous urticarial, pyoderma gangrenosum, focal segmental glomerular sclerosis, membranous nephropathy, aplastic anemia, ulcerative colitis, Crohn’s disease, uveitis, Behcet’s disease, autoimmune hepatitis, ILD-myositis, and inflammation-induced liver injury.
In one aspect, the present invention provides a kit comprising any of the multispecific antibodies or pharmaceutical compositions provided herein. Provided herein are single domain antibodies which bind to suppressor of tumorigenicity 2 (ST2).
In one aspect, the present invention provides a single domain antibody which competes for binding with, binds a same epitope as, or comprises an antigen binding domain having three complementarity determining regions (CDRs) of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 35; an amino acid sequence set forth as SEQ ID NO: 36; an amino acid sequence set forth as SEQ ID NO: 37; an amino acid sequence set forth as SEQ ID NO: 38; an amino acid sequence set forth as SEQ ID NO: 39; an amino acid sequence set forth as SEQ ID NO: 40; an amino acid sequence set forth as SEQ ID NO: 41; an amino acid sequence set forth as SEQ ID NO: 42; an amino acid sequence set forth as SEQ ID NO: 43; an amino acid sequence set forth as SEQ ID NO: 44; an amino acid sequence set forth as SEQ ID NO: 45; an amino acid sequence set forth as SEQ ID NO: 46; an amino acid sequence set forth as SEQ ID NO: 47; an amino acid sequence set forth as SEQ ID NO: 48; an amino acid sequence set forth as SEQ ID NO: 49; an amino acid sequence set forth as SEQ ID NO: 50; an amino acid sequence set forth as SEQ ID NO: 51; an amino acid sequence set forth as SEQ ID NO: 52; an amino acid sequence set forth as SEQ ID NO: 53; an amino acid sequence set forth as SEQ ID NO: 54; or an amino acid sequence set forth as SEQ ID NO: 55.
In one embodiment, the single domain antibody comprises (a) residues 27-38 of SEQ ID NO: 35 for CDR1, residues 56-65 of SEQ ID NO: 35 for CDR2, and residues 105-117 of SEQ ID NO: 35 for CDR3; (b) residues 27-38 of SEQ ID NO: 36 for CDR1, residues 56-65 of SEQ ID NO: 36 for CDR2, and residues 105-117 of SEQ ID NO: 36 for CDR3; (c) residues 27- 38 of SEQ ID NO: 37 for CDR1, residues 56-65 of SEQ ID NO: 37 for CDR2, and residues 105- 117 of SEQ ID NO: 37 for CDR3; (d) residues 27-38 of SEQ ID NO: 38 for CDR1, residues 56- 65 of SEQ ID NO: 38 for CDR2, and residues 105-117 of SEQ ID NO: 38 for CDR3 (e) residues 27-38 of SEQ ID NO: 39 for CDR1, residues 56-65 of SEQ ID NO: 39 for CDR2, and residues 105-117 of SEQ ID NO: 39 for CDR3; (f) residues 27-38 of SEQ ID NO: 40 for CDR1, residues 56-65 of SEQ ID NO: 40 for CDR2, and residues 105-117 of SEQ ID NO: 40 for CDR3; (g) residues 27-38 of SEQ ID NO: 41 for CDR1, residues 56-65 of SEQ ID NO: 41 for CDR2, and residues 105-117 of SEQ ID NO: 41 for CDR3; (h) residues 27-38 of SEQ ID NO: 42 for CDR1, residues 56-65 of SEQ ID NO: 42 for CDR2, and residues 105-117 of SEQ ID NO: 42 for CDR3; (i) residues 27-38 of SEQ ID NO: 43 for CDR1, residues 56-65 of SEQ ID NO: 43 for CDR2, and residues 105-117 of SEQ ID NO: 43 for CDR3; (j) residues 27-38 of SEQ ID NO: 44 for CDR1, residues 56-65 of SEQ ID NO: 44 for CDR2, and residues 105-117 of SEQ ID NO: 44 for CDR3; (k) residues 27-38 of SEQ ID NO: 45 for CDR1, residues 56-65 of SEQ ID NO: 45 for CDR2, and residues 105-117 of SEQ ID NO: 45 for CDR3; (1) residues 27-38 of SEQ ID NO: 46 for CDR1, residues 56-65 of SEQ ID NO: 46 for CDR2, and residues 105-117 of SEQ ID NO: 46 for CDR3; (m) residues 27-38 of SEQ ID NO: 47 for CDR1, residues 56-65 of SEQ ID NO: 47 for CDR2, and residues 105-117 of SEQ ID NO: 47 for CDR3; (n) residues 27-38 of SEQ ID NO: 48 for CDR1, residues 56-65 of SEQ ID NO: 48 for CDR2, and residues 105-117 of SEQ ID NO: 48 for CDR3; (o) residues 27-38 of SEQ ID NO: 49 for CDR1, residues 56-65 of SEQ ID NO: 49 for CDR2, and residues 105-117 of SEQ ID NO: 49 for CDR3; (p) residues 27-38 of SEQ ID NO: 50 for CDR1, residues 56-65 of SEQ ID NO: 50 for CDR2, and residues 105-117 of SEQ ID NO: 50 for CDR3; (q) residues 27-38 of SEQ ID NO: 51 for CDR1, residues 56-65 of SEQ ID NO: 51 for CDR2, and residues 105-117 of SEQ ID NO: 51 for CDR3; (r) residues 27-38 of SEQ ID NO: 52 for CDR1, residues 56-65 of SEQ ID NO: 52 for CDR2, and residues 105-117 of SEQ ID NO: 52 for CDR3; (s) residues 27-38 of SEQ ID NO: 53 for CDR1, residues 56-65 of SEQ ID NO: 53 for CDR2, and residues 105-117 of SEQ ID NO: 53 for CDR3; (t) residues 27-38 of SEQ ID NO: 54 for CDR1, residues 56-65 of SEQ ID NO: 54 for CDR2, and residues 105-117 of SEQ ID NO: 54 for CDR3; or (u) residues 27-38 of SEQ ID NO: 55 for CDR1, residues 56-65 of SEQ ID NO: 55 for CDR2, and residues 105-117 of SEQ ID NO: 55 for CDR3;wherein the residues are numbered according to Lefranc (IMGT).
In one embodiment, the single domain antibody comprises (a) residues 31-35 of
SEQ ID NO: 35 for CDR1, residues 50-65 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 31-35 of SEQ ID NO: 36 for CDR1, residues 50-65 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 31-35 of SEQ ID NO: 37 for CDR1, residues 50-65 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 31-35 of SEQ ID NO: 38 for CDR1, residues 50-65 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 (e) residues 31-35 of SEQ ID NO: 39 for CDR1, residues 50-65 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3; (f) residues 31-35 of SEQ ID NO: 40 for CDR1, residues 50-65 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3; (g) residues 31-35 of SEQ ID NO: 41 for CDR1, residues 50-65 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3; (h) residues 31-35 of SEQ ID NO: 42 for CDR1, residues 50-65 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3; (i) residues 31-35 of SEQ ID NO: 43 for CDR1, residues 50-65 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3; (j) residues 31-35 of SEQ ID NO: 44 for CDR1, residues 50-65 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3; (k) residues 31-35 of SEQ ID NO: 45 for CDR1, residues 50-65 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3; (1) residues 31-35 of SEQ ID NO: 46 for CDR1, residues 50-65 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3; (m) residues 31-35 of SEQ ID NO: 47 for CDR1, residues 50-65 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3; (n) residues 31-35 of SEQ ID NO: 48 for CDR1, residues 50-65 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3; (o) residues 31-35 of SEQ ID NO: 49 for CDR1, residues 50-65 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3; (p) residues 31-35 of SEQ ID NO: 50 for CDR1, residues 50-65 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3; (q) residues 31-35 of SEQ ID NO: 51 for CDR1, residues 50-65 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3; (r) residues 31-35 of SEQ ID NO: 52 for CDR1, residues 50-65 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3; (s) residues 31-35 of SEQ ID NO: 53 for CDR1, residues 50-65 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3; (t) residues 31-35 of SEQ ID NO: 54 for CDR1, residues 50-65 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or (u) residues 31- 35 of SEQ ID NO: 55 for CDR1, residues 50-65 of SEQ ID NO: 55 for CDR2, and residues 95- 102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Kabat.
In one embodiment, the single domain antibody comprises (a) residues 26-32 of
SEQ ID NO: 35 for CDR1, residues 52-56 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 26-32 of SEQ ID NO: 36 for CDR1, residues 52-56 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 26-32 of SEQ ID NO: 37 for CDR1, residues 52-56 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 26-32 of SEQ ID NO: 38 for CDR1, residues 52-56 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3; (e) residues 26-32 of SEQ ID NO: 39 for CDR1, residues 52-56 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3; (f) residues 26-32 of SEQ ID NO: 40 for CDR1, residues 52-56 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3; (g) residues 26-32 of SEQ ID NO: 41 for CDR1, residues 52-56 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3; (h) residues 26-32 of SEQ ID NO: 42 for CDR1, residues 52-56 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3; (i) residues 26-32 of SEQ ID NO: 43 for CDR1, residues 52-56 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3; (j) residues 26-32 of SEQ ID NO: 44 for CDR1, residues 52-56 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3; (k) residues 26-32 of SEQ ID NO: 45 for CDR1, residues 52-56 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3; (1) residues 26-32 of SEQ ID NO: 46 for CDR1, residues 52-56 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3; (m) residues 26-32 of SEQ ID NO: 47 for CDR1, residues 52-56 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3; (n) residues 26-32 of SEQ ID NO: 48 for CDR1, residues 52-56 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3; (o) residues 26-32 of SEQ ID NO: 49 for CDR1, residues 52-56 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3; (p) residues 26-32 of SEQ ID NO: 50 for CDR1, residues 52-56 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3; (q) residues 26-32 of SEQ ID NO: 51 for CDR1, residues 52-56 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3; (r) residues 26-32 of SEQ ID NO: 52 for CDR1, residues 52-56 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3; (s) residues 26-32 of SEQ ID NO: 53 for CDR1, residues 52-56 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3; (t) residues 26-32 of SEQ ID NO: 54 for CDR1, residues 52-56 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or (u) residues 26- 32 of SEQ ID NO: 55 for CDR1, residues 52-56 of SEQ ID NO: 55 for CDR2, and residues 95- 102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Chothia.
In one embodiment, the single domain antibody comprises (a) residues 30-35 of
SEQ ID NO: 35 for CDR1, residues 47-58 of SEQ ID NO: 35 for CDR2, and residues 93-101 of SEQ ID NO: 35 for CDR3; (b) residues 30-35 of SEQ ID NO: 36 for CDR1, residues 47-58 of SEQ ID NO: 36 for CDR2, and residues 93-101 of SEQ ID NO: 36 for CDR3; (c) residues 30-35 of SEQ ID NO: 37 for CDR1, residues 47-58 of SEQ ID NO: 37 for CDR2, and residues 93-101 of SEQ ID NO: 37 for CDR3; (d) residues 30-35 of SEQ ID NO: 38 for CDR1, residues 47-58 of SEQ ID NO: 38 for CDR2, and residues 93-101 of SEQ ID NO: 38 for CDR3 (e) residues 30-35 of SEQ ID NO: 39 for CDR1, residues 47-58 of SEQ ID NO: 39 for CDR2, and residues 93-101 of SEQ ID NO: 39 for CDR3; (f) residues 30-35 of SEQ ID NO: 40 for CDR1, residues 47-58 of SEQ ID NO: 40 for CDR2, and residues 93-101 of SEQ ID NO: 40 for CDR3; (g) residues 30-35 of SEQ ID NO: 41 for CDR1, residues 47-58 of SEQ ID NO: 41 for CDR2, and residues 93-101 of SEQ ID NO: 41 for CDR3; (h) residues 30-35 of SEQ ID NO: 42 for CDR1, residues 47-58 of SEQ ID NO: 42 for CDR2, and residues 93-101 of SEQ ID NO: 42 for CDR3; (i) residues 30-35 of SEQ ID NO: 43 for CDR1, residues 47-58 of SEQ ID NO: 43 for CDR2, and residues 93-101 of SEQ ID NO: 43 for CDR3; (j) residues 30-35 of SEQ ID NO: 44 for CDR1, residues 47-58 of SEQ ID NO: 44 for CDR2, and residues 93-101 of SEQ ID NO: 44 for CDR3; (k) residues 30-35 of SEQ ID NO: 45 for CDR1, residues 47-58 of SEQ ID NO: 45 for CDR2, and residues 93-101 of SEQ ID NO: 45 for CDR3; (1) residues 30-35 of SEQ ID NO: 46 for CDR1, residues 47-58 of SEQ ID NO: 46 for CDR2, and residues 93-101 of SEQ ID NO: 46 for CDR3; (m) residues 30-35 of SEQ ID NO: 47 for CDR1, residues 47-58 of SEQ ID NO: 47 for CDR2, and residues 93-101 of SEQ ID NO: 47 for CDR3; (n) residues 30-35 of SEQ ID NO: 48 for CDR1, residues 47-58 of SEQ ID NO: 48 for CDR2, and residues 93-101 of SEQ ID NO: 48 for CDR3; (o) residues 30-35 of SEQ ID NO: 49 for CDR1, residues 47-58 of SEQ ID NO: 49 for CDR2, and residues 93-101 of SEQ ID NO: 49 for CDR3; (p) residues 30-35 of SEQ ID NO: 50 for CDR1, residues 47-58 of SEQ ID NO: 50 for CDR2, and residues 93-101 of SEQ ID NO: 50 for CDR3; (q) residues 30-35 of SEQ ID NO: 51 for CDR1, residues 47-58 of SEQ ID NO: 51 for CDR2, and residues 93-101 of SEQ ID NO: 51 for CDR3; (r) residues 30-35 of SEQ ID NO: 52 for CDR1, residues 47-58 of SEQ ID NO: 52 for CDR2, and residues 93-101 of SEQ ID NO: 52 for CDR3; (s) residues 30-35 of SEQ ID NO: 53 for CDR1, residues 47-58 of SEQ ID NO: 53 for CDR2, and residues 93-101 of SEQ ID NO: 53 for CDR3; (t) residues 30-35 of SEQ ID NO: 54 for CDR1, residues 47-58 of SEQ ID NO: 54 for CDR2, and residues 93-101 of SEQ ID NO: 54 for CDR3; or (u) residues 30- 35 of SEQ ID NO: 55 for CDR1, residues 47-58 of SEQ ID NO: 55 for CDR2, and residues 93- 101 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to MacCallum.
In one embodiment, the single domain antibody comprises (a) residues 26-35 of SEQ ID NO: 35 for CDR1, residues 50-58 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 26-35 of SEQ ID NO: 36 for CDR1, residues 50-58 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 26-35 of SEQ ID NO: 37 for CDR1, residues 50-58 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 26-35 of SEQ ID NO: 38 for CDR1, residues 50-58 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 (e) residues 26-35 of SEQ ID NO: 39 for CDR1, residues 50-58 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3; (f) residues 26-35 of SEQ ID NO: 40 for CDR1, residues 50-58 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3; (g) residues 26-35 of SEQ ID NO: 41 for CDR1, residues 50-58 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3; (h) residues 26-35 of SEQ ID NO: 42 for CDR1, residues 50-58 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3; (i) residues 26-35 of SEQ ID NO: 43 for CDR1, residues 50-58 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3; (j) residues 26-35 of SEQ ID NO: 44 for CDR1, residues 50-58 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3; (k) residues 26-35 of SEQ ID NO: 45 for CDR1, residues 50-58 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3; (1) residues 26-35 of SEQ ID NO: 46 for CDR1, residues 50-58 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3; (m) residues 26-35 of SEQ ID NO: 47 for CDR1, residues 50-58 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3; (n) residues 26-35 of SEQ ID NO: 48 for CDR1, residues 50-58 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3; (o) residues 26-35 of SEQ ID NO: 49 for CDR1, residues 50-58 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3; (p) residues 26-35 of SEQ ID NO: 50 for CDR1, residues 50-58 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3; (q) residues 26-35 of SEQ ID NO: 51 for CDR1, residues 50-58 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3; (r) residues 26-35 of SEQ ID NO: 52 for CDR1, residues 50-58 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3; (s) residues 26-35 of SEQ ID NO: 53 for CDR1, residues 50-58 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3; (t) residues 26-35 of SEQ ID NO: 54 for CDR1, residues 50-58 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or (u) residues 26- 35 of SEQ ID NO: 55 for CDR1, residues 50-58 of SEQ ID NO: 55 for CDR2, and residues 95- 102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to AbM.
In another embodiment, the single domain antibody comprises (a) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 56, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 57, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 58; (b) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 59, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 60, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 61; (c) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 62, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 63, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 64; (d) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 65, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 66, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 67; (e) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 68, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 70; (f) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 71, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 72, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 73; (g) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 74, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 75, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 76; (h) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 77, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 78, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 79; (i) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 80, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 81, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 82; (j) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 83, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 84, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 85; (k) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 86, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 87, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 88; (1) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 89, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 91; (m) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 92, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 93, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 94; (n) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 95, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 96, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 97; (o) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 98, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 99, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 100; (p) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 101, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 102, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 103; (q) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 104, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 105, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 106; (r) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 107, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 108, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 109; (s) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 110, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 111, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 112; (t) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 113, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 114, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 115; or (u) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 116, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 117, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 118.
In another embodiment, the single domain antibody comprises a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
In one aspect, the present invention provides a nucleic acid encoding any of the heavy chain variable regions of an ST2 antibody provided herein. In another aspect, the present invention provides a vector comprising a nucleic acid encoding any of the heavy chain variable regions of an ST2 antibody provided herein. In one aspect, the present invention provides a host cell comprising a nucleic acid encoding any of the ST2 single domain antibodies provided herein. In another aspect, the present invention provides a host cell comprising a vector comprising a nucleic acid encoding any of the ST2 single domain antibodies provided herein.
In one aspect, a pharmaceutical composition comprising any of the ST2 single domain antibodies provided herein is provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates an exemplary anti-Orail IgG monoclonal antibody (mAb) and an exemplary anti-Orail multispecific antibody (CTAB PR071) comprising an Orail IgG mAb first antigen binding domain and a second antigen binding domain comprising a sdAb (single domain antibody) targeting a T cell antigen.
FIG. 2 depicts the percent inhibition of IL-2 release as measured in an IL-2 ELISA. Human PBMCs were incubated with various concentrations of anti-Orai-1 mAB (2C1.1 human IgG2), Anti-Orail/Anti-PDl VHH multispecific antibody (CTAB PR071), anti-Orail mAB and anti-PDl VHH (equimolar), anti-PDl VHH, or cyclosporine A for 1 hour followed by activation with human anti-CD3/CD28 dynabeads for 24 hours. IL-2 was measured by ELISA in the supernatants.
FIG. 3 shows the annotated sequences for exemplary Orail/PDl multispecific antibody constructs. The Orail antibody variable regions are shown in yellow, the PD1 scFv antibody variable regions are shown in blue, the PD1 VHH variable regions are shown in green, and the linkers are shown in gray. The underlined text indicates the CDR regions as defined by IMGT, the red text indicates the CDR regions as defined by Kabat, and the italic text indicates the CDR regions as defined by Chothia.
FIG. 4 depicts the percent inhibition of NF AT as measured in a Jurkat screening assay. Jurkat T cells containing an NF AT luciferase reporter were cultured with various concentrations of an anti -Orail /anti-PDl VHH multispecific antibody (CTAB PR071; anti-Orail IgG/ anti-PDl VHH) or the anti-Orail mAB alone (BB PR001). Luminescence was measured after 48 hours.
FIG. 5 depicts the percent inhibition of NF AT as measured in a Jurkat screening assay. Jurkat T cells containing an NF AT luciferase reporter were cultured with various dilutions of supernatant containing an anti -Orail /anti-PDl VHH multispecific antibody (CTABs), or anti- Orail antibody and PD1 VHH (control). Various Orail multispecific antibodies were tested.
Luminescence was measured after 48 hours.
DETAILED DESCRIPTION
I. Orail Multispecific Antibodies
Orail is expressed on T cells and is a critical component of a Ca2+ channel that is required for differentiation and function of T cells. The present inventors have discovered that multispecific antibodies comprising an Orail binding domain and a second antigen binding domain that binds to a target on a T cell exhibit enhanced T cell inhibitory activity compared to the parent anti-Orail antibody. The disclosed multispecific antibodies are both highly specific to Orail expressing targets and cell selective based upon the specificity of the second antigen binding domain and have an improved therapeutic index. The Orail multispecific antibodies provided herein are useful for inhibiting T cell activity and/or function and for treating immune disorders, including, T cell- mediated inflammatory diseases, such as autoimmune disease and allergic disease. In particular, Orail multispecific antibodies inhibit T cell activity with a similar efficacy to the standard of care cyclosporine which is a part of the standard of care for a variety of T cell-mediated inflammatory diseases and works downstream of the Orail Ca2+ channel, supporting the therapeutic use of the Orail multispecific antibodies of the invention.
The present invention provides compositions comprising multispecific antibodies comprising at least a first antigen binding domain that binds to Orail and a second binding domain that binds to a target antigen. Such multispecific antibodies are also called “Orail multispecific antibodies” herein. Also provided are methods of using the Orai 1 multispecific antibodies to target cell activity and/or function. For example, where the targeting antigen is a T cell antigen, inhibition of T cell activity and/or function can be a reduction in the release or production of any of various cytokines, such as interleukin-2 (IL-2), as described elsewhere herein.
By “antigen binding domain” is meant a protein or polypeptide sequence that can bind to a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, specific antigenic determinant, epitope, antigen, or protein.
The antigen binding domains used to assemble the Orail multispecific antibody domains of the instant invention can be prepared using any known technique for antibody engineering or antibody selection. In some aspects, the antigen binding domains may be synthesized using known antigen binding domain sequences. In other aspects, the antigen binding domains are selected by screening a library for functional properties.
As one example, the antigen binding domains may be selected from a stabilized synthetic antibody library that was prepared by (a) generating a primary library comprising a plurality of antibodies, wherein each of the antibodies comprises a diverse CDR1, a diverse CDR2, and a neutral CDR3; (b) selecting a subset of the antibodies having improved developability features; and (c) generating the stabilized synthetic antibody library by replacing the neutral CDR3 of each of the antibodies selected in (b) with a diverse CDR3. By “neutral CDR” is meant a CDR3 that is representative of human CDR3 diversity (see e.g, PCT International Publication No. WO 2019/099454, incorporated by reference herein in its entirety), and preferably a neutral CDR3 that is representative of a target scaffold for antibody generation, i.e., a target scaffold for generation of each of the Orail binding domain and the target binding domain. A neutral CDR3 that is representative of a target scaffold includes an amino acid composition that reflects a pattern observed in a plurality of unique CDR3 sequences for a target scaffold. For example, a neutral CDR3 that is representative of a target scaffold may be determined by (i) identifying a plurality of unique CDR3 sequences for a target scaffold, for example by observing unique CDR3 sequences in antibodies comprising the target scaffold; (ii) selecting multiple subsets of the plurality of unique CDR3 sequences, wherein each subset comprises CDR3 sequences of a same length, and wherein subsets are selected for multiple CDR3 lengths, (iii) performing clustering analysis of CDR3 sequences in randomly selected subsets, (iv) selecting multiple clusters for each CDR3 length, and (v) selecting a representative CDR3 sequence from each cluster. Representative target scaffolds include, for example, either in unmutated or mutated configuration, human heavy chain scaffolds, such as those from the human IGHV germline gene family. In particular aspects of the instant invention, the target scaffold is from the human IGHV-3 germline gene family. Representative developability features is based on binding of antibody fragments to Protein A or to Protein L, or based on protein unfolding using treatments selected from high temperature, acid treatment, basic treatment, protease sensitivity, stability in serum, denaturation reagents, urea, and high salt concentration. Increased developability may also include deselection or removal of antibodies characterized as having decreased developability using methods selected from nonspecific absorption, binding to HSP90, and hydrophobic interactions. By “target,” or “target antigen” is meant a molecule of interest that is expressed by a cell. Preferably the target is a non-cytoplasmic target, for example, an extracellular or endosomal target. The target antigen can be any molecule expressed by a cell and the cell can be any cell type of interest. In one aspect, the target is a molecule that is expressed by a particular cell type. In such aspects, the target may not be expressed or has very low expression on other cell types. In one aspect, the cell of interest is a mammalian cell, and preferably a primate cell, and even more preferably a human cell. In certain aspects, the cell of interest is an immune cell. In a particular aspect, the cell of interest is a T cell. In other aspects, the cell of interest is a pancreatic cell (e.g., a pancreatic acinar cell), an epithelia cell (e.g., a lung or intestinal epithelial cell), or a blood cell (e.g., a white blood cell such as a mast cell).
