WO2023235703A1 - Méthodes thérapeutiques utilisant des agents de liaison ilt - Google Patents

Méthodes thérapeutiques utilisant des agents de liaison ilt Download PDF

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Publication number
WO2023235703A1
WO2023235703A1 PCT/US2023/067618 US2023067618W WO2023235703A1 WO 2023235703 A1 WO2023235703 A1 WO 2023235703A1 US 2023067618 W US2023067618 W US 2023067618W WO 2023235703 A1 WO2023235703 A1 WO 2023235703A1
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WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
cdr2
cdr1
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PCT/US2023/067618
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English (en)
Inventor
Ursula Jeffry
Daniel David KAPLAN
Koho IIZUKA
Jeong Kim
Christina SONG
Geoffrey William Stone
Nicole Taylor
Lisa Kay BLUM
Igor MIKAELIAN
Julie Michelle RODA
Vladimir Hanes
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Ngm Biopharmaceuticals, Inc.
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Publication of WO2023235703A1 publication Critical patent/WO2023235703A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the Sequence Listing XML file submitted with this application is entitled “13370-179-228_SEQLISTING.xml”, was created on May 25, 2023, and is 205,024 bytes in size.
  • a binding agent e.g., antibodies and antigen-binding fragments thereof
  • the present disclosure also provides methods of using a biomarker to predict a subject’s responsiveness to the therapy disclosed herein and the efficacy of the therapy.
  • BACKGROUND The basis for immunotherapy is the manipulation and/or modulation of the immune system, including both innate immune responses and adaptive immune responses.
  • the general aim of immunotherapy is to treat diseases by controlling the immune response to a “foreign agent”, for example a pathogen or a tumor cell.
  • immunotherapy is used to treat autoimmune diseases which may arise from an abnormal immune response against proteins, molecules, and/or tissues normally present in the body.
  • Immunotherapy may include methods to induce or enhance specific immune responses or to inhibit or reduce specific immune responses.
  • the immune system is a highly complex system made up of a great number of cell types, including but not limited to, T-cells, T-cell subsets, B-cells, natural killer cells, antigen- presenting cells, dendritic cells, monocytes, and macrophages. These cells possess complex and subtle systems for controlling their interactions and responses.
  • the cells utilize both activating and inhibitory mechanisms and feedback loops to keep responses in check and not allow negative consequences of an uncontrolled immune response (e.g., autoimmune diseases or a cytokine storm).
  • Some of the inhibitory mechanisms of the immune system use proteins from the leukocyte Ig-like receptor (LILR) family.
  • the leukocyte Ig-like receptor subfamily B (LILRB) is a group of type I transmembrane glycoproteins with extracellular Ig-like domains and cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs).
  • This group of ITIM- containing receptors includes 5 members: LILRB1 (also known as CD85J, LIR1, ILT2), LILRB2 (also known as CD85D, LIR2, ILT4), LILRB3 (also known as CD85A, LIR3, ILT5), LILRB4 (also known as CD85K, LIR5, ILT3), and LILRB5 (also known as CD85C, LIR8).
  • LILRB1 also known as CD85J, LIR1, ILT2
  • LILRB2 also known as CD85D, LIR2, ILT4
  • LILRB3 also known as CD85A, LIR3, ILT5
  • LILRB4 also known as CD85K, LIR5, ILT3
  • LILRB5 also known as CD85C, LIR8.
  • LILRA LILR subfamily A
  • ITAMs cytoplasmic immunoreceptor tyrosine-based activating motifs
  • This group of ITAM-containing receptors includes 6 members: LILRA1 (also known as CD85I, LIR6), LILRA2 (also known as CD85H, LIR7, ILT1), LILRA3 (also known as CD85E, LIR4, ILT6, monocyte inhibitory receptor HM43/31), LILRA4 (also known as CD85G, ILT7), LILRA5 (also known as CD85F, LIR9, ILT11), and LILRA6 (also known as ILT8).