A “multispecific antibody” or “multispecific binding agent” is one that binds to more than one antigen or epitope. In some cases, the multispecific antibody binds to two, three, four, five or more target antigens or epitopes. The target antigens or epitopes can be on the same cell or on separate cells. The target antigens can be two or more separate and unique antigens or can be different epitopes of the same antigen. The terms “multispecific polypeptide,” “multispecific antibody,” “multispecific binding agent,” and “multispecific antibody construct” are used interchangeably herein. The multispecific antibodies disclosed herein provide cell selective and disease selective targeting, and as such, a “multispecific antibody” as used herein includes a “cell targeted biologic” or “CTAB”.
In one aspect, the antibodies useful in the invention are bispecific antibodies. A bispecific antibody or antigen binding protein is an antibody having two different antigen binding sites. Bispecific antibodies are a species of multispecific antibody and may be produced by a variety of techniques known in the art including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148: 1547-1553. A non-limiting example of the synthesis of a bispecific antibody is provided in Example 1 herein. In the context of the present invention, the two binding sites of a bispecific antibody will bind to two different epitopes, Orail and a target antigen.
In another aspect, the antibodies useful in the invention are trispecific antibodies. A trispecific antibody or antigen binding protein is an antibody having three different antigen binding sites. In the context of the present invention, two of the binding sites of a trispecific antibody will bind to Orail and a target antigen, and the third antigen binding site can bind to any antigen. In some aspects, the third antigen binding site is a half-life extender.
In some aspects, multispecific antibodies of the invention comprise additional sequences including signal peptides, linkers, half-life extenders, and/or tag sequences, such as a His-tag.
A. Orail
The first antigen binding domain of the disclosed multispecific antibodies binds to Orail and comprises any of the various antigen binding domains provided herein, such as an antibody, including an antibody variable region, or an sdAb. Preferably, the first antigen binding domain binds to the Orai with relatively low affinity.
The affinity is a measure for the binding strength between an antigenic determinant, i.e. the target, and an antigen-binding domain, i.e., an antigen binding domain of any of the multispecific antibodies provided herein. The affinity can be represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding domain (KD or KD): the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding domain (alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/KD). Affinity can be determined by any manner known in the art depending on the specific antigen of interest.
In the present invention, an antigen-binding domain (such as, any of the antigen binding domains of the multispecific antibodies, antibodies, or sdAbs provided herein) are said to bind to their target antigen with a high affinity when the dissociation constant (KD) is 10-9to IO-12 moles/liter or less, and preferably 10“10to IO-12 moles/liter or less and more preferably 10-11 to IO-12 moles/liter (i.e. with an association constant (KA) of 109to 1012 liter/moles or more, and preferably 1010to 1012 liter/moles or more and more preferably 10u to 1012 liter/moles).
In the present invention, an antigen-binding domain (such as, any of the antigen binding domains of the multispecific antibodies, antibodies, or sdAbs provided herein) are said to bind to their target antigen with a low affinity when the dissociation constant (KD) is 10-6to IO-9 moles/liter or more, and preferably 10-6to 10-8 moles/liter or more and more preferably 10-6to IO-7 moles/liter (i.e. with an association constant (KA) of 106to 109 liter/moles or more, and preferably 106to 108 liter/moles or more and more preferably 106to 107 liter/moles). As used herein, the term, “Orai Calcium Release-Activated Calcium Modulator 1” used interchangeably with the term “Orail” refers to the well-known gene and polypeptide encoded by that gene, also known in the art as IMD9, TAM2, ORAT1, CRACM1, TMEM142A, and the ORAI calcium release-activated calcium protein 1.
“Orail” and “anti-Orail”, as used herein to describe antibodies, are used interchangeably.
The Orail gene is located in the chromosomal region 12q24.31 (base pairs 121,626,550- 121,642,040 on chromosome 12) and consists of 2 exons. Orail transcripts are differentially expressed throughout the body, including in immune cells (e.g., T cells, such as CD4 T cells and CD8 T cells), muscle tissues, and the gastrointestinal tract.
Exemplary nucleotide sequences and amino acid sequences of Orail can be found, for example, at GenBank Accession No. NM_032790.3 and UniProt Accession No. Q96D31 for Homo sapiens Orail; GenBank Accession No. NM_175423.3 and UniProt Accession No. Q8BWG9 for Mus musculus Orail; and GenBank Accession No. NM 001013982.1 and UniProt Accession No. Q5M848 for Rattus norvegicus Orai 1. Additional examples of Orai 1 sequences are readily available using, e.g., GenBank, UniProt, and OMIM web site. Further information on Orail is provided, for example, in the NCBI Gene database at http://www.ncbi.nlm.nih.gov/gene/84876. The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.
Orail is a pore-forming subunit of a plasma membrane Ca2+ release-activated Ca2+(CRAC) channel, which mediates store-operated calcium (SOC) influx into cells. Orail is activated by the Ca2+ sensor, stromal interaction molecule 1 (STIM1), localized to the endoplasmic reticulum (ER) when intracellular Ca2+ stores are depleted.
Orail provides the primary pathway for calcium influx into T cells and plays a role in T cell activation and immune function. For example, a form of hereditary severe combined immune deficiency (SCID) in human patients has been linked to a missense mutation in 0RAI1 and abrogation of CRAC channel function (Feske et al., 2005, J. Exptl. Med. 202(5): 651-62; Feske et al., 2006, Nature 441 :179-85). T and B cells from ORAI1 knock-in mice expressing a nonfunctional Orail protein display severely impaired SOCE and CRAC channel function, resulting in a strongly reduced expression of several cytokines including IL-2, IL-4, IL- 17, IFN-y, and TNF-a in CD4 and CD8 T cells. Orail deficient mice exhibited severely attenuated cell- mediated immune responses in vivo that depend on TH1, TH2 and TH I 7 cell function, and absence of contact hypersensitivity responses with tolerance of skin allografts. In addition, T cells from Orail deficient mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease (McCarl C. et al., 2010, J. Immunol. 185(10): 5845-5858). Without wishing to be bound by theory, a sustained calcium influx via CRAC channels into T cells leads to the activation of several transcription factors including nuclear factor of activated T cells (NF AT). In the nucleus, NF AT activates the transcription of a variety of genes encoding for cytokines such as IL-2, IL-4, IL- 17, IFN-y, and TNF-a that are crucial for T cell activation. (Feske et al., 2003 Biochem. Biophys. Res. Comm. 311 : 1117-1132).
Known Orail binders are only weak or partial inhibitors of T cell function (see e.g., Lin et al., 2013, J. Pharmacol. Exp. Ther., 345:225; Gaida et al., 2015, J. Immunotoxicol ., 12: 164) such that they are not useful as stand-alone therapeutic molecules. In particular, the degree of immunosuppression provided by these Orail antibodies is inferior to cyclosporine, a therapeutically relevant comparator which is a part of the standard of care for a variety of T cell- mediated inflammatory diseases and works downstream of the Orail Ca2+ channel.
As used herein, a parent anti-Orail binding domain is a binding domain in monospecific form, that is, not otherwise designed to bind to targets other than Orail. In accordance with the present invention, the parent anti-Orail binding domain e.g., an antibody may or may not be functional as a therapeutic agent. For example, a parent Orail binding domain may or may not show T cell inhibition or CRAC channel inhibition as a single agent. However, in preferred aspects of the invention, the parent Orail antibody has some therapeutic activity as a single agent, such as T cell inhibitory activity and/or CRAC channel inhibitory activity, for example, at least about 50% in routine assays such as any of the assays provided herein, including IL2 reduction and Ca++ influx, and NF AT inhibition. The parent anti-Orail binding domain may inhibit T cell activity and/or CRAC channel activity by about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less.
Orail multispecific antibodies of the present invention demonstrate improved and/or enhanced function as compared to the parent anti-Orail binding domain (e.g., inhibition of CRAC channel activity and/or inhibition of T cell activity). For example, in some aspects, there is at least about a 10% improvement or increase in function, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%, or more improvement or increase in function. In other aspects, there is at least about a 2-fold improvement or increase in Orail multispecific antibody function as compared to the parent Orail binding domain, or at least about 5-fold, or at least about 10-fold, or at least about 20-fold, or at least about 25-fold, or at least about 50-fold, or at least about 100-fold, or at least about 200-fold or more improvement or increase in function. Any of the various assays provided elsewhere herein or known in the art can be used to measure the CRAC channel activity and/or the T cell activity. In some aspects of the invention, the Orail multispecific design can reveal functionality of a parent Orail antibody that is otherwise considered non-functional.
In some aspects, the enhanced activity effect of the Orail multispecific antibodies provided herein may be additive or synergistic in nature as compared to the activity of the parent antigen binding domains alone.
As described further herein, Orail multispecific antibodies of the present invention demonstrate improved T cell function (e.g., IL-2 inhibition, NF AT inhibition, and/or Ca++ influx) as compared to the parent Orail antibody, such that the multispecific antibody achieves a level of efficacy that is comparable to the standard of care cyclosporine.
B. Targeting Antigen
The second antigen binding domain of the disclosed Orail multispecific antibodies binds to a cell type-specific antigen or “targeting antigen” and can comprise any of the various antigen binding domains provided herein, such as an antibody, an antibody variable region, or an sdAb. Preferably, the targeting antigen is expressed on the cell surface of the target cell. Also preferably, the second antigen binding domain binds to the targeting antigen with high affinity. The Orail multispecific antibodies of the invention exploit the avidity of the second antigen binding domain to provide an improved therapeutic index for Orail binders.
In some aspects of the invention, the second antigen binding domain binds to a targeting antigen on a T cell or on a subset of T cells, including activated T cells, CD4+ T cells (helper T cells or Th cells) including T helper type 1 cells (Thl), T helper type 2 cells (Th2), Th2A, Th9, and Thl7 cells, Th22 cells, CD8+ T cells (cytotoxic or killer T cells) including Tel and Tc2 cells, effector T cells, memory T cells, natural killer T cells (NKT cells), regulatory T cells (Treg), and gamma delta T cells. In other aspects of the invention, the targeting antigen is a white blood cell (e.g., mast cells), or epithelial cells (e.g., lung epithelial cells and intestinal epithelial cells), or pancreatic cells (e.g., acinar cells).
Exemplary T cell targeting antigens include, but are not limited to, PD1, CD4, CD3, CTLA4, CXCR4, CD25, ST2, CRTh2, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CLA, IL- 23R, IL-12R, CD52, CD122, CD127, CD27, KLRG-1, ICOS, CD45RA, GITR, 0X40, CD103, LAG3, CD28, CD8, CD2, CD5, CD6, CDl la, CD43, CD132, CD38, CD53, CD69, CD70, CD95, CD45RO, or CD45RB.
In one aspect of the invention, the targeting antigen is PD1. As used herein, the term, “programmed cell death protein 1” is used interchangeably with the term “PD1” refers to the well- known gene and polypeptide encoded by that gene, also known in the art as PDCD1, PD1, CD279, SLEB2, systemic lupus erythematosus susceptibility 2, HPD-L, HPD-1, ALWB2, and hSLEl.
The PD1 gene is located in the chromosomal region 2q37.3 (base pairs 241,848,978- 241,859,811 on chromosome 2) and consists of 6 exons. The PD1 protein is expressed predominantly on immune cells, e.g., T cells.
Exemplary nucleotide sequences and amino acid sequences of PD1 can be found, for example, at GenBank AccessionNo. NM_005018.3 and UniProt Accession No. QI 5116 for Homo sapiensVD ,' GenBank AccessionNo. NM_008798.3 and UniProt Accession No. Q02242 for Mus musculus PD1; and GenBank Accession No. NM 001106927.1 and UniProt Accession No. D3ZIN8 for Rattus norvegicus PD1. Additional examples of PD1 sequences are readily available using, e.g., GenBank, UniProt, and OMIM web site. Further information on PD1 is provided, for example, in the NCBI Gene database at http://www.ncbi.nlm.nih.gov/gene/5133. The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.
PD1 expression is tightly and dynamically regulated, which makes PD1 an effective targeting antigen in the context of the disclosed Orail multispecific antibodies. On resting naive T cells, as well as in certain populations of developing thymocytes, PD1 is expressed at low basal levels. Following an initial immune stimulus, PD1 is transiently expressed on multiple immune cell types, including CD4 and CD8 T cells. During acute antigen exposure, PD1 is down regulated in a time course that is concurrent with or prior to antigen clearance. During chronic immune stimulation, PD1 remains highly expressed. PD1 functions as an immune inhibitory receptor on the cell surface in activated T cells and regulates T cell functions including those of CD8 T cells. Further, PD1 promotes the differentiation of CD4 T cells into T regulatory cells. Follicular helper T (TFH) cells also constitutively express high PD1 (Bally et al., 2016, J Immunol. 196(6): 2431-37). For use in the present invention, a second antigen binding domain that binds PD1 may or may not have antagonistic or agonistic activity, but preferably has high binding affinity.
In another aspect of the invention, the targeting antigen is ST2. As used herein, the term, “suppressor of tumorigenicity 2” is used interchangeably with the term “ST2” refers to the well- known gene and polypeptide encoded by that gene, also known in the art as interleukin 1 receptorlike 1, IL1RL1, DER4, FIT-1, IL33R, ST2L, ST2V, Tl, suppression of tumorigenicity 2, EC 3.2.2.6, protein ST2, growth stimulation-expressed, interleukin 1 receptor-related protein, soluble interleukin 1 receptor like 1, and homolog of mouse growth stimulation expressed.
The ST2 gene is located in the chromosomal region 2ql2.1 (base pairs 102,311,529- 102,352,367 on chromosome 2) and consists of 13 exons. The PD1 protein is expressed in placenta, kidney, adrenal gland, gall bladder, lung, and on immune cells, e.g., T cells.
Exemplary nucleotide sequences and amino acid sequences of ST2 can be found, for example, at NCBI Accession No. NM_016232.5 and UniProt Accession No. Q01638 for Homo sapiens ST2. Additional examples of ST2 sequences are readily available using, e.g., GenBank, UniProt, and OMIM web site. Further information on ST2 is provided, for example, in the NCBI Gene database at http://www.ncbi.nlm.nih.gov/gene/9173. The entire contents of each of the foregoing Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.
ST2 plays a role in T cell mediated allergic responses and is differentially expressed in Th2 cells. ST2 is a receptor for IL-33 which drives Th2 cytokine production that has a significant role in the allergic process, including in asthma, atopic dermatitis, and food allergies. For use in the present invention, a second antigen binding domain that binds ST2 may or may not have antagonistic or agonistic activity, but preferably has high binding affinity.
II. Antigen Binding Domains
The first and/or second antigen binding domains of the disclosed Orail multispecific antibodies can each comprise a full-length antibody, a monoclonal antibody, a humanized antibody, an IgG antibody, an sdAb (e.g., a nanobody or VHH), an scFv, a variable heavy (VH) domain, a variable light (VL) domain, any antigen binding fragment of an antibody, or any combinations thereof.
In some aspects, the first antigen binding domain and/or the second antigen binding domain comprises an antibody or fragment thereof, such as a single domain antibody (sdAb). In other aspects, the first and/or the second antigen binding domains comprise antibody-like formats, such as darpins, anticalins, or fibronectin domains. In one aspect, the first antigen binding domain comprises an IgG monoclonal antibody and the second antigen binding domain comprises an sdAb.
In one aspect, the Orail binding domain is an antibody. Any Orail antibody can be used in the multispecific antibodies provided herein. Exemplary Orail antibodies include those disclosed in US 2012/0231006, US 2017/0226203, WO 2011/063277, and WO 2013/091903, each of which is herein incorporated by reference in their entirety.
A. Antibodies
Antibodies are immunoglobulin molecules that recognize and bind to a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
As used herein, the terms “antibody” and “antibodies” encompass any type of antibody, including but not limited to monoclonal antibodies, polyclonal antibodies, antigen-binding fragments of intact antibodies (e.g., Fab, Fab’, F(ab’)2, Fd, Fv, Fc, etc.) that retain the ability to specifically bind to a given antigen, bispecific antibodies, multispecific antibodies, heteroconjugate antibodies, fusion proteins having an antibody or antigen-binding fragment thereof, (e.g., a domain antibody), single chain antibodies, (scFv), single domain antibodies (sdAbs, also known as nanobodies and VHH antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that includes an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
In some aspects, antibodies useful in the invention are antibodies that share a same antigen binding region with a reference antibody, for example, antibodies that compete for binding with a reference antibody and/or antibodies that bind to a same epitope as a reference antibody. The term “reference antibody” refers to the Orail and target antigen binding domains described herein.
In some aspects, the antibodies useful in the invention are human, mouse, rat, rabbit, donkey, or camelid antibodies. In certain aspects, antibodies useful in the invention are obtained from camelid species such as dromedary camels, Bactrian camels, wild Bactrian camels, llamas, alpacas, vicunas, and guanacos. In particular, antibodies produced in camelid species have only three hypervariable regions (CDRs), compared to antibody formats derived from conventional IgGs.
In some aspects, antibodies useful in the invention are fully human antibodies, humanized antibodies, or chimeric antibodies.
Canonical antibodies comprise two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Each light chain is composed of one variable domain (VL) and one constant domain (CL). Light chains of the antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (X), based on the amino acid sequences of their constant domains. Each heavy chain comprises one variable domain (VH) and a constant region, which in the case of IgG, IgA, and IgD antibodies, comprises three domains termed CHI, CH2, and CH3 (IgM and IgE have a fourth domain, CH4).
The variable domains of antibodies show considerable variation in amino acid composition from one antibody to another and are primarily responsible for antigen recognition and binding. Variable regions of each light/heavy chain pair form the antibody binding site such that an intact IgG antibody has two binding sites (i.e., it is bivalent). VH and VL domains comprise three regions of extreme variability, which are termed hypervariable regions, or more commonly, complementarity-determining regions (CDRs), framed and separated by four less variable regions known as framework regions (FRs). Non-covalent association between the VH and the VL region forms the Fv fragment (for "fragment variable") which contains one of the two antigen-binding sites of the antibody.
As used herein, the assignment of amino acids to each domain, framework region and CDR may be in accordance with one of the schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5th Ed.), US Dept, of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3rd Ed., Wily-VCH Verlag GmbH and Co or AbM (Oxford Molecular/MSI Pharmacopia); or Lefranc et al., 2003 (IMGT numbering) Dev. Comp. Immunol. 27:55-57, unless otherwise noted. As is well known in the art variable region residue numbering is typically as set forth in Chothia or Kabat. Amino acid residues which comprise CDRs as defined by Kabat, Chothia, MacCallum (also known as Contact), Lefranc (also known as IMGT), and AbM as obtained from the Abysis website database (infra.) are set out in Table 1.
Table 1
Figure imgf000046_0001
Variable regions and CDRs in an antibody sequence can be identified according to general rules that have been developed in the art (as set out above, such as, for example, the Kabat numbering system) or by aligning the sequences against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described in, and can be accessed through, the “IMGT” (ImMunoGeneTics) website at www.imgt.org; the “Abysis" website at www.bioinf.org.uk/abs (maintained by A.C. Martin in the Department of Biochemistry & Molecular Biology University College London, London, England) and the VBASE2 website at www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671 -D674 (2005). Unless otherwise indicated, all CDRs set forth herein are derived as per one of the definitions in Table 1.
The term “epitope” refers to the portion of a molecule that is bound by an antigen binding protein (for example, an antibody, an antigen binding domain, or an sdAb). The term includes any determinant capable of specifically binding to an antigen binding protein, such as an antibody an antigen binding domain, or an sdAb. An epitope can be contiguous or non-contiguous (e.g., in a single-chain polypeptide, amino acid residues that are not contiguous to one another in the polypeptide sequence but that within the context of the molecule are bound by the antigen binding protein). In certain aspects, epitopes may be mimetic in that they comprise a three-dimensional structure that is similar to an epitope used to generate the antigen binding protein, yet comprise none or only some of the amino acid residues found in that epitope used to generate the antigen binding protein. Most often, epitopes reside on proteins, but in some instances may reside on other kinds of molecules, such as nucleic acids. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.
The term “compete” when used in the context of antigen binding domains (e.g., antibodies) that compete for the same epitope means competition between antigen binding domains is determined by an assay in which the antigen binding domain (e.g., antibody or fragment thereof) under test prevents or inhibits specific binding of a reference antigen binding domain (e.g., a ligand, or a reference antibody) to a common antigen (e.g., Orail or an epitope thereof). Numerous types of competitive binding assays can be used, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotinavidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antigen binding domain and a labeled reference antigen binding domain. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding domain. Usually, the test antigen binding domain is present in excess. Antigen binding domains identified by competition assay (competing antigen binding domains) include antigen binding domains binding to the same epitope as the reference antigen binding domains and antigen binding domains binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding domain for steric hindrance to occur. Usually, when a competing antigen binding domain is present in excess, it will inhibit specific binding of a reference antigen binding domain to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.
In one aspect of the invention, an Orail multispecific antibody of the invention comprises a single domain antibody (sdAb), also known as a VHH molecule or nanobody.
The term “single domain antibody” or “sdAb” or “single domain antibody fragment” are used interchangeably herein to refer to antigen binding domains or fragments, such as VHH domains or VH or VL domains that have a single antigen binding domain or single variable antibody domain.
The sdAbs can be light chain variable domain sequences (e.g., a VL-sequence), or heavy chain variable domain sequences (e.g., a VH-sequence); more specifically, they can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. Accordingly, the sdAbs can be domain antibodies, or immunoglobulin sequences that are suitable for use as domain antibodies, single domain antibodies, or immunoglobulin sequences that are suitable for use as single domain antibodies, or immunoglobulin sequences that are suitable for use as sdAbs or nanobodies, including but not limited to VHH sequences.
The structure of an sdAb can be considered, but not limited to, to be comprised of four framework regions or “FR's”, which are referred to in the art and herein as “Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”, respectively; which framework regions are interrupted by three complementary determining regions or “CDR's”, which are referred to in the art as “Complementarity Determining Region 1” or “CDR1”; as “Complementarity Determining Region 2” or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3”, respectively.
The amino acid residues of an sdAb can be numbered according to the general numbering for VH or VL domains provided in Table 1. For example, an sdAb comprising a VH domain, as defined by Kabat et al., comprises: FR1 comprising the amino acid residues at positions 1-30, CDR1 comprising the amino acid residues at positions 31-35, FR2 comprising the amino acids at positions 36-49, CDR2 comprising the amino acid residues at positions 50-65, FR3 comprising the amino acid residues at positions 66-94, CDR3 comprising the amino acid residues at positions 95-102, and FR4 comprising the amino acid residues at positions 103-113.