  • LILRA1 also known as CD85I, LIR6
  • LILRA2 also known as CD85H, LIR7, ILT1
  • LILRA3 also known as CD85E, LIR4, ILT6, monocyte inhibitory receptor HM43/31
  • LILRA4 also known as CD85G, ILT7
  • LILRA5 also known as CD85F
  • the inhibitory and activating proteins of the LILR family appear to work in concert to modulate immune homeostasis.
  • the concept of cancer immunosurveillance is based on the theory that the immune system can recognize tumor cells, mount an immune response, and suppress the development and/or growth of a tumor.
  • cancerous/tumor cells have developed mechanisms and/or hijacked normal inhibitory mechanisms to evade the immune system which can allow for uninhibited growth of tumor cells.
  • Cancer/tumor immunotherapy (immuno- oncology) focuses on the development of new and novel agents that can activate and/or boost the immune system to achieve a more effective attack against cancer/tumor cells resulting in increased killing of cancer/tumor cells and/or inhibition of cancer/tumor growth.
  • the present disclosure provides methods of treating cancer in a subject in need thereof, comprising administering to a subject an agent that binds immunoglobulin-like transcript 2 (ILT2) and immunoglobulin-like transcript 4 (ILT4).
  • ILT2 immunoglobulin-like transcript 2
  • ITT4 immunoglobulin-like transcript 4
  • the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of (a) a binding agent comprising (i) a heavy chain variable region comprising a heavy chain variable region CDR1 (VH-CDR1), a heavy chain variable region CDR2 (VH-CDR2), and a heavy chain variable region CDR3 (VH-CDR3) from the amino acid sequence of SEQ ID NO:144; and a light chain variable region comprising a light chain variable region CDR1 (VL-CDR1), a light chain variable region CDR2 (VL-CDR2), and a light chain variable region CDR3 (VL-CDR3) from the amino acid sequence of SEQ ID NO:145; or (ii) a heavy chain variable region comprising a heavy chain variable region CDR1 (VH-CDR1), a heavy chain variable region CDR2 (VH-CDR2), and a heavy chain variable region CDR3 (V
  • the cancer is hepatocellular carcinoma, small cell lung cancer, thyroid cancer, mesothelioma, glioblastoma, renal cell carcinoma, non-small cell lung cancer, melanoma (skin cutaneous and uveal), pancreatic ductal adenocarcinoma, gastric cancer, squamous cell carcinoma of the head and neck, biliary duct cancer, breast cancer, triple-negative breast cancer, ovarian cancer, cervical cancer, endocervical cancer, colorectal cancer, esophageal cancer, gastroesophageal junction adenocarcinoma, squamous cell carcinoma of head and neck, bladder cancer, uterine cancer, cholangiocarcinoma, or prostate cancer.
  • the cancer is a microsatellite instability – high (MSI-H) tumor or a deficient mismatch repair (dMMR) positive tumor.
  • the binding agent is a dose of between about 200 mg and about 1800 mg, including for examples, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, and about 1800 mg.
  • the binding agent is administered at a dose of about 600 mg.
  • the binding agent is administered at a dose of about 1200 mg.
  • the binding agent is administered at a dose of about 1800 mg. [0012] In certain embodiment, the binding agent is administered at a dose that achieves at least about 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of receptor occupancy in immune cells within 24 hours of administration. In certain embodiment, the binding agent is administered at a dose that achieves at least about 75% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more) receptor occupancy in immune cells twenty-one (21) days or three weeks after administration.
  • at least about 95% e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
  • the binding agent is administered at a dose that achieves at least about 75% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more)
  • the immune cells are selected from the group consisting of granulocytes, myeloid antigen presenting cells, natural killer cells, monocytes, CD33+ myeloid cells, T cells, and B cells.
  • the binding agent is administered once every week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiment, the binding agent is administered once every 3 weeks. [0015] In certain embodiment, the binding agent is administered over about 30 minutes, about 60 minutes or about 90 minutes. In certain embodiment, the binding agent is administered over about 30 minutes at a dose of ⁇ 600 mg (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg).