B. ST2 Single Domain Antibodies
Provided herein are single domain antibodies (sdAbs) that specifically bind to ST2. The ST2 sdAbs provided herein can be the second antigen binding domain of the Orail multispecific antibodies. Exemplary ST2 sdAbs comprise an amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
In one embodiment, the ST2 sdAbs provided herein compete for binding with, bind a same epitope as, or comprise an antigen binding domain having three complementarity determining regions (CDRs) of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. The CDRs can be defined by any of the various CDR definitions known in the art, for example, those depicted in Table 1. Exemplary CDR sequences for the ST2 sdAbs provided herein are set forth as SEQ ID NOs 56-118 and are listed in Table 11 in Example 4.
The sdAbs can be synthesized by any method known in the art. An exemplary method is described elsewhere herein.
C. Antigen Binding Domain Variants
Contemplated herein are certain polypeptides (e.g., antigen binding domains, antibodies, multispecific antibodies, sdAbs, or bispecific antibodies) that exhibit “sequence identity”, sequence similarity” or “sequence homology” to the polypeptides of the invention. For example, a derived humanized antibody VH or VL domain may exhibit a sequence similarity with the source (e.g., murine) or acceptor (e.g., human) VH or VL domain. A “homologous” polypeptide may exhibit 65%, 70%, 75%, 80%, 85%, or 90% sequence identity. In other aspects a “homologous” polypeptide may exhibit 93%, 95% or 98% sequence identity. As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting Examples below.
The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. ' l (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. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
Additionally, or alternatively, the protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
Residue positions which are not identical may differ by conservative amino acid substitutions or by non-conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. In cases where there is a substitution with a non-conservative amino acid, in aspects the polypeptide exhibiting sequence identity will retain the desired function or activity of the polypeptide of the invention (e.g., antibody). D. Exemplary Antigen Binding Domains
The first antigen binding domain of the Orail multispecific antibodies provided herein comprises an Orail antibody, and in certain aspects, the first antigen binding domain is an Orail antibody as disclosed in U.S. Publication Number 2012/0231006, which is incorporated herein by reference in its entirety, or antibodies that bind to a same antigen binding site as, or that compete for binding with, or that bind to a same epitope as an Orail antibody as disclosed in U.S. Publication Number 2012/0231006. Additional exemplary anti-Orai 1 antibodies are also contemplated for use in the multispecific antibodies of the present invention. Non-limiting examples include mAb 10F8 (WO2013/091903) and mAb hR198_HGl/LGl (US 2017/0226203).
For example, in one aspect, the first antigen binding domain competes for binding with or binds to a same epitope as an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2.
For example, the first antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, and residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 2 for CDR-H1, residues 50-65 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3, wherein the residues are numbered according to Kabat; (b) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 2 for CDR-H1, residues 52-56 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3, wherein the residues are numbered according to Chothia; (c) residues 30-36 of SEQ ID NO: 5 for CDR-L1, residues 46-55 of SEQ ID NO: 5 for CDR-L2, and residues 89-96 of SEQ ID NO: 5 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 2 for CDR-H1, residues 47-58 of SEQ ID NO: 2 for CDR- H2, and residues 93-101 of SEQ ID NO: 2 for CDR-H3, wherein the residues are numbered according to MacCallum; or (d) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, and residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 2 for CDR-H1, residues 50-58 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3, wherein the residues are numbered according to AbM. In some aspects, the first antigen binding domain of an Orail multispecific antibody comprises a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 18, a CDRL2 comprising the amino acid sequence set forth as SEQ ID NO: 19, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 20; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 21, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 22, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 23.
In a specific aspect, the first antigen binding domain of an Orail multispecific antibody comprises a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5, and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2. In a further aspect, the first antigen binding domain of an Orail multispecific antibody comprises a light chain comprising an amino acid sequence set forth as SEQ ID NO: 15, and/or a heavy chain comprising an amino acid sequence set forth as SEQ ID NO: 14.
In another aspect, the first antigen binding domain competes for binding with or binds to a same epitope as (a) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (b) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or (c) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 151.
In a specific aspect, the first antigen binding domain of the multispecific antibody comprises (a) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (b) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or (c) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 151. The second antigen binding domain can bind to any targeting antigen, and in certain aspects, the targeting antigen is a T cell antigen, for example, PD1. In a particular aspect of the invention, the second antigen binding domain is PD1 antibody, such as a sdAb, including, for example, a PD1 antibody as disclosed in U.S. Patent Number 10,323,090, which is incorporated herein by reference in its entirety, or antibodies that bind to a same antigen binding site as, or that compete for binding with, or that bind to a same epitope as PD1 antibody as disclosed in U.S. Patent Number 10,323,090.
In another aspect, the second antigen binding domain is PD1 antibody, such as a scFv or VHH, including, for example, a PD1 antibody as disclosed in US2019/032749, US2019/032749, US 2020/0347135, or US 2019/0322747, each of which is incorporated herein by reference in its entirety, or antibodies that bind to a same antigen binding site as, or that compete for binding with, or that bind to a same epitope as PD1 antibody as disclosed in US2019/032749, US 2020/0347135, and US 2019/0322747. In another aspect, the second antigen binding domain is PD1 antibody derived from known antibodies, such as Nivolumab, Pembrolizumab, Sintilimab, Spartalizumab, Cemiplimab, Retifanlimab, Dostarlimab, Ezabenlimab, Cetrelimab, Budigalimab, Sasanlimab, and Vudalimab, or antibodies that bind to a same antigen binding site as, or that compete for binding with, or that bind to a same epitope as any of these antibodies. In one aspect, the second antigen binding domain of an Orail multispecific antibody competes for binding with or binds to a same epitope as an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 8.
In another aspect, the second antigen binding domain of the multispecific antibody competes for binding with, binds a same epitope as, or comprises (a) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 170; (b) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 171; (c) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 172; or (d) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 173.
The second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 31-35 of SEQ ID NO: 8 for CDR1, residues 50-65 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3, wherein the residues are numbered according to Kabat; (b) residues 26-32 of SEQ ID NO: 8 for CDR1, residues 52-56 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3, wherein the residues are numbered according to Chothia; (c) residues 30-35 of SEQ ID NO: 8 for CDR1, residues 47-58 of SEQ ID NO: 8 for CDR2, and residues 93-101 of SEQ ID NO: 8 for CDR3, wherein the residues are numbered according to MacCallum; or (d) residues 26-35 of SEQ ID NO: 8 for CDR1, residues 50-58 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3, wherein the residues are numbered according to AbM.
In a specific aspect, the second antigen binding domain of an Orail multispecific antibody comprises a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 24, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 26. In a further aspect, the second antigen binding domain of the multispecific antibody comprises the amino acid sequence set forth as SEQ ID NO: 8.
In a specific aspect, the second antigen binding domain of the multispecific antibody comprises (a) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 314, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 315, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 316; (b) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 322, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 323, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 324; (c) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 330, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 331, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 332; or (d) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 338, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 339, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 340.
In another specific aspect, the second antigen binding domain of the multi specific antibody comprises the amino acid sequence set forth as SEQ ID NO: 8, 170. 171, 172, or 173.
In some aspects, the second antigen binding domain is an scFv. For example, the second antigen binding domain of the multispecific antibody competes for binding with, binds a same epitope as, or comprises (a) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 145 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 143; (b) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 154 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 153; (c) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 158 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 157; (d) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 160 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 169; (e) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 162 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 161; (f) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 164 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 163; (g) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 167 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 165; or (h) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 168 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 169.
In a specific aspect, the second antigen binding domain of the multispecific antibody comprises (a) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 145; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 143; (b) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 154; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 153; (c) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 158; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 157; (d) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 160; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 159; (e) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 162; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 161; (f) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 164; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 163; (g) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 167; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 165; or (h) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 168; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 169.
In another aspect, the second antigen binding domain can bind to ST2 as the T cell targeting antigen. In a particular aspect of the invention, the second antigen binding domain is an ST2 antibody, such as an sdAb, including, for example, the ST2 sdAbs set forth as SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55, or antibodies that bind to a same antigen binding site as, or that compete for binding with, or that bind to a same epitope as the ST2 sdAbs set forth as SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
In one aspect, the ST2 second antigen binding domain of an Orail multispecific antibody competes for binding with or binds to a same epitope as an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
The ST2 second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 27-38 of SEQ ID NO: 35 for CDR1, residues 56-65 of SEQ ID NO: 35 for CDR2, and residues 105-117 of SEQ ID NO: 35 for CDR3; (b) residues 27-38 of SEQ ID NO: 36 for CDR1, residues 56-65 of SEQ ID NO: 36 for CDR2, and residues 105-117 of SEQ ID NO: 36 for CDR3; (c) residues 27-38 of SEQ ID NO: 37 for CDR1, residues 56-65 of SEQ ID NO: 37 for CDR2, and residues 105-117 of SEQ ID NO: 37 for CDR3; (d) residues 27-38 of SEQ ID NO: 38 for CDR1, residues 56-65 of SEQ ID NO: 38 for CDR2, and residues 105-117 of SEQ ID NO: 38 for CDR3 (e) residues 27-38 of SEQ ID NO: 39 for CDR1, residues 56-65 of SEQ ID NO: 39 for CDR2, and residues 105-117 of SEQ ID NO: 39 for CDR3; (f) residues 27-38 of SEQ ID NO: 40 for CDR1, residues 56-65 of SEQ ID NO: 40 for CDR2, and residues 105-117 of SEQ ID NO: 40 for CDR3; (g) residues 27-38 of SEQ ID NO: 41 for CDR1, residues 56-65 of SEQ ID NO: 41 for CDR2, and residues 105-117 of SEQ ID NO: 41 for CDR3; (h) residues 27-38 of SEQ ID NO: 42 for CDR1, residues 56-65 of SEQ ID NO: 42 for CDR2, and residues 105-117 of SEQ ID NO: 42 for CDR3; (i) residues 27-38 of SEQ ID NO: 43 for CDR1, residues 56-65 of SEQ ID NO: 43 for CDR2, and residues 105-117 of SEQ ID NO: 43 for CDR3; (j) residues 27-38 of SEQ ID NO: 44 for CDR1, residues 56-65 of SEQ ID NO: 44 for CDR2, and residues 105-117 of SEQ ID NO: 44 for CDR3; (k) residues 27-38 of SEQ ID NO: 45 for CDR1, residues 56-65 of SEQ ID NO: 45 for CDR2, and residues 105-117 of SEQ ID NO: 45 for CDR3; (1) residues 27-38 of SEQ ID NO: 46 for CDR1, residues 56-65 of SEQ ID NO: 46 for CDR2, and residues 105-117 of SEQ ID NO: 46 for CDR3; (m) residues 27-38 of SEQ ID NO: 47 for CDR1, residues 56-65 of SEQ ID NO: 47 for CDR2, and residues 105-117 of SEQ ID NO: 47 for CDR3; (n) residues 27-38 of SEQ ID NO: 48 for CDR1, residues 56-65 of SEQ ID NO: 48 for CDR2, and residues 105-117 of SEQ ID NO: 48 for CDR3; (o) residues 27-38 of SEQ ID NO: 49 for CDR1, residues 56-65 of SEQ ID NO: 49 for CDR2, and residues 105-117 of SEQ ID NO: 49 for CDR3; (p) residues 27-38 of SEQ ID NO: 50 for CDR1, residues 56-65 of SEQ ID NO: 50 for CDR2, and residues 105-117 of SEQ ID NO: 50 for CDR3; (q) residues 27-38 of SEQ ID NO: 51 for CDR1, residues 56-65 of SEQ ID NO: 51 for CDR2, and residues 105-117 of SEQ ID NO: 51 for CDR3; (r) residues 27-38 of SEQ ID NO: 52 for CDR1, residues 56-65 of SEQ ID NO: 52 for CDR2, and residues 105-117 of SEQ ID NO: 52 for CDR3; (s) residues 27-38 of SEQ ID NO: 53 for CDR1, residues 56-65 of SEQ ID NO: 53 for CDR2, and residues 105-117 of SEQ ID NO: 53 for CDR3; (t) residues 27-38 of SEQ ID NO: 54 for CDR1, residues 56-65 of SEQ ID NO: 54 for CDR2, and residues 105-117 of SEQ ID NO: 54 for CDR3; or (u) residues 27-38 of SEQ ID NO: 55 for CDR1, residues 56-65 of SEQ ID NO: 55 for CDR2, and residues 105-117 of SEQ ID NO: 55 for CDR3;wherein the residues are numbered according to Lefranc (IMGT).
The ST2 second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 31-35 of SEQ ID NO: 35 for CDR1, residues 50-65 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 31-35 of SEQ ID NO: 36 for CDR1, residues 50-65 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 31-35 of SEQ ID NO: 37 for CDR1, residues 50-65 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 31-35 of SEQ ID NO: 38 for CDR1, residues 50-65 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 (e) residues 31-35 of SEQ ID NO: 39 for CDR1, residues 50-65 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3; (f) residues 31-35 of SEQ ID NO: 40 for CDR1, residues 50-65 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3; (g) residues 31-35 of SEQ ID NO: 41 for CDR1, residues 50-65 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3; (h) residues 31-35 of SEQ ID NO: 42 for CDR1, residues 50-65 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3; (i) residues 31-35 of SEQ ID NO: 43 for CDR1, residues 50-65 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3; (j) residues 31-35 of SEQ ID NO: 44 for CDR1, residues 50-65 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3; (k) residues 31-35 of SEQ ID NO: 45 for CDR1, residues 50-65 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3; (1) residues 31-35 of SEQ ID NO: 46 for CDR1, residues 50-65 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3; (m) residues 31-35 of SEQ ID NO: 47 for CDR1, residues 50-65 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3; (n) residues 31-35 of SEQ ID NO: 48 for CDR1, residues 50-65 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3; (o) residues 31-35 of SEQ ID NO: 49 for CDR1, residues 50-65 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3; (p) residues 31-35 of SEQ ID NO: 50 for CDR1, residues 50-65 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3; (q) residues 31-35 of SEQ ID NO: 51 for CDR1, residues 50-65 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3; (r) residues 31-35 of SEQ ID NO: 52 for CDR1, residues 50-65 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3; (s) residues 31-35 of SEQ ID NO: 53 for CDR1, residues 50-65 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3; (t) residues 31-35 of SEQ ID NO: 54 for CDR1, residues 50-65 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or (u) residues 31-35 of SEQ ID NO: 55 for CDR1, residues 50-65 of SEQ ID NO: 55 for CDR2, and residues 95-102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Kabat.
The ST2 second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 26-32 of SEQ ID NO: 35 for CDR1, residues 52-56 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 26-32 of SEQ ID NO: 36 for CDR1, residues 52-56 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 26-32 of SEQ ID NO: 37 for CDR1, residues 52-56 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 26-32 of SEQ ID NO: 38 for CDR1, residues 52-56 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3; (e) residues 26-32 of SEQ ID NO: 39 for CDR1, residues 52-56 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3; (f) residues 26-32 of SEQ ID NO: 40 for CDR1, residues 52-56 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3; (g) residues 26-32 of SEQ ID NO: 41 for CDR1, residues 52-56 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3; (h) residues 26-32 of SEQ ID NO: 42 for CDR1, residues 52-56 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3; (i) residues 26-32 of SEQ ID NO: 43 for CDR1, residues 52-56 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3; (j) residues 26-32 of SEQ ID NO: 44 for CDR1, residues 52-56 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3; (k) residues 26-32 of SEQ ID NO: 45 for CDR1, residues 52-56 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3; (1) residues 26-32 of SEQ ID NO: 46 for CDR1, residues 52-56 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3; (m) residues 26-32 of SEQ ID NO: 47 for CDR1, residues 52-56 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3; (n) residues 26-32 of SEQ ID NO: 48 for CDR1, residues 52-56 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3; (o) residues 26-32 of SEQ ID NO: 49 for CDR1, residues 52-56 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3; (p) residues 26-32 of SEQ ID NO: 50 for CDR1, residues 52-56 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3; (q) residues 26-32 of SEQ ID NO: 51 for CDR1, residues 52-56 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3; (r) residues 26-32 of SEQ ID NO: 52 for CDR1, residues 52-56 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3; (s) residues 26-32 of SEQ ID NO: 53 for CDR1, residues 52-56 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3; (t) residues 26-32 of SEQ ID NO: 54 for CDR1, residues 52-56 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or (u) residues 26-32 of SEQ ID NO: 55 for CDR1, residues 52-56 of SEQ ID NO: 55 for CDR2, and residues 95-102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Chothia.
The ST2 second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 30-35 of SEQ ID NO: 35 for CDR1, residues 47-58 of SEQ ID NO: 35 for CDR2, and residues 93-101 of SEQ ID NO: 35 for CDR3; (b) residues 30-35 of SEQ ID NO: 36 for CDR1, residues 47-58 of SEQ ID NO: 36 for CDR2, and residues 93-101 of SEQ ID NO: 36 for CDR3; (c) residues 30-35 of SEQ ID NO: 37 for CDR1, residues 47-58 of SEQ ID NO: 37 for CDR2, and residues 93-101 of SEQ ID NO: 37 for CDR3; (d) residues 30-35 of SEQ ID NO: 38 for CDR1, residues 47-58 of SEQ ID NO: 38 for CDR2, and residues 93-101 of SEQ ID NO: 38 for CDR3 (e) residues 30-35 of SEQ ID NO: 39 for CDR1, residues 47-58 of SEQ ID NO: 39 for CDR2, and residues 93-101 of SEQ ID NO: 39 for CDR3; (f) residues 30-35 of SEQ ID NO: 40 for CDR1, residues 47-58 of SEQ ID NO: 40 for CDR2, and residues 93-101 of SEQ ID NO: 40 for CDR3; (g) residues 30-35 of SEQ ID NO: 41 for CDR1, residues 47-58 of SEQ ID NO: 41 for CDR2, and residues 93-101 of SEQ ID NO: 41 for CDR3; (h) residues 30-35 of SEQ ID NO: 42 for CDR1, residues 47-58 of SEQ ID NO: 42 for CDR2, and residues 93-101 of SEQ ID NO: 42 for CDR3; (i) residues 30-35 of SEQ ID NO: 43 for CDR1, residues 47-58 of SEQ ID NO: 43 for CDR2, and residues 93-101 of SEQ ID NO: 43 for CDR3; (j) residues 30-35 of SEQ ID NO: 44 for CDR1, residues 47-58 of SEQ ID NO: 44 for CDR2, and residues 93-101 of SEQ ID NO: 44 for CDR3; (k) residues 30-35 of SEQ ID NO: 45 for CDR1, residues 47-58 of SEQ ID NO: 45 for CDR2, and residues 93-101 of SEQ ID NO: 45 for CDR3; (1) residues 30-35 of SEQ ID NO: 46 for CDR1, residues 47-58 of SEQ ID NO: 46 for CDR2, and residues 93-101 of SEQ ID NO: 46 for CDR3; (m) residues 30-35 of SEQ ID NO: 47 for CDR1, residues 47-58 of SEQ ID NO: 47 for CDR2, and residues 93-101 of SEQ ID NO: 47 for CDR3; (n) residues 30-35 of SEQ ID NO: 48 for CDRl, residues 47-58 of SEQ ID NO: 48 for CDR2, and residues 93-101 of SEQ ID NO: 48 for CDR3; (o) residues 30-35 of SEQ ID NO: 49 for CDR1, residues 47-58 of SEQ ID NO: 49 for CDR2, and residues 93-101 of SEQ ID NO: 49 for CDR3; (p) residues 30-35 of SEQ ID NO: 50 for CDR1, residues 47-58 of SEQ ID NO: 50 for CDR2, and residues 93-101 of SEQ ID NO: 50 for CDR3; (q) residues 30-35 of SEQ ID NO: 51 for CDR1, residues 47-58 of SEQ ID NO: 51 for CDR2, and residues 93-101 of SEQ ID NO: 51 for CDR3; (r) residues 30-35 of SEQ ID NO: 52 for CDR1, residues 47-58 of SEQ ID NO: 52 for CDR2, and residues 93-101 of SEQ ID NO: 52 for CDR3; (s) residues 30-35 of SEQ ID NO: 53 for CDR1, residues 47-58 of SEQ ID NO: 53 for CDR2, and residues 93-101 of SEQ ID NO: 53 for CDR3; (t) residues 30-35 of SEQ ID NO: 54 for CDR1, residues 47-58 of SEQ ID NO: 54 for CDR2, and residues 93-101 of SEQ ID NO: 54 for CDR3; or (u) residues 30-35 of SEQ ID NO: 55 for CDR1, residues 47-58 of SEQ ID NO: 55 for CDR2, and residues 93-101 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to MacCallum.
The ST2 second antigen binding domain of an Orail multispecific antibody may comprise: (a) residues 26-35 of SEQ ID NO: 35 for CDR1, residues 50-58 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3; (b) residues 26-35 of SEQ ID NO: 36 for CDR1, residues 50-58 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3; (c) residues 26-35 of SEQ ID NO: 37 for CDR1, residues 50-58 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3; (d) residues 26-35 of SEQ ID NO: 38 for CDR1, residues 50-58 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3 (e) residues 26-35 of SEQ ID NO: 39 for CDR1, residues 50-58 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3; (f) residues 26-35 of SEQ ID NO: 40 for CDR1, residues 50-58 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3; (g) residues 26-35 of SEQ ID NO: 41 for CDR1, residues 50-58 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3; (h) residues 26-35 of SEQ ID NO: 42 for CDR1, residues 50-58 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3; (i) residues 26-35 of SEQ ID NO: 43 for CDR1, residues 50-58 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3; (j) residues 26-35 of SEQ ID NO: 44 for CDR1, residues 50-58 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3; (k) residues 26-35 of SEQ ID NO: 45 for CDR1, residues 50-58 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3; (1) residues 26-35 of SEQ ID NO: 46 for CDR1, residues 50-58 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3; (m) residues 26-35 of SEQ ID NO: 47 for CDR1, residues 50-58 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3; (n) residues 26-35 of SEQ ID NO: 48 for CDR1, residues 50-58 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3; (o) residues 26-35 of SEQ ID NO: 49 for CDR1, residues 50-58 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3; (p) residues 26-35 of SEQ ID NO: 50 for CDR1, residues 50-58 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3; (q) residues 26-35 of SEQ ID NO: 51 for CDR1, residues 50-58 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3; (r) residues 26-35 of SEQ ID NO: 52 for CDR1, residues 50-58 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3; (s) residues 26-35 of SEQ ID NO: 53 for CDR1, residues 50-58 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3; (t) residues 26-35 of SEQ ID NO: 54 for CDR1, residues 50-58 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or (u) residues 26-35 of SEQ ID NO: 55 for CDR1, residues 50-58 of SEQ ID NO: 55 for CDR2, and residues 95-102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to AbM.
The Orail multispecific antibodies provided herein can comprise any combination of the first and the second antigen binding domains provided herein. An exemplary Orail multispecific antibody of the invention comprises a second antigen binding domain that binds to PD1, for example, a multispecific antibody having the sequence set forth as SEQ ID NO: 17.
Additional Orail multispecific antibodies of the invention that comprise a second antigen binding domain that binds to PD1 include those set forth as SEQ ID NOs: 175-195.
III. Preparation and Expression of Orail Multispecific Antibody Constructs
The design of the Orail multispecific antibodies provided herein can include any of the first and second antigen binding domains as described above and optionally can include other components, such as, a signal peptide, a linker, a half-life extender, and/or a tag.
A. Linkers
The Orail multispecific antibodies provided herein may comprise one or more linkers to connect the various antigen binding domains. Any suitable linker known in the art can be used to connect the antigen binding domains of the Orail multispecific antibodies. Non-limiting examples of a linker include a peptide linker, a chemical linker, a PEG linker, or a genetic linker. The first antigen binding domain and the second antigen binding domain may be covalently linked.