  • the binding agent is administered over about 60 minutes at a dose of ⁇ 600 mg (e.g., about 600 mg, about 800 mg, about 1000 mg, about 1200 mg, about 1400 mg, about 1600 mg, and about 1800 mg).
  • the binding agent is administered intravenously.
  • the binding agent is administered intravenously at a dose of about 600 mg once every 3 weeks.
  • the binding agent is administered intravenously at a dose of about 1200 mg once every 3 weeks.
  • the binding agent is administered intravenously at a dose of about 1800 mg once every 3 weeks.
  • the heavy chain variable region of the binding agent comprises the VH-CDR1 having the amino acid sequence of SEQ ID NO:115, the VH-CDR2 having the amino acid sequence of SEQ ID NO:112, and the VH-CDR3 having the amino acid sequence of SEQ ID NO:107; and the light chain variable region of the binding agent comprises the VL-CDR1 having the amino acid sequence of SEQ ID NO:89, the VL-CDR2 having the amino acid sequence of SEQ ID NO:90, and the VL-CDR3 having the amino acid sequence of SEQ ID NO:91;
  • the heavy chain variable region of the binding agent comprises the VH- CDR1 having the amino acid sequence of SEQ ID NO:111, the VH-CDR2 having the amino acid sequence of SEQ ID NO:112, and the VH-CDR3 having the amino acid sequence of SEQ ID NO:107; and the light chain variable region of the binding agent comprises the VL-CDR1 having the amino acid sequence of SEQ ID NO:89,
  • the heavy chain variable region of the binding agent comprises the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2, and the VL-CDR3 comprising amino acid sequences of any one of the five CDR definitions set forth in Table 8A; or (2) the heavy chain variable region of the binding agent comprises the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2, and the VL-CDR3 comprising amino acid sequences of any one of the five CDR definitions set forth in Table 8B.
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:144;
  • the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:145; or
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:144 and the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:145.
  • the heavy chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:144;
  • the light chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:145; and/or (c) the heavy chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:144 and the light chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:145.
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:143;
  • the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:142; and/or (c) the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:143 and the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:142.
  • the heavy chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:143;
  • the light chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:142; and/or (c) the heavy chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:143 and the light chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:142.
  • the present disclosure provides a method for: (a) inhibiting tumor growth in a subject; (b) increasing or enhancing an immune response to a tumor or tumor cells in a subject; (c) activating or enhancing a persistent or long-term immune response to a tumor or tumor cells in a subject; (d) inhibiting tumor relapse or tumor regrowth in a subject; or (e) inducing a persistent or long-term immunity that inhibits tumor relapse or tumor regrowth in a subject; the method comprising administering to the subject a therapeutically effective amount of a binding agent comprising (i) a heavy chain variable region comprising a heavy chain variable region CDR1 (VH-CDR1), a heavy chain variable region CDR2 (VH-CDR2), and a heavy chain variable region CDR3 (VH-CDR3) from the amino acid sequence of SEQ ID NO:144; and a light chain variable region comprising a light chain variable region CDR1 (VL- CDR1),
  • the heavy chain variable region of the binding agent comprises the VH-CDR1 having the amino acid sequence of SEQ ID NO:115, the VH-CDR2 having the amino acid sequence of SEQ ID NO:112, and the VH-CDR3 having the amino acid sequence of SEQ ID NO:107; and the light chain variable region of the binding agent comprises the VL-CDR1 having the amino acid sequence of SEQ ID NO:89, the VL-CDR2 having the amino acid sequence of SEQ ID NO:90, and the VL-CDR3 having the amino acid sequence of SEQ ID NO:91;
  • the heavy chain variable region of the binding agent comprises the VH- CDR1 having the amino acid sequence of SEQ ID NO:111, the VH-CDR2 having the amino acid sequence of SEQ ID NO:112, and the VH-CDR3 having the amino acid sequence of SEQ ID NO:107; and the light chain variable region of the binding agent comprises the VL-CDR1 having the amino acid sequence of SEQ ID NO:89,
  • the heavy chain variable region of the binding agent comprises the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2, and the VL-CDR3 comprising amino acid sequences of any one of the five CDR definitions set forth in Table 8A; or (2) the heavy chain variable region of the binding agent comprises the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2, and the VL-CDR3 comprising amino acid sequences of any one of the five CDR definitions set forth in Table 8B.