In some aspects, the first antigen binding domain and the second antigen binding domain of the Orail multispecific antibodies provided herein are connected via a peptide linker, i.e., any chain of amino acids. In a specific aspect, the peptide linker comprises a glycine/ serine linker.
A peptide linker can be any length of amino acids, including at least 60 amino acids, at least 55 amino acids, at least 50 amino acids, at least 45 amino acids, at least 40 amino acids, at least 35 amino acids, at least 30 amino acids, at least 25 amino acids, at least 20 amino acids, at least 15 amino acids, at least 10 amino acids, at least 9 amino acids, at least 8 amino acids, at least 7 amino acids, at least 6 amino acids, at least 5 amino acids, at least 4 amino acids, at least 3 amino acids, at least 2 amino acids, or at least 1 amino acid in length. In one aspect, the linker is about 35 amino acids in length. In a specific aspect, the linker comprises the sequence set forth as SEQ ID NOs: 9, 11, 144, or 174.
B. Half-Life Extenders
In another aspect of the invention, the Orail multispecific antibodies of the invention comprise a half-life extender. A “half-life extender” refers to any modification that increases the serum half-life of the Orai 1 multispecific antibody as compared to the Orai 1 multispecific antibody without the half-life extender. For example, an Orail multispecific antibody of the invention may be linked (chemically or otherwise) to one or more groups or moieties that extend the half-life (such as PEG), so as to provide Orail multispecific antibodies of the invention with increased halflife.
Non-limiting examples of such Orail multispecific antibodies comprising half-life extenders include chemically modified multispecific antibodies (for example, pegylation); Orail multispecific antibodies that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); and Orail multispecific antibodies of the invention which comprise at least one amino acid sequence that is linked to at least one moiety (and in particular at least one amino acid sequence) which increases the half-life of the Orail multispecific antibodies of the invention. For example, the Orail multispecific antibodies of the invention are suitably linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, antibodies, domain antibodies, single domain antibodies (sdAbs), VHH antibodies, or nanobodies that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrin.
In one aspect, the half-life extender binds to the neonatal Fc receptor (FcRn). The half-life extender can bind to the FcRn directly or indirectly. For example, a multispecific antibody may be directly fused or conjugated to an Fc sequence capable of binding to the FcRn or can bind indirectly via an FcRn binding protein.
In another aspect, the half-life extender is an Fc sequence comprising a YTE mutation. Mutations at positions 252, 254, and 256 of human IgGl to a Tyr (Y), Thr (T), and Glu (E), respectively, result in increased serum half-life as described in Dall'acqua WF, et al., Properties of human IgGls engineered for enhanced binding to the neonatal Fc receptor (FcRn), J Biol Chem. (2006) 281 :23514-24, which is incorporated herein in its entirety. See also U.S. Patent Nos. 9,562,100; 8,795,661; 8,475,792; 8,323,962; 8,012,476; 7,704,497; 7,670,600; 7658,921; and 7,083,784, each also incorporated herein in its entirety.
The Orail multi specific antibodies of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding Orail multispecific antibody without the half-life extender. For example, the Orail multispecific antibodies of the invention with increased half-life may have a half-life e.g., in humans that is increased more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding Orail multispecific antibody without the half-life extender.
In another aspect of the invention, such Orail multispecific antibodies of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, multispecific antibodies of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).
C. Representative Orail Multispecific Antibody Constructs
The Orail multispecific antibody constructs of the invention can comprise any first antigen binding domain that bind to Orail, any second antigen binding domain that binds to a targeting antigen, and any linkers and/or half-life extenders provided herein.
In one aspect, the Orail multispecific antibody comprises (a) a first antigen binding domain that competes for binding with or binds a same epitope as an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5 and three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2; (b) a linker, for example a linker comprising the sequence set forth as SEQ ID NO: 11; and (c) a second antigen binding domain that competes for binding with or binds a same epitope as an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 8.
In another aspect, the Orail the Orail multispecific antibody comprises (a) a first antigen binding domain that competes for binding with or binds a same epitope as (i) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (ii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or (iii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 151; (b) a linker, for example a linker comprising the sequence set forth as SEQ ID NO: 11; and (c) a second antigen binding domain that competes for binding with or binds a same epitope as (i) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 8, (ii) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 170, (iii) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 171, (iv) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO : 172; or (v) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 173.
In another aspect, the Orail the Orail multispecific antibody comprises (a) a first antigen binding domain that competes for binding with or binds a same epitope as (i) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140; (ii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or (iii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 151; (b) a linker, for example a linker comprising the sequence set forth as SEQ ID NO: 11; and (c) a second antigen binding domain that competes for binding with or binds a same epitope as (i) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 145 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 143; (ii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 154 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 153; (iii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 158 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 157; (iv) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 160 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 169; (v) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 162 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 161; (vi) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 164 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 163; (vii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 167 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 165; or (viii) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 168 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 169.
In another aspect, the Orail multispecific antibody comprises (a) a first antigen binding domain comprising three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5 and three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2; (b) a linker, for example a linker comprising the sequence set forth as SEQ ID NO: 11; and (c) a second antigen binding domain comprising three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 8.
In another aspect, the Orail multispecific antibody comprises (a) a first antigen binding domain comprising a heavy chain sequence comprising the sequence set forth as SEQ ID NO: 12 and a light chain sequence comprising the sequence set forth as SEQ ID NO: 13; (c) a linker comprising the sequence set forth as SEQ ID NO: 11; and (c) a second antigen binding domain comprising the sequence set forth as SEQ ID NO: 16.
In a specific aspect, the Orail multispecific antibody comprises the sequence set forth as SEQ ID NO: 17.
In specific aspects, the Orail multispecific antibody comprises (a) a heavy chain (HC)- scFv set forth as SEQ ID NO: 175 and a light chain (LC) set forth as SEQ ID NO: 176; (b) a heavy chain (HC)-scFv set forth as SEQ ID NO: 177 and a light chain (LC) set forth as SEQ ID NO: 178; (c) a heavy chain (HC)-scFv set forth as SEQ ID NO: 179 and a light chain (LC) set forth as SEQ ID NO: 180; (d) a heavy chain (HC)-scFv set forth as SEQ ID NO: 181 and a light chain (LC) set forth as SEQ ID NO: 180; (e) a heavy chain (HC)-scFv set forth as SEQ ID NO: 182 and a light chain (LC) set forth as SEQ ID NO: 180; (f) a heavy chain (HC)-scFv set forth as SEQ ID NO: 183 and a light chain (LC) set forth as SEQ ID NO: 180; (g) a heavy chain (HC)-scFv set forth as SEQ ID NO: 184 and a light chain (LC) set forth as SEQ ID NO: 180; (h) a heavy chain (HC)-scFv set forth as SEQ ID NO: 185 and a light chain (LC) set forth as SEQ ID NO: 180; (i) a heavy chain (HC)-scFv set forth as SEQ ID NO: 186 and a light chain (LC) set forth as SEQ ID NO: 180; (j) a heavy chain (HC)-scFv set forth as SEQ ID NO: 187 and a light chain (LC) set forth as SEQ ID NO: 180; (k) a heavy chain (HC)-VHH set forth as SEQ ID NO: 188 and a light chain (LC) set forth as SEQ ID NO: 180; (1) a heavy chain (HC)-VHH set forth as SEQ ID NO: 189 and a light chain (LC) set forth as SEQ ID NO: 180; (m) a heavy chain (HC)-VHH set forth as SEQ ID NO: 190 and a light chain (LC) set forth as SEQ ID NO: 180; (n) a heavy chain (HC)- VHH set forth as SEQ ID NO: 191 and a light chain (LC) set forth as SEQ ID NO: 180; (o) a heavy chain (HC)-VHH set forth as SEQ ID NO: 192 and a light chain (LC) set forth as SEQ ID NO: 180; (p) a heavy chain (HC)-VHH set forth as SEQ ID NO: 193 and a light chain (LC) set forth as SEQ ID NO: 180; (q) a heavy chain (HC)-VHH set forth as SEQ ID NO: 194 and a light chain (LC) set forth as SEQ ID NO: 180; or (r) a heavy chain (HC)-VHH set forth as SEQ ID NO: 195 and a light chain (LC) set forth as SEQ ID NO: 178.
In one aspect, the Orail multispecific antibody comprises (a) a first antigen binding domain that competes for binding with or binds a same epitope as an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5 and three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2; (b) a linker, for example a linker comprising the sequence set forth as SEQ ID NO: 11; and (c) a second antigen binding domain that competes for binding with or binds a same epitope as an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
D. Nucleic Acids, Vectors, and Host Cells
The present invention provides nucleic acid molecules that encode the Orail multispecific antibodies or the ST2 sdAbs provided herein and vectors and host cells comprising the nucleic acid molecules. The nucleic acid molecules, vectors, and host cells can be used for expressing any of the Orail multispecific antibodies or the ST2 sdAbs provided herein.
Also contemplated herein are nucleic acids that that exhibit “sequence identity”, sequence similarity” or “sequence homology” to the nucleic acids that encode the Orail multispecific antibodies or ST2 sdAbs of the invention, wherein the encoded Orail multispecific antibody or the ST2 sdAb exhibits the desired functionality. A “homologous sequence” means a sequence of nucleic acid molecules exhibiting at least about 65%, 70%, 75%, 80%, 85%, or 90% sequence identity. In other aspects, a “homologous sequence” of nucleic acids may exhibit 93%, 95% or 98% sequence identity to the reference nucleic acid.
The instant invention also provides vectors comprising nucleic acid molecules that encode the Orail multispecific antibodies or ST2 sdAbs provided herein, which may be operably linked to a promoter (see, e.g., WO 86/05807; WO 89/01036; and U.S.P.N. 5,122,464); and other transcriptional regulatory and processing control elements of the eukaryotic secretory pathway. The invention also provides host cells harboring those vectors and host-expression systems.
As used herein, the term “host-expression system” includes any kind of cellular system that can be engineered to generate the nucleic acids, the Orail multispecific antibodies, or the ST2 sdAbs of the invention. Such host-expression systems include, but are not limited to microorganisms (e.g., E. coli or B. subtilis) transformed or transfected with recombinant bacteriophage DNA or plasmid DNA; yeast (e.g., Saccharomyces) transfected with recombinant yeast expression vectors; or mammalian cells (e.g., COS, CHO-S, HEK293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells or viruses (e.g., the adenovirus late promoter). The host cell may be co-transfected with two or more expression vectors, for example, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
Methods of transforming mammalian cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. The host cell may also be engineered to allow the production of an antigen binding molecule with various characteristics (e.g., modified glycoforms or proteins having GnTIII activity).
For long-term, high-yield production of recombinant proteins stable expression is preferred. Accordingly, cell lines that stably express the selected antibody, ST2 sdAb, or Orail multispecific antibody may be engineered using standard art recognized techniques and form part of the invention. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter or enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Any of the selection systems well known in the art may be used, including the glutamine synthetase gene expression system (the GS system) which provides an efficient approach for enhancing expression under selected conditions. The GS system is discussed in whole or part in connection with EP 0 216 846, EP 0 256 055, EP 0 323 997 and EP 0 338 841 and U.S. Patent Nos. 5,591,639 and 5,879,936. Another compatible expression system for the development of stable cell lines is the Freedom™ CHO-S Kit (Life Technologies).
Once an Orail multispecific antibody or ST2 sdAb of the invention has been produced by recombinant expression or any other of the disclosed techniques, it may be purified or isolated by methods known in the art in that it is identified and separated and/or recovered from its natural environment and separated from contaminants that would interfere with therapeutic uses for the multispecific antibody.
These isolated preparations may be purified using various art-recognized techniques, such as, for example, ion exchange and size exclusion chromatography, dialysis, diafiltration, and affinity chromatography, particularly Protein A or Protein G affinity chromatography. Compatible methods are discussed more fully in the Examples below.
IV. Methods and Uses of Orail Multispecific Antibodies
A. Inhibiting T Cell Activity
The Orail multispecific antibodies of the present invention are designed to specifically target T cells and inhibit the activity and/or function of T cells, either in vitro and in vivo. The Orail first antigen binding domain and the second antigen binding domain of the disclosed multispecific antibodies both bind to targets on T cells. Without wishing to be bound by any theory, the first antigen binding domain is specific for a functional target on T cells (i.e., Orail) and binding of the first antigen binding domain to Orail on the T cell inhibits or reduces T cell activity. The second antigen binding domain functions as a targeting component and directs the Orail multispecific antibody to a T cell by binding to a target molecule on the T cell.
The term “inhibit,” as used herein, is used interchangeably with “reduce,” “decrease,” and other similar terms, and includes any level of inhibition of T cell activity.
Inhibiting or reducing T cell activity can be any decrease in any T cell activity, including, for example, a decrease of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95% or greater from T cell activity observed in the absence of the Orail multispecific antibody. Inhibiting or reducing T cell activity can also be an inhibition of any T cell activity by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, about 50-fold, about 100-fold or more from T cell activity observed in the absence of the Orail multispecific antibody.
Any T cell activity can be inhibited by the Orail multispecific antibodies disclosed herein, including, but not limited to, cytokine release, such as IL-2, IFN-y, or TNF-a, T cell proliferation, up-regulation/expression of T cell activation markers (e.g., IL2RA/CD25), nuclear translocation and/or activity of NF AT, or intracellular calcium influx as described in, for example, U.S. Patent No. 10,981,995; U.S. Application Nos. 2017/0226203, 2012/0231006; Zappasodi R. et al., 2020, Methods EnzymoL 631 :43-59; Maguire, O. et al., 2013 Cytometry A. 83(12): 1096-1104; McCarl C. et al., 2010 J. Immunol. 185(10):5845-5858; Bercovici N. et al., 2000, Clin. Diag. Lab. Immunol. 7(6):859-864. Each of these references is incorporated herein by reference in its entirety.
The inhibition of T cell activity may be specific to particular subsets of T cells as described herein. In particular aspects, the Orail multispecific constructs inhibit Th2 helper cell activity.
T cell function and/or activity may be assessed by a variety of methods known in the art, and exemplary assays are provided herein in Examples 2 and 3.
To evaluate T cell function, in some aspects, T cell proliferation is assessed by measuring incorporation of a radioactive tracer (e.g., [3H]thymidine) or a fluorescent dye (e.g., bromodeoxyuridine) into T cells, which reflects the total amount of DNA synthesized in the T cell population. In some other aspects, T cell proliferation is assessed by flow cytometry. In this method, T cells are loaded with a dye such as fluorescent 5,6-carboxyfluorescein succinimidyl ester (CFSE), cell trace violet (CTV), and violet proliferation dye 450 (VPD-450), followed by incubation with ligands such as anti-CD3/anti-CD28 antibodies and assessment of cell divisions using a flow cytometer. When the labeled cells divide, half of the labeled proteins are distributed to each daughter cell, resulting in a decrease in dye intensity and a peak that is shifted to the left on a histogram of fluorescence intensity.
To evaluate T cell function, in some aspects, upon stimulation of T cells with a ligand or ionophore [e.g., anti-CD3 antibody, anti-CD28 antibody, glucocorticoid-induced TNFR-related protein (GITR) ligand, thapsigargin, phorbol myristate acetate (PMA), ionomycin], levels of effector cytokines (e.g., FFNy, TNFa, IL-2, IL-4, IL-10, IL-17) are assessed. In certain aspects, cytokine secretion/release is measured by assays including, for example, enzyme-linked immunosolvent assay (ELISA), enzyme-linked immunospot (ELISPOT), or intracellular cytokine staining (ICS).
In another aspect, T cell function and/or activity is evaluated by analyzing subcellular localization and/or activity of NF AT. In another aspect, T cell function and/or activity is evaluated by assessing calcium influx. Assays for assessing calcium influx are known in the art and include utilizing a calcium-binding dye (e.g., Fura-2) in combination with a fluorescent imaging plate reader (FLIPR) assay. B. Treatment of Orail-Associated Diseases and Disorders
It will be appreciated that the Orail multispecific antibodies of the present invention may be used for treating or preventing diseases or disorders that can benefit from Orail inhibition, i.e., Orail -associated diseases and disorders. The disclosed Orail multispecific antibodies are particularly useful for treating subjects with immune disorders including T cell mediated inflammatory disease, such as autoimmune disease and allergic disease. In such cases, the Orail multispecific antibodies can be administered alone or in combination with additional antiinflammatory or other agents.
As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more signs or symptoms associated with and Orail -associated disease or disorder, for example, an immune disease or disorder such as a T cell-mediated inflammatory disease, autoimmune disease and allergic disease. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.
An “effective amount” is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remediate the damage that results from or is associated with an Orail -associated disease or disorder. In some aspects, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” is an amount sufficient to remedy a disease state (e.g., transplant rejection or autoimmune disease) or symptom(s), particularly a state or symptom(s) associated with the disease state, or otherwise prevent, hinder, retard or reverse the progression of the disease state or any other undesirable symptom associated with the disease in any way whatsoever (i.e., that provides “therapeutic efficacy”). A “prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of symptoms associated with a disease state, or reducing the likelihood of the onset (or reoccurrence) of symptoms. The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.
The therapeutically or prophylactically effective amount may vary depending on the Orail multispecific antibody, how the Orail multispecific antibody is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated. Determination of an effective amount is within the skill of a trained clinician.
C. Subjects
The Orail multispecific antibodies disclosed herein may be used to treat or prevent any of the various immune disorders or diseases as are recognized in the art. In some aspects, the Orail multispecific antibodies are used to treat immune disorders or diseases. In certain aspects, the immune disorder is associated with T cells, including, for example, autoimmune disease and allergic disease. In some aspects, the immune disease is a T cell-mediated inflammatory disease. In a specific aspect the T cell-mediated inflammatory disease is a chronic disease.
Non-limiting examples of immune disorders or diseases include, T cell-mediated chronic inflammatory diseases, autoimmune diseases, allergic diseases, rheumatoid arthritis, multiple sclerosis, type-1 diabetes, systemic lupus erythematosus (SLE), vitiligo, autoimmune thyroid disease (e.g., Graves’ disease, Hashimoto’s thyroiditis), pernicious anemia, alopecia areata, immune (idiopathic) thrombocytopenic purpura, celiac disease, atopic dermatitis (AD), eosinophilic esophagitis, nasal polyps, chronic rhinosinusitis with nasal polyps (CRSwNP), psoriasis, chronic plaque psoriasis (PsO), psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis), asthma, allergic rhinitis, eosinophilic disease, transplant rejection (e.g., allograft rejection), graft versus host disease (GvHD), lupus nephritis, urticaria, chronic spontaneous urticarial, ANCA vasculitis, neuromyelitis optical, myasthenia gravis, systemic sclerosis, autoimmune glomerulonephritis, Sjogren’s syndrome, IgG4-related disease, IgA vasculitis, warm autoimmune hemolytic anemia, immune related adverse events (irAEs), pancreatitis, autoimmune central nervous system (CNS) inflammation, inflammation-induced liver injury, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), T cell mediated venous or arterial thrombosis, autoimmune encephalomyelitis (AT-EAE), chronic viral infection, psoriatic arthritis, atopic dermatitis, alopecia aerata, vitiligo, IgA nephropathy, lupus nephritis, chronic spontaneous urticarial, pyoderma gangrenosum, focal segmental glomerular sclerosis, membranous nephropathy, aplastic anemia, ulcerative colitis, Crohn’s disease, uveitis, Behcet’s disease, autoimmune hepatitis, ILD-myositis,.
As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g. , a monkey, and a chimpanzee), or a non-primate (such as a rat, or a mouse). In one aspect, the subject is a human. In some aspects, the human subject is being treated or assessed for an immune disorder or disease, or is at risk for developing an immune disorder or disease, that would benefit from reduction in T cell activity.
D. Combination Therapies
In some aspects, an Orail multispecific antibody disclosed herein is administered in combination with one or more additional therapies known to be effective in treating immune disorders (e.g., a T cell-mediated inflammatory disease, such as an autoimmune disease, or an allergic disease, or a symptom of such a disorder). The Orail multispecific antibody may be administered before, after, or concurrent with the one or more additional therapies. In some aspects, the Orail multispecific antibody is administered before the one or more additional therapies. In some aspects, the Orail multispecific antibody is administered after the one or more additional therapies. In some aspects, the Orail multispecific antibody is administered concurrent with the one or more additional therapies. In some aspects, the Orail multispecific antibody is administered in conjunction with the one or more additional therapies.
The one or more additional therapies may be an additional therapeutic agent(s). The Orail multispecific antibody and the additional therapeutic agent(s) can be administered in combination in the same composition or the additional therapeutic agent(s) can be administered as part of a separate composition.
In some aspects, the one or more additional therapies is a non-antibody therapeutic agent that is effective to treat the disorder or symptoms of the disorder. In some aspects, the one or more additional therapies is an antibody therapeutic agent that is effective to treat the disorder or symptoms of the disorder.
The Orail multispecific antibodies provided herein may, in certain aspects provide an enhanced effect (e.g., additive or synergistic in nature) that potentiates the mode of action of another administered therapeutic agent. There is no requirement for the combined results to be additive of the effects observed when each treatment (e.g., Orail multispecific antibody and additional therapeutic agent) is conducted separately. Although at least additive effects are generally desirable, any increased effect above one of the single therapies is beneficial. Furthermore, the invention does not require the combined treatment to exhibit synergistic effects. However, those skilled in the art will appreciate that with certain selected combinations that comprise preferred aspects, synergism may be observed.
The one or more additional therapies may be a compound that also inhibits Orail. Such compounds include, but are not limited to, CM4620, DS-2741a monoclonal antibody, CM6325, VV2003, SYL116011, SPLUNC peptide, PRCL-02, RP4010, RP3128, CM2489, CM3457, GSK- 7975A, RO2959, 10F8 monoclonal antibody.
In some aspects, the additional therapeutic agent(s) is an immunosuppressive agent, an immunomodulatory agent, an anti-inflammatory agent, or a biologic. Exemplary additional agents include, but are not limited to, corticosteroids (e.g., prednisone, prednisolone, methylprednisolone, budesonide, beclomethasone), calcineurin inhibitors [e.g., cyclosporine, also known as cyclosporin A (Sandimmune, Neoral, Gengraf), tacrolimus (Protopic, Prograf, FK506), pimecrolimus (Elidel)], methotrexate (Trexall, Otrexup, Xatmep, others), azathioprine (Azasan, Imuran), mercaptopurine (Purinethol, Purixan), leflunomide (Arava), hydroxychloroquine (Plaquenil), 5-aminosalicylates [e.g., sulfasalazine (Azulfidine), mesalamine (Asacol HD, Delzicol, others)], abatacept (Orencia), anakinra (Kineret), certolizumab (Cimzia), rituximab (Rituxan, Truxima), sarilumab (Kevzara), tocilizumab (Actemra), TNF inhibitors [e.g., infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), etanercept (Enbrel), golimumab (Simponi)], ustekinumab (Stelara), natalizumab (Tysabri), vedolizumab (Entyvio), dupilumab (Dupixent), omalizumab (Xolair), mepolizumab (Nucala), reslizumab (Cinqair), benralizumab (Fasenra), retinoids [tazarotene (Tazorac, Avage), acitretin (Soriatane)], thioguanine (Tabloid), hydroxyurea (Droxia, Hydrea), apremilast (Otezla), IL-17 inhibitors [e.g., secukinumab (Cosentyx), ixekizumab (Taltz)], Janus kinase (JAK) inhibitors [e.g., tofacitinib (Xeljanz), baricitinib (Olumiant), upadacitinib (Rinvoq)], denileukin diftitox (Ontak), alemtuzumab (Campath), belimumab (Benlysta), mycophenolate mofetil (CellCept), mycophenolate sodium (Myfortic), sirolimus (Rapamune), pentostatin (Nipent), thalidomide (Thalomid), imatinibmesylate (Gleevec), cyclophosphamide, IL-2 inhibitors [e.g., basiliximab (Simulect), daclizumab (Zenapax)], IL-22 agonists, leukotriene modifiers [e.g., montelukast (Singulair), zafirlukast (Accolate) and zileuton (Zyflo)], theophylline (Theo-24, Elixophyllin, Theochron), anticholinergic agents [e.g., ipratropium (Atrovent HF A), tiotropium (Spiriva, Spiriva Respimat)], beta agonists [e.g., albuterol (ProAir HF A, Ventolin HF A, others) and levalbuterol (Xopenex, Xopenex HF A)], antithymocyte globulin (rabbit ATG; Thymoglobulin), intravenous immune globulin (IVIg), and nonsteroidal anti-inflammatory drugs [NSAIDs, e.g., acetaminophen (Tylenol, others), ibuprofen (Advil, Motrin IB), naproxen (Aleve, Naprosyn), indomethacin (Indocin, Tivorbex)]. Although some of these agents can be categorized as disease modifying antirheumatic drugs (DMARDs), DMARDs may be used for treatment of diseases, disorders, or pathological processes other than rheumatoid arthritis in accordance with the present disclosure.
In some aspects, the additional therapeutic agent(s) is a blocker of T cell costimulatory signals, such as a blocker of CD28, CTLA-4, CD80, and CD86, or a blocker of interactions between the receptor (e.g., CD28, CTLA-4) and the ligands (e.g., CD80, CD86). In some aspects, the additional therapeutic agent is CTLA-4-IgG.
In some aspects, the additional therapeutic agent is an antibiotic, such as ciprofloxacin and metronidazole. In some aspects, the additional therapeutic agent is intestinal microbiome.
In some aspects, the additional therapeutic agent(s) aims to replenish cellular components or functions depleted or affected by the disease, the disorder, or the pathological process. For example, in some aspects, the additional therapeutic agent is thyroid hormone (to treat autoimmune thyroid disease), insulin (to treat type-1 diabetes), or vitamin B12 (to treat pernicious anemia).
In some aspects, the additional therapeutic agent aims to relieve symptoms or signs. Exemplary additional agents include, but are not limited to: pain relievers (see NSAIDs above); nutritional supplements [e.g., fish oil, dehydroepiandrosterone (DHEA)]; ointment, wet dressings, vitamin D analogs [e.g., calcipotriene, calcitriol (Vectical)], coal tar, anthralin (for diseases, disorders, and pathological processes involving skin conditions, e.g., atopic dermatitis, chronic plaque psoriasis); anti-diarrheals [e.g., psyllium powder (Metamucil), methylcellulose (Citrucel), loperamide (Imodium A-D)] (for diseases, disorders, and pathological processes involving bowel conditions, e.g., Crohn’s disease); proton pump inhibitor (for diseases, disorders, and pathological processes involving acid reflux, e.g., eosinophilic esophagitis); inhalers (for diseases, disorders, and pathological processes involving respiratory conditions, e.g., asthma).
In some aspects, the one or more additional therapies is one or more of regulatory T cell (Treg) augmentation therapy, extracorporeal photopheresis, plasmapheresis, physical therapy, occupational therapy, acupuncture, psychological therapy, nutrition/dietary therapy, phototherapy (for skin conditions), renal replacement therapy (e.g., dialysis, for nephritis and renal failure), or surgery. Surgery to treat diseases involving arthritis (e.g., rheumatoid arthritis, ankylosing spondylitis) includes synovectomy, tendon repair, j oint fusion, and total j oint replacement. Surgery for inflammatory bowel disease includes partial or total bowel resection. Surgery for eosinophilic esophagitis includes dilatation of esophagus. Surgery for asthma includes bronchial thermoplasty. The second therapy may comprise transplant, e.g., kidney transplant (for renal failure, e.g., lupus nephritis), or pancreatic or islet cell transplant (for type-1 diabetes).
E. Formulation, Administration., and Dose
Pharmaceutical compositions or medicaments comprising the Orail multispecific antibodies or the ST2 sdAbs are also provided herein. The disclosed Orail multispecific antibodies or the ST2 sdAbs of the invention may be formulated as pharmaceutical compositions as desired using art-recognized techniques. In some aspects, the pharmaceutical compositions of the invention may be administered neat or with a minimum of additional components while others may optionally be formulated to contain suitable pharmaceutically acceptable carriers e.g., vehicles, adjuvants, and diluents) comprising excipients and auxiliaries that are well known in the art, including for example, pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, radioprotectants and the like. Certain non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Certain nonlimiting exemplary radioprotectants include ascorbic acid, gentisic acid, ethanol, and combinations thereof.
In general, the compounds and compositions of the invention may be administered to a subject by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation. The subject compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms suitable for the particular mode of administration, including, for example, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. The particular dosage regimen for administering Orail multispecific antibodies of the invention, z.e., dose, timing and repetition, will depend on the particular subject and that subject’s medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.). Frequency of administration may be determined and adjusted over the course of therapy. A therapeutically effective dose is a dose sufficient to provide a clinical benefit to the subject, including for example, a dose sufficient to reduce swelling and/or redness, reduce skin rashes, hives and/or itching, reduce wheezing and/or shortness of breath, reduce sneezing and/or runny/stuffy nose, reduce anaphylaxis, decrease fatigue, reduce fever, reduce abdominal pain or digestive issues, decrease muscle aches, and/or reduce swelling in glands. Dosage administered may be adjusted or attenuated to manage potential side effects and/or toxicity.
V. Kits
In certain aspects, the instant disclosure provides kits that include a suitable container containing a Orail multispecific antibody or ST2 sdAb of the invention or a pharmaceutical composition of a Orail multispecific antibody or ST2 sdAb provided herein.
Such kits include one or more Orail multispecific antibodies or ST2 sdAbs and instructions for use, e.g., instructions for administering a therapeutically or prophylactically effective amount of a Orail multispecific antibody. The Orail multispecific antibody or ST2 sdAb may be in a vial or a pre-filled syringe. The kits may optionally further comprise means for administering the Orai 1 multispecific antibody or ST2 sdAb (e.g., an injection device, such as a pre-filled syringe or an intrathecal pump) or means for measuring the inhibition of T cell activity (e.g, means for measuring the inhibition of cytokine release). Such means for measuring the inhibition of T cells may comprise a means for obtaining a sample from a subject, such as, e.g., a blood or plasma sample. The kits of the invention may optionally further comprise means for determining the therapeutically effective amount.
In certain aspects the individual components of the pharmaceutical composition may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical composition separately in two or more containers, e.g., one container for an Orail multispecific antibody or ST2 sdAb preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.
VI. Miscellaneous
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.
The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to". The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.
The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain aspects, about means ±10%. In certain aspects, about means ±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.
The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.
The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like.
EXAMPLES
The following examples are included to demonstrate certain aspects of the invention.
Example 1. Orail Multispecific Antibody Synthesis
Overview
An anti-Orail monoclonal human IgG2 antibody with a lambda light chain (mAb 2C1.1; US 2012/0231006) was used as the Orail/first antigen binding domain in the synthesis of Orail multispecific antibodies as described herein. As one example, an Orail multispecific antibody was prepared comprising a second antigen binding domain that is an anti-PDl VHH (sdAb) as disclosed in U.S. Patent Number 10,323,090. This exemplary multispecific antibody is also referred to herein as “CTAB PR071” or “PR071”. See, Table 2 below for the components of the antibody constructs, Table 3 for the sequences of the full-length constructs, including the Orail multispecific antibody constructs, and Table 4 for exemplary CDR sequences of the Orail multispecific antibody. Additional exemplary anti-Orai 1 antibodies are also contemplated for use in the multispecific antibodies of the present invention. Non-limiting examples include mAb 10F8 (Novo Nordisk, W02013/091903) and mAb hR198_HGl/LGl (Daiichi Sankyo Company, US 2017/0226203). The sequences of these additional exemplary antibodies are indicated in Table 5. The sequences of the 2C 1.1, 10F8, hR198, and PD1 VHH antibodies, as disclosed in the abovenoted patent publications, are herein incorporated by reference.
The expression constructs for the anti-Orai 1 antibody and the anti-PDl single chain VHH variants were cloned into a high expression mammalian vector and the DNA sequences of the gene insert were confirmed. Each DNA construct was scaled up for transfection and the DNA sequence was confirmed after DNA scale-up. A 0.1 Liter transient production was completed in CHO cells (TunaCHO™ extended 14-day process) for each. The anti-Orai 1 antibody variants were purified by Protein A chromatography, and a final yield range of 6.93 mg to 30.95 mg was obtained. The anti-PDl single chain VHH variants were purified by IMAC, and a final yield range of 0.72 mg to 30.34 mg was obtained. CE-SDS analysis was performed. Endotoxin measurements confirmed all samples met the <1 EU/mg requirement. SEC polishing and post polishing SE-UPLC analysis were only performed for three anti-Orai 1 antibody variants. An exemplary anti-Orai 1 mAb and an exemplary anti-Orai 1 multispecific antibody are illustrated in FIG. 1.
Additional exemplary anti-Orail/PDl multispecific antibodies (i.e., CTABs) were generated and the sequences indicated in Tables 6, 7, 8, and 9. Tables 6-9 set forth sequences of Orail multispecific antibodies as shown in FIG. 3. To the extent that there is an inadvertent discrepancy as to the sequence in Tables 6-9 and in FIG. 3, the annotation as shown in FIG. 3 is correct.
Tables 6 and 7 provide the components, including variable regions, linkers, and constant region sequences for the multispecific antibodies. The Orail antibody components were from Orail antibodies disclosed in US 2012/0231006, W02013/091903, and US 2017/0226203. The PD1 portion of the exemplary multispecific antibody constructs comprises a VHH or an scFv binding domain sequence. The PD1 sequences are as disclosed in U.S. Patent Number 10,323,090, US2019/032749, US2019/032749, US 2020/0347135, and US 2019/0322747, and were also derived from known antibodies, such as Nivolumab, Pembrolizumab, Sintilimab, Spartalizumab, Cemiplimab, Retifanlimab, Dostarlimab, Ezabenlimab, Cetrelimab, Budigalimab, Sasanlimab, and Vudalimab. Table 8 illustrates the sequences of the full-length constructs, including the Orail multispecific antibody constructs, and Table 9 provides exemplary CDR sequences of the Orail/PDl multispecific antibody constructs.
Molecular Construction of Expression Vector
Each gene sequence was cloned into a high expression mammalian vector. Each completed construct was sequence-confirmed before proceeding to DNA scale up.
Plasmid DNA Scale-up
Each DNA expression construct was scaled-up for transfection. Uncut plasmid DNA was analyzed by agarose gel electrophoresis and quality was assessed. Plasmid DNA was sequence- confirmed before proceeding to transfection.
CHO Transient Transfection (TunaCHO™ Process)
Suspension CHO cells were seeded in a shake flask and expanded using a serum-free and chemically defined medium. On the day of transfection, the expanded cells were seeded into a new vessel with fresh medium. After transfection, the cells were maintained as a batch-fed culture until the end of the production run.
Purification by Protein A Chromatography
Clarified conditioned media from the production run was loaded onto a Protein A column pre-equilibrated with binding buffer. Washing buffer was passed through the column until the OD280 value returned to baseline. The protein was eluted with a low pH buffer, fractions were collected, buffered, and the OD280 value of each fraction was recorded. Fractions containing the protein of interest were pooled and filtered through a 0.2 pm membrane filter. The protein concentration was calculated from the OD280 value and the calculated extinction coefficient. IMAC (Immobilized Metal Affinity Chromatography) Purification of His Tagged Protein
Clarified and buffered conditioned media from the production run was loaded onto an IMAC column preequilibrated with binding buffer. Washing buffer containing 40 mM imidazole was passed through the column until the OD280 value returned to baseline. The protein was eluted with a linear gradient of increasing imidazole concentration up to 0.5 M. The eluate was collected in fractions, and the OD280 value of each fraction was recorded. Denaturing capillary electrophoresis (CE-SDS, LabChip GXII, Perkin Elmer) of each fraction was performed and analyzed. Fractions containing the protein of interest were pooled and dialyzed into buffer. The protein was filtered through a 0.2 pm membrane filter and the protein concentration was calculated using the OD280 value and the calculated extinction coefficient.
CE-SDS (Capillary Electrophoresis using Sodium Dodecyl Sulfate) Analysis
CE-SDS analysis of the antibody protein was performed using a LabChip GXII (Perkin Elmer).
Intact Mass Analysis
Intact mass analysis by mass spectrometry was performed (Xevo G2-XS Qtof, Waters). The observed molecular weights for the IgG were within the expected range.
Endotoxin Measurement
Endotoxin in a sample of the purified product was quantified using the chromogenic Limulus Amebocyte Lysate method (Endosafe-MCS, Charles River). The sample was run in duplicate. Endotoxin measurements confirmed all samples met the <1 EU/mg requirement.
SE-UPLC (Size Exclusion-Ultra High Pressure Liquid Chromatography) Analysis
SE-UPLC analysis of the protein was performed. All proteins were observed to contain >95% monomers. SEC standards (Medna, Y3101) were chromatographed as a reference for protein sizes.
Polishing by Size Exclusion Chromatography
If necessary, size exclusion chromatography (SEC) was performed using a HiLoad 26/600 Superdex 200 column (GE Healthcare Life Sciences) using the mobile phase. Collected fractions were analyzed by Capillary Electrophoresis- Sodium Dodecyl Sulfate (CE-SDS, LabChip GXII, Perkin Elmer) analysis. Fractions containing the antibody protein were pooled and analyzed by Size Exclusion-Ultra High Pressure Liquid Chromatography (SE-UPLC).
Table 2. Sequences for the Components of OrailMultispecific Antibody Constructs
Figure imgf000084_0001
Figure imgf000085_0001
Table 3. Sequence of Full-Length Parent Antibody Constructs
Figure imgf000086_0001
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Table 4: Exemplary CDR Sequences for Orail and Target Antigen Binding Domains
Figure imgf000087_0002
Table 5: Additional Exemplary Orail Antibody Sequences
Figure imgf000088_0001
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Table 6. Sequences for the Components of Additional Exemplary Orail Multispecific Antibody Constructs
Figure imgf000090_0001
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Table 7. Components of an Additional Exemplary Orail Multispecific Antibody Construct
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Table 8. Sequences of Additional Full-Length Multispecific Antibody Constructs
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Table 9. Additional Exemplary CDR Sequences for Orail and Target Antigen Binding Domains
Figure imgf000110_0001
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Example 2. T Cell Functional Assay
The Orail/PDl multispecific antibodies provided herein were tested in an IL-2 cytokine release assay to determine the effect on T cell activity. Human PBMCs were seeded in 24-well tissue culture plates at a density of 106 cells/ml in a volume of 900 pL. The PBMCs were treated for 1 hour at 37°C with various concentrations of Anti-Orai-1 mAB (2C1.1 human IgG2), Anti- Orail/Anti-PDl VHH multispecific antibody, anti-Orail antibody and anti-PDl VHH (equimolar), anti-PDl VHH, or cyclosporine A. Twenty -five pL of human Anti-CD3/CD28 dynabeads were added per well and the PBMCs were incubated at 37°C for 24 hours. The cells were pelleted and the supernatants collected and stored at -80°C. The supernatants were assayed for IL-2 using a human IL-2 ELISA kit from BD (Catalog #555190).
FIG. 2 depicts the percent inhibition of IL-2 release with the various treatments. The graph in FIG. 2 shows that the Orail/PDl VHH multispecific antibody inhibited IL-2 release. Importantly, the dose-response curve in FIG. 2 demonstrates that the anti -Orail/PDl VHH multispecific antibody exhibited enhanced inhibition of IL-2 release as compared to the parent anti-Orail antibody. Moreover, the level of IL-2 cytokine suppression achieved with the anti- Orail/PDl VHH multispecific antibody was equivalent to therapeutically relevant levels of the immunosuppressant agent cyclosporine.
Example 3. NFAT Jurkat Screening Assay
PD1 -expressing Jurkat T cells containing a nuclear factor of activated T-cells (NFAT) luciferase reporter (Promega, J1250) were utilized to assess activity of Cell Targeting Biologies (CTABs). Reporter cells were cultured in assay media consisting of RPMI (Gibco, 72400-047) supplemented with 10% FBS (Gibco, J1211). Supernatants from CHO cells secreting CTAB proteins targeting PD1 and Orail were diluted 1 : 10, 1 :20, or 1 : 50 in assay media and 50uL of each was dispensed in wells of a white, flat-bottom 96 well plate (Thermo, 136102). Reporter cells were next thawed and diluted in assay media according to manufacturer’s instructions and 50uL of cell suspension was added to each well. Cells and test supernatants were next mixed by pipette and preincubated for 1 hour at 37C at 5% CO2. After incubation, cells were mixed by pipetting and 80uL was transferred to a lug/mL anti-CD3 (Biolegend, 300465) coated 96 well plate for stimulation. The coated stimulation plates were white, flat-bottomed plates coated overnight with lug/mL anti-CD3 diluted in PBS (Gibco, 14190-144). Plates were washed three times with PBS and aspirated immediately prior to the addition of reporter cells. After 48 hours at 37C and 5% CO2, plates were allowed to equilibrate to room temperature for 30 minutes. 80uL of Bioglo (Promega, G7940) was then added to each well, mixed by pipette, and protected from light for 15 minutes at room temperature. Luminescence was quantified with a plate reader (Molecular Devices, SpectraMax i3x) with an integration time of 500ms.
FIG. 4 depicts the percent inhibition of NF AT at various nM concentrations of the anti- Orail/PDl VHH multispecific antibody (CTAB PR071; anti-Orail IgG/anti-PDl VHH). The dose-response curve in FIG. 4 demonstrates that the anti-Orail/PDl VHH multispecific antibody exhibited enhanced inhibition of NF AT as compared to the parent anti-Orail antibody (BB PR001; anti-Orail IgG).
FIG. 5 depicts the percent inhibition of NF AT at various dilutions of various anti- Orail/PDl multispecific antibodies (CTABs). The results show that the Orail/PDl multispecific antibodies exhibited enhanced inhibition of NF AT as compared to the Orail and PD1 individual antibody components.
Example 4: ST2 Single Domain Antibodies (VHH)
ST2 single domain (VHH) antigen binding domains were prepared as described in methods provided elsewhere herein. Exemplary sequences of ST2 VHH antibodies are depicted as SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 in Table 10. Exemplary CDR sequences for the ST2 VHH antibodies are illustrated in Table 11. Orail multispecific antibodies comprising any of the various ST2 VHH antigen binding domains provided herein can be synthesized as described in Example 1.
Table 10: Sequences for ST2 VHH Antigen Binding Domains
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Table 11: Exemplary CDR sequences for ST2 VHH Antigen Binding Domains
Figure imgf000126_0002
Figure imgf000127_0001
Example 5: Functional Assay for Anti-Orail-Anti-ST2 Multispecific Antibodies
ST2-expressing T cells are generated essentially as described in Calise et al. (2021, J. Allergy Clin. Immunol. 148(3):867-875), which is incorporated herein by reference in its entirety. In brief, human PBMCs are obtained from subjects with allergy to peanut, alder pollen, grass pollen, or dust mite. The PBMCs are stimulated with allergen plus or minus recombinant human IL-33 for 18 hours. The cells are enriched for CD154 expressing cells using anti-CD154 antibody and magnetic beads. ST2 expression is monitored by flow cytometry.
The ST2-expressing T cells are treated with any of the anti-Orail/anti-ST2 multispecific antibodies provided herein and a cytokine release assay is performed as described in Example 2.

Claims

WE CLAIM:
1. A multispecific antibody comprising
(a) a first antigen binding domain that binds to Orai 1 ; and
(b) a second antigen binding domain that binds to a target on a T cell; wherein the multispecific antibody inhibits T cell activity.
2. The multispecific antibody of claim 1, wherein the first antigen binding domain competes for binding with, binds a same epitope as, or comprises
(a) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2;
(b) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140;
(c) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or
(d) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 151.
3. The multispecific antibody of claim 1 or 2, wherein the first antigen binding domain comprises
(a) residues 27-38 of SEQ ID NO: 5 for CDR-L1, residues 56-65 of SEQ ID NO: 5 for CDR-L2, and residues 105-117 of SEQ ID NO: 5 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 2 for CDR-H1, residues 56-65 of SEQ ID NO: 2 for CDR-H2, and residues 105-117 of SEQ ID NO: 2 for CDR-H3;
(b) residues 27-38 of SEQ ID NO: 146 for CDR-L1, residues 56-65 of SEQ ID NO: 146 for CDR-L2, and residues 105-117 of SEQ ID NO: 146 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 140 for CDR-H1, residues 56-65 of SEQ ID NO: 140 for CDR-H2, and residues 1 OS- 117 of SEQ ID NO: 140 for CDR-H3;
(c) residues 27-38 of SEQ ID NO: 149 for CDR-L1, residues 56-65 of SEQ ID NO: 149 for CDR-L2, and residues 105-117 of SEQ ID NO: 149 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 147 for CDR-H1, residues 56-65 of SEQ ID NO: 147 for CDR-H2, and residues 1 OS- 117 of SEQ ID NO: 147 for CDR-H3; or
(d) residues 27-38 of SEQ ID NO: 155 for CDR-L1, residues 56-65 of SEQ ID NO: 155 for CDR-L2, and residues 105-117 of SEQ ID NO: 155 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 151 for CDR-H1, residues 56-65 of SEQ ID NO: 151 for CDR-H2, and residues 105- 117 of SEQ ID NO: 151 for CDR-H3; wherein the residues are numbered according to Lefranc (IMGT).
4. The multispecific antibody of claim 1 or 2, wherein the first antigen binding domain comprises
(a) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, and residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 2 for CDR-H1, residues 50-65 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3;
(b) residues 24-34 of SEQ ID NO: 146 for CDR-L1, residues 50-56 of SEQ ID NO: 146 for CDR-L2, and residues 89-97 of SEQ ID NO: 146 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 140 for CDR-H1, residues 50-65 of SEQ ID NO: 140 for CDR-H2, and residues 95-102 of SEQ ID NO: 140 for CDR-H3;
(c) residues 24-34 of SEQ ID NO: 149 for CDR-L1, residues 50-56 of SEQ ID NO: 149 for CDR-L2, and residues 89-97 of SEQ ID NO: 149 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 147 for CDR-H1, residues 50-65 of SEQ ID NO: 147 for CDR-H2, and residues 95-102 of SEQ ID NO: 147 for CDR-H3; or (d) residues 24-34 of SEQ ID NO: 155 for CDR-L1, residues 50-56 of SEQ ID NO: 155 for CDR-L2, and residues 89-97 of SEQ ID NO: 155 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 151 for CDR-H1, residues 50-65 of SEQ ID NO: 151 for CDR-H2, and residues 95-102 of SEQ ID NO : 151 for CDR-H3 ; wherein the residues are numbered according to Kabat.
5. The multispecific antibody of claim 1 or 2, wherein the first antigen binding domain comprises
(a) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 2 for CDR-H1, residues 52-56 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3;
(b) residues 24-34 of SEQ ID NO: 146 for CDR-L1, residues 50-56 of SEQ ID NO: 146 for CDR-L2, residues 89-97 of SEQ ID NO: 146 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 140 for CDR-H1, residues 52-56 of SEQ ID NO: 140 for CDR-H2, and residues 95-102 of SEQ ID NO: 140 for CDR-H3;
(c) residues 24-34 of SEQ ID NO: 149 for CDR-L1, residues 50-56 of SEQ ID NO: 149 for CDR-L2, residues 89-97 of SEQ ID NO: 149 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 147 for CDR-H1, residues 52-56 of SEQ ID NO: 147 for CDR-H2, and residues 95-102 of SEQ ID NO: 147 for CDR-H3; or
(d) residues 24-34 of SEQ ID NO: 155 for CDR-L1, residues 50-56 of SEQ ID NO: 155 for CDR-L2, residues 89-97 of SEQ ID NO: 155 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 151 for CDR-H1, residues 52-56 of SEQ ID NO: 151 for CDR-H2, and residues 95-102 of SEQ ID NO: 151 for CDR-H3; wherein the residues are numbered according to Chothia.