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:144;
  • the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:145; or
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:144 and the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:145.
  • the heavy chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:144;
  • the light chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:145; and/or (c) the heavy chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:144 and the light chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:145.
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:143;
  • the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:142; and/or (c) the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:143 and the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:142.
  • the heavy chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:143;
  • the light chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:142; and/or (c) the heavy chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:143 and the light chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:142.
  • the tumor comprises a microsatellite instability – high (MSI-H) tumor or a deficient mismatch repair (dMMR) positive tumor.
  • the tumor is a hepatocellular carcinoma, a small cell lung tumor, a thyroid tumor, a mesothelioma, a glioblastoma, a renal cell carcinoma, a non-small cell lung tumor, a melanoma (skin cutaneous and uveal), a pancreatic ductal adenocarcinoma, a gastric tumor, a squamous cell carcinoma of the head and neck, a biliary duct tumor, a breast tumor, a triple-negative breast tumor, an ovarian tumor, a cervical tumor, an endocervical tumor, a colorectal tumor, an esophageal tumor, a gastroesophageal junction adenocarcinoma, a squamous cell carcinoma of head and neck, a bladder tumor, an uterine tumor, a cholangiocarcinoma, or a prostate tumor.
  • a pancreatic ductal adenocarcinoma
  • the binding agent is (a) an antibody; (b) a recombinant antibody; (c) an antibody fragment comprising at least one antigen-binding site; (d) a chimeric antibody; (e) a humanized antibody; (f) a bispecific or multispecific antibody; or (g) attached to a half-life extending moiety.
  • the binding agent is an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody; optionally wherein the binding agent is a human IgG1 antibody, a human IgG2 antibody, or a human IgG4 antibody; further optionally the human IgG1 antibody has reduced or no effector function.
  • the binding agent comprises a kappa light chain or a lambda light chain, optionally wherein the antibody comprises a human kappa light chain or a human lambda light chain.
  • the antibody fragment is a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, a (scFv)2, a single chain antibody, a dual variable region antibody, a diabody, or a nanobody.
  • the binding agent comprises a heavy chain comprising an amino acid sequence with 80% identity to the sequence of SEQ ID NO:156 and a light chain comprising an amino acid sequence with 80% identity to the sequence of SEQ ID NO:157. [0038] In certain embodiment, the binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:156 and a light chain comprising the amino acid sequence of SEQ ID NO:157.
  • the binding agent has one or more of the following properties: (1) binds rhesus ILT2; (2) binds cyno ILT2; (3) does not bind ILT3, ILT5, and LILRB5; (4) does not bind LILRA2, LILRA4, LILRA5, and LILRA6; (5) is an ILT2 antagonist; (6) is an ILT4 antagonist, (7) inhibits ILT2 activity; (8) inhibits ILT4 activity; (9) inhibits ILT2 signaling in cells that express ILT2; (10) inhibits ILT4 signaling in cells that express ILT4; (11) inhibits binding of ILT2 to MHC I molecules; (12) inhibits binding of ILT4 to MHC I molecules; (13) inhibits ILT2-induced suppression of myeloid cells; (14) inhibits ILT4-induced suppression of myeloid cells; (15) inhibits ILT2-induced suppression of myeloid cell activity; (16) inhibits ILT4-induced suppression of myeloid cell activity; (17) restores
  • the subject is a human.