6. The multispecific antibody of claim 1 or 2, wherein the first antigen binding domain comprises
(a) residues 30-36 of SEQ ID NO: 5 for CDR-L1, residues 46-55 of SEQ ID NO: 5 for CDR-L2, and residues 89-96 of SEQ ID NO: 5 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 2 for CDR-H1, residues 47-58 of SEQ ID NO: 2 for CDR-H2, and residues 93-101 of SEQ ID NO: 2 for CDR-H3;
(b) residues 30-36 of SEQ ID NO: 146 for CDR-L1, residues 46-55 of SEQ ID NO: 146 for CDR-L2, and residues 89-96 of SEQ ID NO: 146 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 140 for CDR-H1, residues 47-58 of SEQ ID NO: 140 for CDR-H2, and residues 93-101 of SEQ ID NO: 140 for CDR-H3;
(c) residues 30-36 of SEQ ID NO: 149 for CDR-L1, residues 46-55 of SEQ ID NO: 149 for CDR-L2, and residues 89-96 of SEQ ID NO: 149 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 147 for CDR-H1, residues 47-58 of SEQ ID NO: 147 for CDR-H2, and residues 93-101 of SEQ ID NO: 147 for CDR-H3; or
(d) residues 30-36 of SEQ ID NO: 155 for CDR-L1, residues 46-55 of SEQ ID NO: 155 for CDR-L2, and residues 89-96 of SEQ ID NO: 155 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 151 for CDR-H1, residues 47-58 of SEQ ID NO: 151 for CDR-H2, and residues 93-101 of SEQ ID NO : 151 for CDR-H3 ; wherein the residues are numbered according to MacCallum.
7. The multispecific antibody of claim 1 or 2, wherein the first antigen binding domain comprises
(a) residues 24-34 of SEQ ID NO: 5 for CDR-L1, residues 50-56 of SEQ ID NO: 5 for CDR-L2, and residues 89-97 of SEQ ID NO: 5 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 2 for CDR-H1, residues 50-58 of SEQ ID NO: 2 for CDR-H2, and residues 95-102 of SEQ ID NO: 2 for CDR-H3;
(b) residues 24-34 of SEQ ID NO: 146 for CDR-L1, residues 50-56 of SEQ ID NO: 146 for CDR-L2, and residues 89-97 of SEQ ID NO: 146 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 140 for CDR-H1, residues 50-58 of SEQ ID NO: 140 for CDR-H2, and residues 95-102 of SEQ ID NO: 140 for CDR-H3;
(c) residues 24-34 of SEQ ID NO: 149 for CDR-L1, residues 50-56 of SEQ ID NO: 149 for CDR-L2, and residues 89-97 of SEQ ID NO: 149 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 147 for CDR-H1, residues 50-58 of SEQ ID NO: 147 for CDR-H2, and residues 95-102 of SEQ ID NO: 147 for CDR-H3; or (d) residues 24-34 of SEQ ID NO: 155 for CDR-L1, residues 50-56 of SEQ ID NO: 155 for CDR-L2, and residues 89-97 of SEQ ID NO: 155 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 151 for CDR-H1, residues 50-58 of SEQ ID NO: 151 for CDR-H2, and residues 95-102 of SEQ ID NO : 151 for CDR-H3 ; wherein the residues are numbered according to AbM.
8. The multispecific antibody of claim 1 or 2, wherein the first antigen binding domain comprises
(a) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 18, a CDRL2 comprising the amino acid sequence set forth as SEQ ID NO: 19, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 20; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 21, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 22, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 23;
(b) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 199, a CDRL2 comprising the amino acid sequence VYN, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 200; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 196, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 197, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 198;
(c) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 223, a CDRL2 comprising the amino acid sequence STS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 224; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 220, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 221, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 222; or
(d) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 235, a CDRL2 comprising the amino acid sequence WAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 236; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 232, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 233, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 234.
9. The multispecific antibody of any one of claims 1-8, wherein the first antigen binding domain comprises
131 (a) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2;
(b) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 146; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 140;
(c) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 149; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 147; or
(d) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 151.
10. The multispecific antibody of any one of claims 1-9, wherein the first antigen binding domain comprises a light chain comprising an amino acid sequence set forth as SEQ ID NO: 15; and/or a heavy chain comprising an amino acid sequence set forth as SEQ ID NO: 14.
11. The multispecific antibody of any one of claims 1-10, wherein the first antigen binding domain comprises a single domain antibody (sdAb).
12. The multispecific antibody of any one of claims 1-11, wherein the second antigen binding domain binds to a target on activated T cells.
13. The multispecific antibody of any one of claims 1-12, wherein the second antigen binding domain comprises a single domain antibody (sdAb).
14. The multispecific antibody of any one of claims 1-12, wherein the second antigen binding domain comprises an scFv.
15. The multispecific antibody of any one of claims 1-14, wherein the second antigen binding domain binds to programmed cell death protein 1 (PD1).
132
16. The multispecific antibody of claim 15, wherein the second antigen binding domain competes for binding with, binds a same epitope as, or comprises
(a) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 8;
(b) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 170;
(c) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 171;
(d) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 172; or
(e) an antigen binding domain having three CDRs of a variable region sequence comprising an amino acid sequence set forth as SEQ ID NO: 173.
17. The multispecific antibody of claim 16, wherein the second antigen binding domain comprises
(a) residues 31-35 of SEQ ID NO: 8 for CDR1, residues 50-65 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3;
(b) residues 31-35 of SEQ ID NO: 170 for CDR1, residues 50-65 of SEQ ID NO: 170 for CDR2, and residues 95-102 of SEQ ID NO: 170 for CDR3;
(c) residues 31-35 of SEQ ID NO: 171 for CDR1, residues 50-65 of SEQ ID NO: 171 for CDR2, and residues 95-102 of SEQ ID NO: 171 for CDR3;
(d) residues 31-35 of SEQ ID NO: 172 for CDR1, residues 50-65 of SEQ ID NO: 172 for CDR2, and residues 95-102 of SEQ ID NO: 172 for CDR3; or
(e) residues 31-35 of SEQ ID NO: 173 for CDR1, residues 50-65 of SEQ ID NO: 173 for CDR2, and residues 95-102 of SEQ ID NO: 173 for CDR3; wherein the residues are numbered according to Kabat.
18. The multispecific antibody of claim 16, wherein the second antigen binding domain comprises
133 (a) residues 26-32 of SEQ ID NO: 8 for CDR1, residues 52-56 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3;
(b) residues 26-32 of SEQ ID NO: 170 for CDR1, residues 52-56 of SEQ ID NO: 170 for CDR2, and residues 95-102 of SEQ ID NO: 170 for CDR3;
(c) residues 26-32 of SEQ ID NO: 171 for CDR1, residues 52-56 of SEQ ID NO: 171 for CDR2, and residues 95-102 of SEQ ID NO: 171 for CDR3;
(d) residues 26-32 of SEQ ID NO: 172 for CDR1, residues 52-56 of SEQ ID NO: 172 for CDR2, and residues 95-102 of SEQ ID NO: 172 for CDR3; or
(e) residues 26-32 of SEQ ID NO: 173 for CDR1, residues 52-56 of SEQ ID NO: 173 for CDR2, and residues 95-102 of SEQ ID NO: 173 for CDR3; wherein the residues are numbered according to Chothia.
19. The multispecific antibody of claim 16, wherein the second antigen binding domain comprises
(a) residues 30-35 of SEQ ID NO: 8 for CDR1, residues 47-58 of SEQ ID NO: 8 for CDR2, and residues 93-101 of SEQ ID NO: 8 for CDR3;
(b) residues 30-35 of SEQ ID NO: 170 for CDR1, residues 47-58 of SEQ ID NO: 170 for CDR2, and residues 93-101 of SEQ ID NO: 170 for CDR3;
(c) residues 30-35 of SEQ ID NO: 171 for CDR1, residues 47-58 of SEQ ID NO: 171 for CDR2, and residues 93-101 of SEQ ID NO: 171 for CDR3;
(d) residues 30-35 of SEQ ID NO: 172 for CDR1, residues 47-58 of SEQ ID NO: 172 for CDR2, and residues 93-101 of SEQ ID NO: 172 for CDR3; or
(e) residues 30-35 of SEQ ID NO: 173 for CDR1, residues 47-58 of SEQ ID NO: 173 for CDR2, and residues 93-101 of SEQ ID NO: 173 for CDR3; wherein the residues are numbered according to MacCallum.
20. The multispecific antibody of claim 16, wherein the second antigen binding domain comprises
(a) residues 26-35 of SEQ ID NO: 8 for CDR1, residues 50-58 of SEQ ID NO: 8 for CDR2, and residues 95-102 of SEQ ID NO: 8 for CDR3;
134 (b) residues 26-35 of SEQ ID NO: 170 for CDR1, residues 50-58 of SEQ ID NO: 170 for CDR2, and residues 95-102 of SEQ ID NO: 170 for CDR3;
(c) residues 26-35 of SEQ ID NO: 171 for CDR1, residues 50-58 of SEQ ID NO: 171 for CDR2, and residues 95-102 of SEQ ID NO: 171 for CDR3;
(d) residues 26-35 of SEQ ID NO: 172 for CDR1, residues 50-58 of SEQ ID NO: 172 for CDR2, and residues 95-102 of SEQ ID NO: 172 for CDR3; or
(e) residues 26-35 of SEQ ID NO: 173 for CDR1, residues 50-58 of SEQ ID NO: 173 for CDR2, and residues 95-102 of SEQ ID NO: 173 for CDR3; wherein the residues are numbered according to AbM.
21. The multispecific antibody of claim 16, wherein the second antigen binding domain comprises
(a) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 24, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 25, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 26;
(b) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 314, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 315, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 316;
(c) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 322, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 323, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 324;
(d) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 330, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 331, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 332; or
(e) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 338, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 339, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 340.
22. The multispecific antibody of any one of claims 1-21, wherein the second antigen binding domain comprises the amino acid sequence set forth as SEQ ID NO: 8, 170, 171, 172, or 173.
135
23. The multispecific antibody of any one of claims 1-15, wherein the second antigen binding domain competes for binding with, binds a same epitope as, or comprises
(a) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 145 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 143;
(b) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 154 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 153;
(c) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 158 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 157;
(d) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 160 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 169;
(e) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 162 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 161;
(f) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 164 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 163;
(g) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 167 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 165; or
136 (h) an antigen binding domain having three complementarity determining regions (CDRs) of a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 168 and/or three CDRs of a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 169.
24. The multispecific antibody of claim 23, wherein the second antigen binding domain comprises
(a) residues 27-38 of SEQ ID NO: 145 for CDR-L1, residues 56-65 of SEQ ID NO: 145 for CDR-L2, and residues 105-117 of SEQ ID NO: 145 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 143 for CDR-H1, residues 56-65 of SEQ ID NO: 143 for CDR-H2, and residues 1 OS- 117 of SEQ ID NO: 143 for CDR-H3;
(b) residues 27-38 of SEQ ID NO: 154 for CDR-L1, residues 56-65 of SEQ ID NO: 154 for CDR-L2, and residues 105-117 of SEQ ID NO: 154 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 153 for CDR-H1, residues 56-65 of SEQ ID NO: 153 for CDR-H2, and residues 105- 117 of SEQ ID NO: 153 for CDR-H3;
(c) residues 27-38 of SEQ ID NO: 158 for CDR-L1, residues 56-65 of SEQ ID NO: 158 for CDR-L2, and residues 105-117 of SEQ ID NO: 158 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 157 for CDR-H1, residues 56-65 of SEQ ID NO: 157 for CDR-H2, and residues 105- 117 of SEQ ID NO: 157 for CDR-H3;
(d) residues 27-38 of SEQ ID NO: 160 for CDR-L1, residues 56-65 of SEQ ID NO: 160 for CDR-L2, and residues 105-117 of SEQ ID NO: 160 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 159 for CDR-H1, residues 56-65 of SEQ ID NO: 159 for CDR-H2, and residues 105- 117 of SEQ ID NO: 159 for CDR-H3;
(e) residues 27-38 of SEQ ID NO: 162 for CDR-L1, residues 56-65 of SEQ ID NO: 162 for CDR-L2, and residues 105-117 of SEQ ID NO: 162 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 161 for CDR-H1, residues 56-65 of SEQ ID NO: 161 for CDR-H2, and residues 105- 117 of SEQ ID NO: 161 for CDR-H3;
(f) residues 27-38 of SEQ ID NO: 164 for CDR-L1, residues 56-65 of SEQ ID NO: 164 for CDR-L2, and residues 105-117 of SEQ ID NO: 164 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 163 for CDR-H1, residues 56-65 of SEQ ID NO: 163 for CDR-H2, and residues 105- 117 of SEQ ID NO: 163 for CDR-H3;
137 (g) residues 27-38 of SEQ ID NO: 167 for CDR-L1, residues 56-65 of SEQ ID NO: 167 for CDR-L2, and residues 105-117 of SEQ ID NO: 167 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 165 for CDR-H1, residues 56-65 of SEQ ID NO: 165 for CDR-H2, and residues 1 OS- 117 of SEQ ID NO: 165 for CDR-H3; or
(h) residues 27-38 of SEQ ID NO: 168 for CDR-L1, residues 56-65 of SEQ ID NO: 168 for CDR-L2, and residues 105-117 of SEQ ID NO: 168 for CDR-L3; and/or residues 27-38 of SEQ ID NO: 169 for CDR-H1, residues 56-65 of SEQ ID NO: 169 for CDR-H2, and residues 1 OS- 117 of SEQ ID NO: 169 for CDR-H3; wherein the residues are numbered according to Lefranc (IMGT).
25. The multispecific antibody of claim 23, wherein the second antigen binding domain comprises
(a) residues 24-34 of SEQ ID NO: 145 for CDR-L1, residues 50-56 of SEQ ID NO: 145 for CDR-L2, and residues 89-97 of SEQ ID NO: 145 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 143 for CDR-H1, residues 50-65 of SEQ ID NO: 143 for CDR-H2, and residues 95-102 of SEQ ID NO: 143 for CDR-H3;
(b) residues 24-34 of SEQ ID NO: 154 for CDR-L1, residues 50-56 of SEQ ID NO: 154 for CDR-L2, and residues 89-97 of SEQ ID NO: 154 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 153 for CDR-H1, residues 50-65 of SEQ ID NO: 153 for CDR-H2, and residues 95-102 of SEQ ID NO: 153 for CDR-H3;
(c) residues 24-34 of SEQ ID NO: 158 for CDR-L1, residues 50-56 of SEQ ID NO: 158 for CDR-L2, and residues 89-97 of SEQ ID NO: 158 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 157 for CDR-H1, residues 50-65 of SEQ ID NO: 157 for CDR-H2, and residues 95-102 of SEQ ID NO: 157 for CDR-H3;
(d) residues 24-34 of SEQ ID NO: 160 for CDR-L1, residues 50-56 of SEQ ID NO: 160 for CDR-L2, and residues 89-97 of SEQ ID NO: 160 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 159 for CDR-H1, residues 50-65 of SEQ ID NO: 159 for CDR-H2, and residues 95-102 of SEQ ID NO: 159 for CDR-H3;
(e) residues 24-34 of SEQ ID NO: 162 for CDR-L1, residues 50-56 of SEQ ID NO: 162 for CDR-L2, and residues 89-97 of SEQ ID NO: 162 for CDR-L3; and/or residues 31-35 of SEQ
138 ID NO: 161 for CDR-H1, residues 50-65 of SEQ ID NO: 161 for CDR-H2, and residues 95-102 of SEQ ID NO: 161 for CDR-H3;
(f) residues 24-34 of SEQ ID NO: 164 for CDR-L1, residues 50-56 of SEQ ID NO: 164 for CDR-L2, and residues 89-97 of SEQ ID NO: 164 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 163 for CDR-H1, residues 50-65 of SEQ ID NO: 163 for CDR-H2, and residues 95-102 of SEQ ID NO: 163 for CDR-H3;
(g) residues 24-34 of SEQ ID NO: 167 for CDR-L1, residues 50-56 of SEQ ID NO: 167 for CDR-L2, and residues 89-97 of SEQ ID NO: 167 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 165 for CDR-H1, residues 50-65 of SEQ ID NO: 165 for CDR-H2, and residues 95-102 of SEQ ID NO: 165 for CDR-H3; or
(h) residues 24-34 of SEQ ID NO: 168 for CDR-L1, residues 50-56 of SEQ ID NO: 168 for CDR-L2, and residues 89-97 of SEQ ID NO: 168 for CDR-L3; and/or residues 31-35 of SEQ ID NO: 169 for CDR-H1, residues 50-65 of SEQ ID NO: 169 for CDR-H2, and residues 95-102 of SEQ ID NO: 169 for CDR-H3; wherein the residues are numbered according to Kabat.
26. The multispecific antibody of claim 23, wherein the second antigen binding domain comprises
(a) residues 24-34 of SEQ ID NO: 145 for CDR-L1, residues 50-56 of SEQ ID NO: 145 for CDR-L2, residues 89-97 of SEQ ID NO: 145 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 143 for CDR-H1, residues 52-56 of SEQ ID NO: 143 for CDR-H2, and residues 95-102 of SEQ ID NO: 143 for CDR-H3;
(b) residues 24-34 of SEQ ID NO: 154 for CDR-L1, residues 50-56 of SEQ ID NO: 154 for CDR-L2, residues 89-97 of SEQ ID NO: 154 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 153 for CDR-H1, residues 52-56 of SEQ ID NO: 153 for CDR-H2, and residues 95-102 of SEQ ID NO: 153 for CDR-H3;
(c) residues 24-34 of SEQ ID NO: 158 for CDR-L1, residues 50-56 of SEQ ID NO: 158 for CDR-L2, residues 89-97 of SEQ ID NO: 158 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 157 for CDR-H1, residues 52-56 of SEQ ID NO: 157 for CDR-H2, and residues 95-102 of SEQ ID NO: 157 for CDR-H3; (d) residues 24-34 of SEQ ID NO: 160 for CDR-L1, residues 50-56 of SEQ ID NO: 160 for CDR-L2, residues 89-97 of SEQ ID NO: 160 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 159 for CDR-H1, residues 52-56 of SEQ ID NO: 159 for CDR-H2, and residues 95-102 of SEQ ID NO: 159 for CDR-H3;
(e) residues 24-34 of SEQ ID NO: 162 for CDR-L1, residues 50-56 of SEQ ID NO: 162 for CDR-L2, residues 89-97 of SEQ ID NO: 162 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 161 for CDR-H1, residues 52-56 of SEQ ID NO: 161 for CDR-H2, and residues 95-102 of SEQ ID NO: 161 for CDR-H3;
(f) residues 24-34 of SEQ ID NO: 164 for CDR-L1, residues 50-56 of SEQ ID NO: 164 for CDR-L2, residues 89-97 of SEQ ID NO: 164 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 163 for CDR-H1, residues 52-56 of SEQ ID NO: 163 for CDR-H2, and residues 95-102 of SEQ ID NO: 163 for CDR-H3;
(g) residues 24-34 of SEQ ID NO: 167 for CDR-L1, residues 50-56 of SEQ ID NO: 167 for CDR-L2, residues 89-97 of SEQ ID NO: 167 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 165 for CDR-H1, residues 52-56 of SEQ ID NO: 165 for CDR-H2, and residues 95-102 of SEQ ID NO: 165 for CDR-H3; or
(h) residues 24-34 of SEQ ID NO: 168 for CDR-L1, residues 50-56 of SEQ ID NO: 168 for CDR-L2, residues 89-97 of SEQ ID NO: 168 for CDR-L3; and/or residues 26-32 of SEQ ID NO: 169 for CDR-H1, residues 52-56 of SEQ ID NO: 169 for CDR-H2, and residues 95-102 of SEQ ID NO: 169 for CDR-H3; wherein the residues are numbered according to Chothia.
27. The multispecific antibody of claim 23, wherein the second antigen binding domain comprises
(a) residues 30-36 of SEQ ID NO: 145 for CDR-L1, residues 46-55 of SEQ ID NO: 145 for CDR-L2, and residues 89-96 of SEQ ID NO: 145 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 143 for CDR-H1, residues 47-58 of SEQ ID NO: 143 for CDR-H2, and residues 93-101 of SEQ ID NO: 143 for CDR-H3;
(b) residues 30-36 of SEQ ID NO: 154 for CDR-L1, residues 46-55 of SEQ ID NO: 154 for CDR-L2, and residues 89-96 of SEQ ID NO: 154 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 153 for CDR-H1, residues 47-58 of SEQ ID NO: 153 for CDR-H2, and residues 93-101 of SEQ ID NO: 153 for CDR-H3;
(c) residues 30-36 of SEQ ID NO: 158 for CDR-L1, residues 46-55 of SEQ ID NO: 158 for CDR-L2, and residues 89-96 of SEQ ID NO: 158 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 157 for CDR-H1, residues 47-58 of SEQ ID NO: 157 for CDR-H2, and residues 93-101 of SEQ ID NO: 157 for CDR-H3;
(d) residues 30-36 of SEQ ID NO: 160 for CDR-L1, residues 46-55 of SEQ ID NO: 160 for CDR-L2, and residues 89-96 of SEQ ID NO: 160 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 159 for CDR-H1, residues 47-58 of SEQ ID NO: 159 for CDR-H2, and residues 93-101 of SEQ ID NO: 159 for CDR-H3;
(e) residues 30-36 of SEQ ID NO: 162 for CDR-L1, residues 46-55 of SEQ ID NO: 162 for CDR-L2, and residues 89-96 of SEQ ID NO: 162 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 161 for CDR-H1, residues 47-58 of SEQ ID NO: 161 for CDR-H2, and residues 93-101 of SEQ ID NO: 161 for CDR-H3;
(f) residues 30-36 of SEQ ID NO: 164 for CDR-L1, residues 46-55 of SEQ ID NO: 164 for CDR-L2, and residues 89-96 of SEQ ID NO: 164 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 163 for CDR-H1, residues 47-58 of SEQ ID NO: 163 for CDR-H2, and residues 93-101 of SEQ ID NO: 163 for CDR-H3;
(g) residues 30-36 of SEQ ID NO: 167 for CDR-L1, residues 46-55 of SEQ ID NO: 167 for CDR-L2, and residues 89-96 of SEQ ID NO: 167 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 165 for CDR-H1, residues 47-58 of SEQ ID NO: 165 for CDR-H2, and residues 93-101 of SEQ ID NO: 165 for CDR-H3; or
(h) residues 30-36 of SEQ ID NO: 168 for CDR-L1, residues 46-55 of SEQ ID NO: 168 for CDR-L2, and residues 89-96 of SEQ ID NO: 168 for CDR-L3; and/or residues 30-35 of SEQ ID NO: 169 for CDR-H1, residues 47-58 of SEQ ID NO: 169 for CDR-H2, and residues 93-101 of SEQ ID NO: 169 for CDR-H3; wherein the residues are numbered according to MacCallum.
28. The multispecific antibody of claim 23, wherein the second antigen binding domain comprises (a) residues 24-34 of SEQ ID NO: 145 for CDR-L1, residues 50-56 of SEQ ID NO: 145 for CDR-L2, and residues 89-97 of SEQ ID NO: 145 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 143 for CDR-H1, residues 50-58 of SEQ ID NO: 143 for CDR-H2, and residues 95-102 of SEQ ID NO: 143 for CDR-H3;
(b) residues 24-34 of SEQ ID NO: 154 for CDR-L1, residues 50-56 of SEQ ID NO: 154 for CDR-L2, and residues 89-97 of SEQ ID NO: 154 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 153 for CDR-H1, residues 50-58 of SEQ ID NO: 153 for CDR-H2, and residues 95-102 of SEQ ID NO: 153 for CDR-H3;
(c) residues 24-34 of SEQ ID NO: 158 for CDR-L1, residues 50-56 of SEQ ID NO: 158 for CDR-L2, and residues 89-97 of SEQ ID NO: 158 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 157 for CDR-H1, residues 50-58 of SEQ ID NO: 157 for CDR-H2, and residues 95-102 of SEQ ID NO: 157 for CDR-H3;
(d) residues 24-34 of SEQ ID NO: 160 for CDR-L1, residues 50-56 of SEQ ID NO: 160 for CDR-L2, and residues 89-97 of SEQ ID NO: 160 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 159 for CDR-H1, residues 50-58 of SEQ ID NO: 159 for CDR-H2, and residues 95-102 of SEQ ID NO: 159 for CDR-H3;
(e) residues 24-34 of SEQ ID NO: 162 for CDR-L1, residues 50-56 of SEQ ID NO: 162 for CDR-L2, and residues 89-97 of SEQ ID NO: 162 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 161 for CDR-H1, residues 50-58 of SEQ ID NO: 161 for CDR-H2, and residues 95-102 of SEQ ID NO: 161 for CDR-H3;
(f) residues 24-34 of SEQ ID NO: 164 for CDR-L1, residues 50-56 of SEQ ID NO: 164 for CDR-L2, and residues 89-97 of SEQ ID NO: 164 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 163 for CDR-H1, residues 50-58 of SEQ ID NO: 163 for CDR-H2, and residues 95-102 of SEQ ID NO: 163 for CDR-H3;
(g) residues 24-34 of SEQ ID NO: 167 for CDR-L1, residues 50-56 of SEQ ID NO: 167 for CDR-L2, and residues 89-97 of SEQ ID NO: 167 for CDR-L3; and/or residues 26-35 of SEQ ID NO: 165 for CDR-H1, residues 50-58 of SEQ ID NO: 165 for CDR-H2, and residues 95-102 of SEQ ID NO: 165 for CDR-H3; or
(h) residues 24-34 of SEQ ID NO: 168 for CDR-L1, residues 50-56 of SEQ ID NO: 168 for CDR-L2, and residues 89-97 of SEQ ID NO: 168 for CDR-L3; and/or residues 26-35 of SEQ
142 ID NO: 169 for CDR-H1, residues 50-58 of SEQ ID NO: 169 for CDR-H2, and residues 95-102 of SEQ ID NO: 169 for CDR-H3; wherein the residues are numbered according to AbM.
29. The multispecific antibody of claim 23, wherein the second antigen binding domain comprises
(a) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 211, a CDRL2 comprising the amino acid sequence AAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 212; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 208, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 209, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 210;
(b) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 246, a CDRL2 comprising the amino acid sequence DAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 247; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 243, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 244, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 245;
(c) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 258, a CDRL2 comprising the amino acid sequence WAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 259; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 255, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 256, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 257;
(d) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 270, a CDRL2 comprising the amino acid sequence VAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 271; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 267, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 268, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 269;
(e) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 282, a CDRL2 comprising the amino acid sequence DAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 283; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 279, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 280, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 281;
143 (f) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 292, a CDRL2 comprising the amino acid sequence AAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 293; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 243, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 290, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 291;
(g) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 302, a CDRL2 comprising the amino acid sequence YAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 303; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 299, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 300, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 301; or
(h) a CDRL1 comprising the amino acid sequence set forth as SEQ ID NO: 311, a CDRL2 comprising the amino acid sequence YAS, and a CDRL3 comprising the amino acid sequence set forth as SEQ ID NO: 312; and/or a CDRH1 comprising the amino acid sequence set forth as SEQ ID NO: 299, a CDRH2 comprising the amino acid sequence set forth as SEQ ID NO: 300, and a CDRH3 comprising the amino acid sequence set forth as SEQ ID NO: 301.
30. The multispecific antibody of any one of claims 1-15 or 23-29, wherein the second antigen binding domain comprises
(a) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 145; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 143;
(b) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 154; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 153;
(c) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 158; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 157;
(d) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 160; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 159;
144 (e) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 162; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 161;
(f) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 164; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 163;
(g) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 167; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 165; or
(h) a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 168; and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 169.
31. The multispecific antibody of any one of claims 1-15, wherein the second antigen binding domain binds to suppressor of tumorigenicity 2 (ST2).
32. The multispecific antibody of claim 31, wherein the second antigen binding domain competes for binding with, binds a same epitope as, or comprises an antigen binding domain having three CDRs of a variable region sequence comprising
(a) an amino acid sequence set forth as SEQ ID NO: 35;
(b) an amino acid sequence set forth as SEQ ID NO: 36;
(c) an amino acid sequence set forth as SEQ ID NO: 37;
(d) an amino acid sequence set forth as SEQ ID NO: 38;
(e) an amino acid sequence set forth as SEQ ID NO: 39;
(f) an amino acid sequence set forth as SEQ ID NO: 40;
(g) an amino acid sequence set forth as SEQ ID NO: 41;
(h) an amino acid sequence set forth as SEQ ID NO: 42;
(i) an amino acid sequence set forth as SEQ ID NO: 43;
(j) an amino acid sequence set forth as SEQ ID NO: 44;
(k) an amino acid sequence set forth as SEQ ID NO: 45;
(l) an amino acid sequence set forth as SEQ ID NO: 46;
145 (m) an amino acid sequence set forth as SEQ ID NO: 47;
(n) an amino acid sequence set forth as SEQ ID NO: 48;
(o) an amino acid sequence set forth as SEQ ID NO: 49;
(p) an amino acid sequence set forth as SEQ ID NO: 50;
(q) an amino acid sequence set forth as SEQ ID NO: 51;
(r) an amino acid sequence set forth as SEQ ID NO: 52;
(s) an amino acid sequence set forth as SEQ ID NO: 53;
(t) an amino acid sequence set forth as SEQ ID NO: 54; or
(u) an amino acid sequence set forth as SEQ ID NO: 55.
33. The multispecific antibody of claim 32, wherein the second antigen binding domain comprises
(a) residues 27-38 of SEQ ID NO: 35 for CDR1, residues 56-65 of SEQ ID NO: 35 for CDR2, and residues 105-117 of SEQ ID NO: 35 for CDR3;
(b) residues 27-38 of SEQ ID NO: 36 for CDR1, residues 56-65 of SEQ ID NO: 36 for CDR2, and residues 105-117 of SEQ ID NO: 36 for CDR3;
(c) residues 27-38 of SEQ ID NO: 37 for CDR1, residues 56-65 of SEQ ID NO: 37 for CDR2, and residues 105-117 of SEQ ID NO: 37 for CDR3;
(d) residues 27-38 of SEQ ID NO: 38 for CDR1, residues 56-65 of SEQ ID NO: 38 for
CDR2, and residues 105-117 of SEQ ID NO: 38 for CDR3
(e) residues 27-38 of SEQ ID NO: 39 for CDR1, residues 56-65 of SEQ ID NO: 39 for CDR2, and residues 105-117 of SEQ ID NO: 39 for CDR3;
(f) residues 27-38 of SEQ ID NO: 40 for CDR1, residues 56-65 of SEQ ID NO: 40 for CDR2, and residues 105-117 of SEQ ID NO: 40 for CDR3;
(g) residues 27-38 of SEQ ID NO: 41 for CDR1, residues 56-65 of SEQ ID NO: 41 for CDR2, and residues 105-117 of SEQ ID NO: 41 for CDR3;
(h) residues 27-38 of SEQ ID NO: 42 for CDR1, residues 56-65 of SEQ ID NO: 42 for CDR2, and residues 105-117 of SEQ ID NO: 42 for CDR3;
(i) residues 27-38 of SEQ ID NO: 43 for CDR1, residues 56-65 of SEQ ID NO: 43 for CDR2, and residues 105-117 of SEQ ID NO: 43 for CDR3;
(j) residues 27-38 of SEQ ID NO: 44 for CDR1, residues 56-65 of SEQ ID NO: 44 for
146 CDR2, and residues 105-117 of SEQ ID NO: 44 for CDR3;
(k) residues 27-38 of SEQ ID NO: 45 for CDR1, residues 56-65 of SEQ ID NO: 45 for CDR2, and residues 105-117 of SEQ ID NO: 45 for CDR3;
(l) residues 27-38 of SEQ ID NO: 46 for CDR1, residues 56-65 of SEQ ID NO: 46 for CDR2, and residues 105-117 of SEQ ID NO: 46 for CDR3;
(m) residues 27-38 of SEQ ID NO: 47 for CDR1, residues 56-65 of SEQ ID NO: 47 for CDR2, and residues 105-117 of SEQ ID NO: 47 for CDR3;
(n) residues 27-38 of SEQ ID NO: 48 for CDR1, residues 56-65 of SEQ ID NO: 48 for CDR2, and residues 105-117 of SEQ ID NO: 48 for CDR3;
(o) residues 27-38 of SEQ ID NO: 49 for CDR1, residues 56-65 of SEQ ID NO: 49 for CDR2, and residues 105-117 of SEQ ID NO: 49 for CDR3;
(p) residues 27-38 of SEQ ID NO: 50 for CDR1, residues 56-65 of SEQ ID NO: 50 for CDR2, and residues 105-117 of SEQ ID NO: 50 for CDR3;
(q) residues 27-38 of SEQ ID NO: 51 for CDR1, residues 56-65 of SEQ ID NO: 51 for CDR2, and residues 105-117 of SEQ ID NO: 51 for CDR3;
(r) residues 27-38 of SEQ ID NO: 52 for CDR1, residues 56-65 of SEQ ID NO: 52 for CDR2, and residues 105-117 of SEQ ID NO: 52 for CDR3;
(s) residues 27-38 of SEQ ID NO: 53 for CDR1, residues 56-65 of SEQ ID NO: 53 for CDR2, and residues 105-117 of SEQ ID NO: 53 for CDR3;
(t) residues 27-38 of SEQ ID NO: 54 for CDR1, residues 56-65 of SEQ ID NO: 54 for CDR2, and residues 105-117 of SEQ ID NO: 54 for CDR3; or
(u) residues 27-38 of SEQ ID NO: 55 for CDR1, residues 56-65 of SEQ ID NO: 55 for CDR2, and residues 105-117 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Lefranc (IMGT).
34. The multispecific antibody of claim 32, wherein the second antigen binding domain comprises
(a) residues 31-35 of SEQ ID NO: 35 for CDR1, residues 50-65 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3;
(b) residues 31-35 of SEQ ID NO: 36 for CDR1, residues 50-65 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3;
147 (c) residues 31-35 of SEQ ID NO: 37 for CDR1, residues 50-65 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3;
(d) residues 31-35 of SEQ ID NO: 38 for CDR1, residues 50-65 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3
(e) residues 31-35 of SEQ ID NO: 39 for CDR1, residues 50-65 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3;
(f) residues 31-35 of SEQ ID NO: 40 for CDR1, residues 50-65 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3;
(g) residues 31-35 of SEQ ID NO: 41 for CDR1, residues 50-65 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3;
(h) residues 31-35 of SEQ ID NO: 42 for CDR1, residues 50-65 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3;
(i) residues 31-35 of SEQ ID NO: 43 for CDR1, residues 50-65 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3;
(j) residues 31-35 of SEQ ID NO: 44 for CDR1, residues 50-65 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3;
(k) residues 31-35 of SEQ ID NO: 45 for CDR1, residues 50-65 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3;
(l) residues 31-35 of SEQ ID NO: 46 for CDR1, residues 50-65 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3;
(m) residues 31-35 of SEQ ID NO: 47 for CDR1, residues 50-65 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3;
(n) residues 31-35 of SEQ ID NO: 48 for CDR1, residues 50-65 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3;
(o) residues 31-35 of SEQ ID NO: 49 for CDR1, residues 50-65 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3;
(p) residues 31-35 of SEQ ID NO: 50 for CDR1, residues 50-65 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3;
(q) residues 31-35 of SEQ ID NO: 51 for CDR1, residues 50-65 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3;
(r) residues 31-35 of SEQ ID NO: 52 for CDR1, residues 50-65 of SEQ ID NO: 52 for
148 CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3;
(s) residues 31-35 of SEQ ID NO: 53 for CDR1, residues 50-65 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3;
(t) residues 31-35 of SEQ ID NO: 54 for CDR1, residues 50-65 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or
(u) residues 31-35 of SEQ ID NO: 55 for CDR1, residues 50-65 of SEQ ID NO: 55 for CDR2, and residues 95-102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Kabat.
35. The multispecific antibody of claim 32, wherein the second antigen binding domain comprises
(a) residues 26-32 of SEQ ID NO: 35 for CDR1, residues 52-56 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3;
(b) residues 26-32 of SEQ ID NO: 36 for CDR1, residues 52-56 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3;
(c) residues 26-32 of SEQ ID NO: 37 for CDR1, residues 52-56 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3;
(d) residues 26-32 of SEQ ID NO: 38 for CDR1, residues 52-56 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3
(e) residues 26-32 of SEQ ID NO: 39 for CDR1, residues 52-56 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3;
(f) residues 26-32 of SEQ ID NO: 40 for CDR1, residues 52-56 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3;
(g) residues 26-32 of SEQ ID NO: 41 for CDR1, residues 52-56 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3;
(h) residues 26-32 of SEQ ID NO: 42 for CDR1, residues 52-56 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3;
(i) residues 26-32 of SEQ ID NO: 43 for CDR1, residues 52-56 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3;
(j) residues 26-32 of SEQ ID NO: 44 for CDR1, residues 52-56 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3;
149 (k) residues 26-32 of SEQ ID NO: 45 for CDR1, residues 52-56 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3;
(l) residues 26-32 of SEQ ID NO: 46 for CDR1, residues 52-56 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3;
(m) residues 26-32 of SEQ ID NO: 47 for CDR1, residues 52-56 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3;
(n) residues 26-32 of SEQ ID NO: 48 for CDR1, residues 52-56 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3;
(o) residues 26-32 of SEQ ID NO: 49 for CDR1, residues 52-56 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3;
(p) residues 26-32 of SEQ ID NO: 50 for CDR1, residues 52-56 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3;
(q) residues 26-32 of SEQ ID NO: 51 for CDR1, residues 52-56 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3;
(r) residues 26-32 of SEQ ID NO: 52 for CDR1, residues 52-56 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3;
(s) residues 26-32 of SEQ ID NO: 53 for CDR1, residues 52-56 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3;
(t) residues 26-32 of SEQ ID NO: 54 for CDR1, residues 52-56 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or
(u) residues 26-32 of SEQ ID NO: 55 for CDR1, residues 52-56 of SEQ ID NO: 55 for CDR2, and residues 95-102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Chothia.
36. The multispecific antibody of claim 32, wherein the second antigen binding domain comprises
(a) residues 30-35 of SEQ ID NO: 35 for CDR1, residues 47-58 of SEQ ID NO: 35 for CDR2, and residues 93-101 of SEQ ID NO: 35 for CDR3;
(b) residues 30-35 of SEQ ID NO: 36 for CDR1, residues 47-58 of SEQ ID NO: 36 for CDR2, and residues 93-101 of SEQ ID NO: 36 for CDR3;
(c) residues 30-35 of SEQ ID NO: 37 for CDR1, residues 47-58 of SEQ ID NO: 37 for
150 CDR2, and residues 93-101 of SEQ ID NO: 37 for CDR3;
(d) residues 30-35 of SEQ ID NO: 38 for CDR1, residues 47-58 of SEQ ID NO: 38 for CDR2, and residues 93-101 of SEQ ID NO: 38 for CDR3
(e) residues 30-35 of SEQ ID NO: 39 for CDR1, residues 47-58 of SEQ ID NO: 39 for CDR2, and residues 93-101 of SEQ ID NO: 39 for CDR3;
(f) residues 30-35 of SEQ ID NO: 40 for CDR1, residues 47-58 of SEQ ID NO: 40 for CDR2, and residues 93-101 of SEQ ID NO: 40 for CDR3;
(g) residues 30-35 of SEQ ID NO: 41 for CDR1, residues 47-58 of SEQ ID NO: 41 for CDR2, and residues 93-101 of SEQ ID NO: 41 for CDR3;
(h) residues 30-35 of SEQ ID NO: 42 for CDR1, residues 47-58 of SEQ ID NO: 42 for CDR2, and residues 93-101 of SEQ ID NO: 42 for CDR3;
(i) residues 30-35 of SEQ ID NO: 43 for CDR1, residues 47-58 of SEQ ID NO: 43 for CDR2, and residues 93-101 of SEQ ID NO: 43 for CDR3;
(j) residues 30-35 of SEQ ID NO: 44 for CDR1, residues 47-58 of SEQ ID NO: 44 for CDR2, and residues 93-101 of SEQ ID NO: 44 for CDR3;
(k) residues 30-35 of SEQ ID NO: 45 for CDR1, residues 47-58 of SEQ ID NO: 45 for CDR2, and residues 93-101 of SEQ ID NO: 45 for CDR3;
(l) residues 30-35 of SEQ ID NO: 46 for CDR1, residues 47-58 of SEQ ID NO: 46 for CDR2, and residues 93-101 of SEQ ID NO: 46 for CDR3;
(m) residues 30-35 of SEQ ID NO: 47 for CDR1, residues 47-58 of SEQ ID NO: 47 for CDR2, and residues 93-101 of SEQ ID NO: 47 for CDR3;
(n) residues 30-35 of SEQ ID NO: 48 for CDR1, residues 47-58 of SEQ ID NO: 48 for CDR2, and residues 93-101 of SEQ ID NO: 48 for CDR3;
(o) residues 30-35 of SEQ ID NO: 49 for CDR1, residues 47-58 of SEQ ID NO: 49 for CDR2, and residues 93-101 of SEQ ID NO: 49 for CDR3;
(p) residues 30-35 of SEQ ID NO: 50 for CDR1, residues 47-58 of SEQ ID NO: 50 for CDR2, and residues 93-101 of SEQ ID NO: 50 for CDR3;
(q) residues 30-35 of SEQ ID NO: 51 for CDR1, residues 47-58 of SEQ ID NO: 51 for CDR2, and residues 93-101 of SEQ ID NO: 51 for CDR3;
(r) residues 30-35 of SEQ ID NO: 52 for CDR1, residues 47-58 of SEQ ID NO: 52 for CDR2, and residues 93-101 of SEQ ID NO: 52 for CDR3;
151 (s) residues 30-35 of SEQ ID NO: 53 for CDR1, residues 47-58 of SEQ ID NO: 53 for CDR2, and residues 93-101 of SEQ ID NO: 53 for CDR3;
(t) residues 30-35 of SEQ ID NO: 54 for CDR1, residues 47-58 of SEQ ID NO: 54 for CDR2, and residues 93-101 of SEQ ID NO: 54 for CDR3; or
(u) residues 30-35 of SEQ ID NO: 55 for CDR1, residues 47-58 of SEQ ID NO: 55 for CDR2, and residues 93-101 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to MacCallum.
37. The multispecific antibody of claim 32, wherein the second antigen binding domain comprises
(a) residues 26-35 of SEQ ID NO: 35 for CDR1, residues 50-58 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3;
(b) residues 26-35 of SEQ ID NO: 36 for CDR1, residues 50-58 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3;
(c) residues 26-35 of SEQ ID NO: 37 for CDR1, residues 50-58 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3;
(d) residues 26-35 of SEQ ID NO: 38 for CDR1, residues 50-58 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3
(e) residues 26-35 of SEQ ID NO: 39 for CDR1, residues 50-58 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3;
(f) residues 26-35 of SEQ ID NO: 40 for CDR1, residues 50-58 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3;
(g) residues 26-35 of SEQ ID NO: 41 for CDR1, residues 50-58 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3;
(h) residues 26-35 of SEQ ID NO: 42 for CDR1, residues 50-58 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3;
(i) residues 26-35 of SEQ ID NO: 43 for CDR1, residues 50-58 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3;
(j) residues 26-35 of SEQ ID NO: 44 for CDR1, residues 50-58 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3;
(k) residues 26-35 of SEQ ID NO: 45 for CDR1, residues 50-58 of SEQ ID NO: 45 for
152 CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3;
(l) residues 26-35 of SEQ ID NO: 46 for CDR1, residues 50-58 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3;
(m) residues 26-35 of SEQ ID NO: 47 for CDR1, residues 50-58 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3;
(n) residues 26-35 of SEQ ID NO: 48 for CDR1, residues 50-58 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3;
(o) residues 26-35 of SEQ ID NO: 49 for CDR1, residues 50-58 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3;
(p) residues 26-35 of SEQ ID NO: 50 for CDR1, residues 50-58 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3;
(q) residues 26-35 of SEQ ID NO: 51 for CDR1, residues 50-58 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3;
(r) residues 26-35 of SEQ ID NO: 52 for CDR1, residues 50-58 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3;
(s) residues 26-35 of SEQ ID NO: 53 for CDR1, residues 50-58 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3;
(t) residues 26-35 of SEQ ID NO: 54 for CDR1, residues 50-58 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or
(u) residues 26-35 of SEQ ID NO: 55 for CDR1, residues 50-58 of SEQ ID NO: 55 for CDR2, and residues 95-102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to AbM.
38. The multispecific antibody of claim 32, wherein the second antigen binding domain comprises
(a) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 56, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 57, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 58;
(b) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 59, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 60, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 61;
153 (c) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 62, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 63, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 64;
(d) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 65, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 66, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 67;
(e) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 68, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 70;
(f) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 71, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 72, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 73;
(g) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 74, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 75, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 76;
(h) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 77, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 78, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 79;
(i) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 80, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 81, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 82;
(j) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 83, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 84, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 85;
(k) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 86, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 87, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 88;
(l) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 89, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 91;
154 (m) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 92, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 93, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 94;
(n) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 95, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 96, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 97;
(o) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 98, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 99, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 100;
(p) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 101, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 102, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 103;
(q) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 104, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 105, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 106;
(r) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 107, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 108, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 109;
(s) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 110, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 111, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 112;
(t) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 113, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 114, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 115; or
(u) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 116, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 117, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 118.
39. The multispecific antibody of claim 32, wherein the second antigen binding domain comprises the amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
155
40. The multispecific antibody of any one of claims 1-39, wherein the first antigen binding domain and the second antigen binding domain are linked by a linker.
41. The multispecific antibody of claim 40, wherein the linker is a peptide linker.
42. The multispecific antibody of claim 41, wherein the peptide linker comprises a glycine/serine linker.
43. The multispecific antibody of claim 41, wherein the linker comprises the amino acid sequence set forth as SEQ ID NO: 11, 144, or 174.
44. The multispecific antibody of any one of claims 1-43, wherein the multispecific antibody further comprises a half-life extender.
45. The multispecific antibody of claim 44, wherein the half-life extender binds to human serum albumin, binds directly to FcRn, or binds indirectly to FcRn.
46. The multispecific antibody of any one of claims 1-45, wherein inhibiting T cell activity comprises inhibiting cytokine release and/or inhibiting nuclear factor of activated T cells (NF AT).
47. The multispecific antibody of claim 46, wherein the cytokine is interleukin-2 (IL-2), interferon gamma (IFNy), and/or tumor necrosis factor alpha (TNFa).
48. The multispecific antibody of any one of claims 1-47, wherein the multispecific antibody is a bispecific antibody.
49. The multispecific antibody of any one of claims 1-47, wherein the multispecific antibody is a trispecific antibody.
50. A nucleic acid encoding the multispecific antibody of any one of claims 1-49.
156
51. A vector comprising the nucleic acid of claim 50.
52. A host cell comprising the nucleic acid of claim 50 or the vector of claim 51.
53. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1-49.
54. A method of inhibiting T cell activity comprising contacting a T cell with an effective amount of the multi specific antibody of any one of claims 1-49 or the pharmaceutical composition of claim 53.
55. A method of treating or preventing an immune disorder in a subject, comprising administering to the subject an effective amount of the multispecific antibody of any one of claimsl-49 or the pharmaceutical composition of claim 53.
56. The method of claim 55, wherein the immune disorder is a T cell mediated inflammatory disease.
57. The method of claim 55 or 56, wherein the immune disorder is selected from the group consisting of a T cell mediated autoimmune disease, allergic disease, idiopathic thrombocytopenic purpura, warm autoimmune hemolytic anemia, autoimmune glomerulonephritis, autoimmune thyroid disease, systemic sclerosis, immune related adverse events (irAEs), transplant rejection, graft versus host disease (GVHD), rheumatoid arthritis, multiple sclerosis, type-1 diabetes, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, asthma, eosinophilic disease, autoimmune central nervous system (CNS) inflammation, psoriatic arthritis, atopic dermatitis, alopecia aerata, vitiligo, IgA nephropathy, lupus nephritis, chronic spontaneous urticarial, pyoderma gangrenosum, focal segmental glomerular sclerosis, membranous nephropathy, aplastic anemia, ulcerative colitis, Crohn’s disease, uveitis, Behcet’s disease, autoimmune hepatitis, ILD-myositis, and inflammation-induced liver injury.
157
58. A kit comprising the multispecific antibody of any one of claims 1-49 or the pharmaceutical composition of claim 53.
59. A single domain antibody which competes for binding with, binds a same epitope as, or comprises an antigen binding domain having three complementarity determining regions (CDRs) of a heavy chain variable region sequence comprising:
(a) an amino acid sequence set forth as SEQ ID NO: 35;
(b) an amino acid sequence set forth as SEQ ID NO: 36;
(c) an amino acid sequence set forth as SEQ ID NO: 37;
(d) an amino acid sequence set forth as SEQ ID NO: 38;
(e) an amino acid sequence set forth as SEQ ID NO: 39;
(f) an amino acid sequence set forth as SEQ ID NO: 40;
(g) an amino acid sequence set forth as SEQ ID NO: 41;
(h) an amino acid sequence set forth as SEQ ID NO: 42;
(i) an amino acid sequence set forth as SEQ ID NO: 43;
(j) an amino acid sequence set forth as SEQ ID NO: 44;
(k) an amino acid sequence set forth as SEQ ID NO: 45;
(l) an amino acid sequence set forth as SEQ ID NO: 46;
(m) an amino acid sequence set forth as SEQ ID NO: 47;
(n) an amino acid sequence set forth as SEQ ID NO: 48;
(o) an amino acid sequence set forth as SEQ ID NO: 49;
(p) an amino acid sequence set forth as SEQ ID NO: 50;
(q) an amino acid sequence set forth as SEQ ID NO: 51;
(r) an amino acid sequence set forth as SEQ ID NO: 52;
(s) an amino acid sequence set forth as SEQ ID NO: 53;
(t) an amino acid sequence set forth as SEQ ID NO: 54; or
(u) an amino acid sequence set forth as SEQ ID NO: 55.
60. The single domain antibody of claim 59, wherein the single domain antibody comprises (a) residues 27-38 of SEQ ID NO: 35 for CDR1, residues 56-65 of SEQ ID NO: 35 for
CDR2, and residues 105-117 of SEQ ID NO: 35 for CDR3;
158 (b) residues 27-38 of SEQ ID NO: 36 for CDR1, residues 56-65 of SEQ ID NO: 36 for CDR2, and residues 105-117 of SEQ ID NO: 36 for CDR3;
(c) residues 27-38 of SEQ ID NO: 37 for CDR1, residues 56-65 of SEQ ID NO: 37 for CDR2, and residues 105-117 of SEQ ID NO: 37 for CDR3;
(d) residues 27-38 of SEQ ID NO: 38 for CDR1, residues 56-65 of SEQ ID NO: 38 for CDR2, and residues 105-117 of SEQ ID NO: 38 for CDR3
(e) residues 27-38 of SEQ ID NO: 39 for CDR1, residues 56-65 of SEQ ID NO: 39 for CDR2, and residues 105-117 of SEQ ID NO: 39 for CDR3;
(f) residues 27-38 of SEQ ID NO: 40 for CDR1, residues 56-65 of SEQ ID NO: 40 for CDR2, and residues 105-117 of SEQ ID NO: 40 for CDR3;
(g) residues 27-38 of SEQ ID NO: 41 for CDR1, residues 56-65 of SEQ ID NO: 41 for CDR2, and residues 105-117 of SEQ ID NO: 41 for CDR3;
(h) residues 27-38 of SEQ ID NO: 42 for CDR1, residues 56-65 of SEQ ID NO: 42 for CDR2, and residues 105-117 of SEQ ID NO: 42 for CDR3;
(i) residues 27-38 of SEQ ID NO: 43 for CDR1, residues 56-65 of SEQ ID NO: 43 for CDR2, and residues 105-117 of SEQ ID NO: 43 for CDR3;
(j) residues 27-38 of SEQ ID NO: 44 for CDR1, residues 56-65 of SEQ ID NO: 44 for CDR2, and residues 105-117 of SEQ ID NO: 44 for CDR3;
(k) residues 27-38 of SEQ ID NO: 45 for CDR1, residues 56-65 of SEQ ID NO: 45 for CDR2, and residues 105-117 of SEQ ID NO: 45 for CDR3;
(l) residues 27-38 of SEQ ID NO: 46 for CDR1, residues 56-65 of SEQ ID NO: 46 for CDR2, and residues 105-117 of SEQ ID NO: 46 for CDR3;
(m) residues 27-38 of SEQ ID NO: 47 for CDR1, residues 56-65 of SEQ ID NO: 47 for CDR2, and residues 105-117 of SEQ ID NO: 47 for CDR3;
(n) residues 27-38 of SEQ ID NO: 48 for CDR1, residues 56-65 of SEQ ID NO: 48 for CDR2, and residues 105-117 of SEQ ID NO: 48 for CDR3;
(o) residues 27-38 of SEQ ID NO: 49 for CDR1, residues 56-65 of SEQ ID NO: 49 for CDR2, and residues 105-117 of SEQ ID NO: 49 for CDR3;
(p) residues 27-38 of SEQ ID NO: 50 for CDR1, residues 56-65 of SEQ ID NO: 50 for CDR2, and residues 105-117 of SEQ ID NO: 50 for CDR3;
(q) residues 27-38 of SEQ ID NO: 51 for CDR1, residues 56-65 of SEQ ID NO: 51 for
159 CDR2, and residues 105-117 of SEQ ID NO: 51 for CDR3;
(r) residues 27-38 of SEQ ID NO: 52 for CDR1, residues 56-65 of SEQ ID NO: 52 for CDR2, and residues 105-117 of SEQ ID NO: 52 for CDR3;
(s) residues 27-38 of SEQ ID NO: 53 for CDR1, residues 56-65 of SEQ ID NO: 53 for CDR2, and residues 105-117 of SEQ ID NO: 53 for CDR3;
(t) residues 27-38 of SEQ ID NO: 54 for CDR1, residues 56-65 of SEQ ID NO: 54 for CDR2, and residues 105-117 of SEQ ID NO: 54 for CDR3; or
(u) residues 27-38 of SEQ ID NO: 55 for CDR1, residues 56-65 of SEQ ID NO: 55 for CDR2, and residues 105-117 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Lefranc (IMGT).
61. The single domain antibody of claim 59, wherein the single domain antibody comprises
(a) residues 31-35 of SEQ ID NO: 35 for CDR1, residues 50-65 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3;
(b) residues 31-35 of SEQ ID NO: 36 for CDR1, residues 50-65 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3;
(c) residues 31-35 of SEQ ID NO: 37 for CDR1, residues 50-65 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3;
(d) residues 31-35 of SEQ ID NO: 38 for CDR1, residues 50-65 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3
(e) residues 31-35 of SEQ ID NO: 39 for CDR1, residues 50-65 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3;
(f) residues 31-35 of SEQ ID NO: 40 for CDR1, residues 50-65 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3;
(g) residues 31-35 of SEQ ID NO: 41 for CDR1, residues 50-65 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3;
(h) residues 31-35 of SEQ ID NO: 42 for CDR1, residues 50-65 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3;
(i) residues 31-35 of SEQ ID NO: 43 for CDR1, residues 50-65 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3;
(j) residues 31-35 of SEQ ID NO: 44 for CDR1, residues 50-65 of SEQ ID NO: 44 for
160 CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3;
(k) residues 31-35 of SEQ ID NO: 45 for CDR1, residues 50-65 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3;
(l) residues 31-35 of SEQ ID NO: 46 for CDR1, residues 50-65 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3;
(m) residues 31-35 of SEQ ID NO: 47 for CDR1, residues 50-65 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3;
(n) residues 31-35 of SEQ ID NO: 48 for CDR1, residues 50-65 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3;
(o) residues 31-35 of SEQ ID NO: 49 for CDR1, residues 50-65 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3;
(p) residues 31-35 of SEQ ID NO: 50 for CDR1, residues 50-65 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3;
(q) residues 31-35 of SEQ ID NO: 51 for CDR1, residues 50-65 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3;
(r) residues 31-35 of SEQ ID NO: 52 for CDR1, residues 50-65 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3;
(s) residues 31-35 of SEQ ID NO: 53 for CDR1, residues 50-65 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3;
(t) residues 31-35 of SEQ ID NO: 54 for CDR1, residues 50-65 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or
(u) residues 31-35 of SEQ ID NO: 55 for CDR1, residues 50-65 of SEQ ID NO: 55 for CDR2, and residues 95-102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Kabat.
62. The single domain antibody of claim 59, wherein the single domain antibody comprises
(a) residues 26-32 of SEQ ID NO: 35 for CDR1, residues 52-56 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3;
(b) residues 26-32 of SEQ ID NO: 36 for CDR1, residues 52-56 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3;
(c) residues 26-32 of SEQ ID NO: 37 for CDR1, residues 52-56 of SEQ ID NO: 37 for
161 CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3;
(d) residues 26-32 of SEQ ID NO: 38 for CDR1, residues 52-56 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3
(e) residues 26-32 of SEQ ID NO: 39 for CDR1, residues 52-56 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3;
(f) residues 26-32 of SEQ ID NO: 40 for CDR1, residues 52-56 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3;
(g) residues 26-32 of SEQ ID NO: 41 for CDR1, residues 52-56 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3;
(h) residues 26-32 of SEQ ID NO: 42 for CDR1, residues 52-56 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3;
(i) residues 26-32 of SEQ ID NO: 43 for CDR1, residues 52-56 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3;
(j) residues 26-32 of SEQ ID NO: 44 for CDR1, residues 52-56 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3;
(k) residues 26-32 of SEQ ID NO: 45 for CDR1, residues 52-56 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3;
(l) residues 26-32 of SEQ ID NO: 46 for CDR1, residues 52-56 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3;
(m) residues 26-32 of SEQ ID NO: 47 for CDR1, residues 52-56 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3;
(n) residues 26-32 of SEQ ID NO: 48 for CDR1, residues 52-56 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3;
(o) residues 26-32 of SEQ ID NO: 49 for CDR1, residues 52-56 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3;
(p) residues 26-32 of SEQ ID NO: 50 for CDR1, residues 52-56 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3;
(q) residues 26-32 of SEQ ID NO: 51 for CDR1, residues 52-56 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3;
(r) residues 26-32 of SEQ ID NO: 52 for CDR1, residues 52-56 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3;
162 (s) residues 26-32 of SEQ ID NO: 53 for CDR1, residues 52-56 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3;
(t) residues 26-32 of SEQ ID NO: 54 for CDR1, residues 52-56 of SEQ ID NO: 54 for CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or
(u) residues 26-32 of SEQ ID NO: 55 for CDR1, residues 52-56 of SEQ ID NO: 55 for CDR2, and residues 95-102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to Chothia.
63. The single domain antibody of claim 59, wherein the single domain antibody comprises
(a) residues 30-35 of SEQ ID NO: 35 for CDR1, residues 47-58 of SEQ ID NO: 35 for CDR2, and residues 93-101 of SEQ ID NO: 35 for CDR3;
(b) residues 30-35 of SEQ ID NO: 36 for CDR1, residues 47-58 of SEQ ID NO: 36 for CDR2, and residues 93-101 of SEQ ID NO: 36 for CDR3;
(c) residues 30-35 of SEQ ID NO: 37 for CDR1, residues 47-58 of SEQ ID NO: 37 for CDR2, and residues 93-101 of SEQ ID NO: 37 for CDR3;
(d) residues 30-35 of SEQ ID NO: 38 for CDR1, residues 47-58 of SEQ ID NO: 38 for CDR2, and residues 93-101 of SEQ ID NO: 38 for CDR3
(e) residues 30-35 of SEQ ID NO: 39 for CDR1, residues 47-58 of SEQ ID NO: 39 for CDR2, and residues 93-101 of SEQ ID NO: 39 for CDR3;
(f) residues 30-35 of SEQ ID NO: 40 for CDR1, residues 47-58 of SEQ ID NO: 40 for CDR2, and residues 93-101 of SEQ ID NO: 40 for CDR3;
(g) residues 30-35 of SEQ ID NO: 41 for CDR1, residues 47-58 of SEQ ID NO: 41 for CDR2, and residues 93-101 of SEQ ID NO: 41 for CDR3;
(h) residues 30-35 of SEQ ID NO: 42 for CDR1, residues 47-58 of SEQ ID NO: 42 for CDR2, and residues 93-101 of SEQ ID NO: 42 for CDR3;
(i) residues 30-35 of SEQ ID NO: 43 for CDR1, residues 47-58 of SEQ ID NO: 43 for CDR2, and residues 93-101 of SEQ ID NO: 43 for CDR3;
(j) residues 30-35 of SEQ ID NO: 44 for CDR1, residues 47-58 of SEQ ID NO: 44 for CDR2, and residues 93-101 of SEQ ID NO: 44 for CDR3;
(k) residues 30-35 of SEQ ID NO: 45 for CDR1, residues 47-58 of SEQ ID NO: 45 for CDR2, and residues 93-101 of SEQ ID NO: 45 for CDR3;
163 (l) residues 30-35 of SEQ ID NO: 46 for CDR1, residues 47-58 of SEQ ID NO: 46 for CDR2, and residues 93-101 of SEQ ID NO: 46 for CDR3;
(m) residues 30-35 of SEQ ID NO: 47 for CDR1, residues 47-58 of SEQ ID NO: 47 for CDR2, and residues 93-101 of SEQ ID NO: 47 for CDR3;
(n) residues 30-35 of SEQ ID NO: 48 for CDR1, residues 47-58 of SEQ ID NO: 48 for CDR2, and residues 93-101 of SEQ ID NO: 48 for CDR3;
(o) residues 30-35 of SEQ ID NO: 49 for CDR1, residues 47-58 of SEQ ID NO: 49 for CDR2, and residues 93-101 of SEQ ID NO: 49 for CDR3;
(p) residues 30-35 of SEQ ID NO: 50 for CDR1, residues 47-58 of SEQ ID NO: 50 for CDR2, and residues 93-101 of SEQ ID NO: 50 for CDR3;
(q) residues 30-35 of SEQ ID NO: 51 for CDR1, residues 47-58 of SEQ ID NO: 51 for CDR2, and residues 93-101 of SEQ ID NO: 51 for CDR3;
(r) residues 30-35 of SEQ ID NO: 52 for CDR1, residues 47-58 of SEQ ID NO: 52 for CDR2, and residues 93-101 of SEQ ID NO: 52 for CDR3;
(s) residues 30-35 of SEQ ID NO: 53 for CDR1, residues 47-58 of SEQ ID NO: 53 for CDR2, and residues 93-101 of SEQ ID NO: 53 for CDR3;
(t) residues 30-35 of SEQ ID NO: 54 for CDR1, residues 47-58 of SEQ ID NO: 54 for CDR2, and residues 93-101 of SEQ ID NO: 54 for CDR3; or
(u) residues 30-35 of SEQ ID NO: 55 for CDR1, residues 47-58 of SEQ ID NO: 55 for CDR2, and residues 93-101 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to MacCallum.
64. The single domain antibody of claim 59, wherein the single domain antibody comprises
(a) residues 26-35 of SEQ ID NO: 35 for CDR1, residues 50-58 of SEQ ID NO: 35 for CDR2, and residues 95-102 of SEQ ID NO: 35 for CDR3;
(b) residues 26-35 of SEQ ID NO: 36 for CDR1, residues 50-58 of SEQ ID NO: 36 for CDR2, and residues 95-102 of SEQ ID NO: 36 for CDR3;
(c) residues 26-35 of SEQ ID NO: 37 for CDR1, residues 50-58 of SEQ ID NO: 37 for CDR2, and residues 95-102 of SEQ ID NO: 37 for CDR3;
(d) residues 26-35 of SEQ ID NO: 38 for CDR1, residues 50-58 of SEQ ID NO: 38 for CDR2, and residues 95-102 of SEQ ID NO: 38 for CDR3
164 (e) residues 26-35 of SEQ ID NO: 39 for CDR1, residues 50-58 of SEQ ID NO: 39 for CDR2, and residues 95-102 of SEQ ID NO: 39 for CDR3;
(f) residues 26-35 of SEQ ID NO: 40 for CDR1, residues 50-58 of SEQ ID NO: 40 for CDR2, and residues 95-102 of SEQ ID NO: 40 for CDR3;
(g) residues 26-35 of SEQ ID NO: 41 for CDR1, residues 50-58 of SEQ ID NO: 41 for CDR2, and residues 95-102 of SEQ ID NO: 41 for CDR3;
(h) residues 26-35 of SEQ ID NO: 42 for CDR1, residues 50-58 of SEQ ID NO: 42 for CDR2, and residues 95-102 of SEQ ID NO: 42 for CDR3;
(i) residues 26-35 of SEQ ID NO: 43 for CDR1, residues 50-58 of SEQ ID NO: 43 for CDR2, and residues 95-102 of SEQ ID NO: 43 for CDR3;
(j) residues 26-35 of SEQ ID NO: 44 for CDR1, residues 50-58 of SEQ ID NO: 44 for CDR2, and residues 95-102 of SEQ ID NO: 44 for CDR3;
(k) residues 26-35 of SEQ ID NO: 45 for CDR1, residues 50-58 of SEQ ID NO: 45 for CDR2, and residues 95-102 of SEQ ID NO: 45 for CDR3;
(l) residues 26-35 of SEQ ID NO: 46 for CDR1, residues 50-58 of SEQ ID NO: 46 for CDR2, and residues 95-102 of SEQ ID NO: 46 for CDR3;
(m) residues 26-35 of SEQ ID NO: 47 for CDR1, residues 50-58 of SEQ ID NO: 47 for CDR2, and residues 95-102 of SEQ ID NO: 47 for CDR3;
(n) residues 26-35 of SEQ ID NO: 48 for CDR1, residues 50-58 of SEQ ID NO: 48 for CDR2, and residues 95-102 of SEQ ID NO: 48 for CDR3;
(o) residues 26-35 of SEQ ID NO: 49 for CDR1, residues 50-58 of SEQ ID NO: 49 for CDR2, and residues 95-102 of SEQ ID NO: 49 for CDR3;
(p) residues 26-35 of SEQ ID NO: 50 for CDR1, residues 50-58 of SEQ ID NO: 50 for CDR2, and residues 95-102 of SEQ ID NO: 50 for CDR3;
(q) residues 26-35 of SEQ ID NO: 51 for CDR1, residues 50-58 of SEQ ID NO: 51 for CDR2, and residues 95-102 of SEQ ID NO: 51 for CDR3;
(r) residues 26-35 of SEQ ID NO: 52 for CDR1, residues 50-58 of SEQ ID NO: 52 for CDR2, and residues 95-102 of SEQ ID NO: 52 for CDR3;
(s) residues 26-35 of SEQ ID NO: 53 for CDR1, residues 50-58 of SEQ ID NO: 53 for CDR2, and residues 95-102 of SEQ ID NO: 53 for CDR3;
(t) residues 26-35 of SEQ ID NO: 54 for CDR1, residues 50-58 of SEQ ID NO: 54 for
165 CDR2, and residues 95-102 of SEQ ID NO: 54 for CDR3; or
(u) residues 26-35 of SEQ ID NO: 55 for CDR1, residues 50-58 of SEQ ID NO: 55 for CDR2, and residues 95-102 of SEQ ID NO: 55 for CDR3; wherein the residues are numbered according to AbM.
65. The single domain antibody of claim 59, wherein the single domain antibody comprises
(a) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 56, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 57, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 58;
(b) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 59, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 60, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 61;
(c) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 62, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 63, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 64;
(d) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 65, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 66, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 67;
(e) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 68, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 70;
(f) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 71, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 72, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 73;
(g) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 74, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 75, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 76;
(h) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 77, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 78, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 79;
166 (i) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 80, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 81, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 82;
(j) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 83, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 84, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 85;
(k) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 86, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 87, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 88;
(l) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 89, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 90, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 91;
(m) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 92, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 93, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 94;
(n) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 95, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 96, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 97;
(o) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 98, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 99, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 100;
(p) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 101, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 102, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 103;
(q) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 104, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 105, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 106;
(r) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 107, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 108, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 109;
167 (s) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 110, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 111, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 112;
(t) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 113, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 114, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 115; or
(u) a CDR1 comprising the amino acid sequence set forth as SEQ ID NO: 116, a CDR2 comprising the amino acid sequence set forth as SEQ ID NO: 117, and a CDR3 comprising the amino acid sequence set forth as SEQ ID NO: 118.
66. The single domain antibody of claim 59, wherein the single domain antibody comprises a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55.
67. A nucleic acid encoding the heavy chain variable region of any one of claims 59-66.
68. A vector comprising the nucleic acid of claim 67.
69. A host cell comprising the nucleic acid of claim 67 or the vector of claim 68.
70. A pharmaceutical composition comprising the single domain antibody of any one of claims
59-66.
168
PCT/US2022/076974 2021-09-24 2022-09-23 Orai1 multispecific antibodies and methods of use WO2023049867A1 (en)

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US5502167A (en) * 1991-12-06 1996-03-26 Waldmann; Herman CDR grafted humanised chimeric T-cell antibodies
US20120231006A1 (en) * 2009-11-20 2012-09-13 Amgen Inc. Anti-orai1 antigen binding proteins and uses thereof
US20170226203A1 (en) * 2014-08-07 2017-08-10 Daiichi Sankyo Company, Limited Anti-Orai1 Antibody
US20200246382A1 (en) * 2018-12-12 2020-08-06 Kite Pharma, Inc. Chimeric antigen and t cell receptors and methods of use

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5502167A (en) * 1991-12-06 1996-03-26 Waldmann; Herman CDR grafted humanised chimeric T-cell antibodies
US20120231006A1 (en) * 2009-11-20 2012-09-13 Amgen Inc. Anti-orai1 antigen binding proteins and uses thereof
US20170226203A1 (en) * 2014-08-07 2017-08-10 Daiichi Sankyo Company, Limited Anti-Orai1 Antibody
US20200246382A1 (en) * 2018-12-12 2020-08-06 Kite Pharma, Inc. Chimeric antigen and t cell receptors and methods of use

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