  • the present disclosure provides a method for identifying a subject having a cancer or a tumor as more likely to respond to a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, the method comprising comparing the expression level of CD163 in a sample of the subject before receiving the therapy to a CD163 reference level, wherein the subject is identified as more likely to respond to the therapy if the expression level of CD163 is higher than the CD163 reference level.
  • the present disclosure provides a method for identifying a subject having a cancer or a tumor as more likely to respond to a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, the method comprising: (a) measuring the expression level of CD163 in a sample of the subject before receiving the therapy; (b) comparing the expression level of CD163 in (a) to a CD163 reference level; and (c) identifying the subject as more likely to respond to the therapy if the expression level of CD163 is higher than the CD163 reference level.
  • the present disclosure provides a method for selectively treating a cancer or a tumor in a subject identified to have a higher expression level of CD163 than a CD163 reference level, the method comprising administering a therapeutically effective amount of a binding agent specifically binding to human ILT2 and/or human ILT4 to the subject.
  • the present disclosure provides a method for treating a subject having a cancer or a tumor, comprising: (a) comparing the expression level of CD163 in a sample of the subject before receiving a therapy to a CD163 reference level, wherein the therapy comprises a binding agent specifically binding to human ILT2 and/or human ILT4; and (b) administering a therapeutically effective amount of the binding agent to the subject if the expression level of CD163 is higher than the CD163 reference level.
  • the present disclosure provides a method for treating a subject having a cancer or a tumor, comprising: (a) measuring the expression level of CD163 in a sample obtained from the subject before receiving a therapy, wherein the therapy comprises a binding agent specifically binding to human ILT2 and/or human ILT4; (b) comparing the expression level of CD163 to a CD163 reference level; and (c) administering the therapy to the subject if the expression level of CD163 is higher than the CD163 reference level.
  • the CD163 reference level is the expression level of CD163 in a sample from a healthy or non-diseased subject, or the expression level of CD163 in samples from a population of healthy or non-diseased subjects, wherein the sample from the healthy or non-diseased subject or the samples from a population of healthy or non-diseased subjects are from the same source as the sample from the subject having the cancer or tumor.
  • the present disclosure provides a method for determining the efficacy of a therapy in a subject having a cancer or a tumor, wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, the method comprising comparing (a) the expression level of CD163 in a sample of the subject before receiving the therapy and (b) the expression level of CD163 in a sample of the subject after receiving the therapy, wherein the therapy is determined to be effective if the expression level of CD163 in (b) is reduced as compared to the expression level of CD163 in (a).
  • the present disclosure provides a method for determining the efficacy of a therapy in a subject having a cancer or a tumor, wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, the method comprising: (a) measuring (i) the expression level of CD163 in a sample of the subject before receiving the therapy, and (ii) the expression level of CD163 in a sample of the subject after receiving the therapy; (b) comparing the expression level of CD163 in (a-i) to the expression level of CD163 in (a-ii); and (c) determining the therapy is effective if the expression level of CD163 in (a-ii) is reduced as compared to the expression level of CD163 in (a-i).
  • the present disclosure provides a method of treating a subject having a cancer or a tumor, wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, and the method comprises: (a) comparing (i) the expression level of CD163 in a sample of the subject before receiving the therapy and (ii) the expression level of CD163 in a sample of the subject after receiving the therapy; and (b) continuing administering the therapy to the subject if the expression level of CD163 in (a-ii) is reduced as compared to the expression level of CD163 in (a-i).
  • the present disclosure provides a method of treating a subject having a cancer or a tumor, comprising: (a) measuring (i) the expression level of CD163 in a sample of the subject before receiving a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, and (ii) the expression level of CD163 in a sample of the subject after receiving the therapy; (b) comparing the expression level of CD163 in (a-i) to the expression level of CD163 in (a-ii); and (c) continuing administering the therapy to the subject if the expression level of CD163 in (a-ii) is reduced as compared to the expression level of CD163 in (a-i).
  • the present disclosure provides a method of treating a subject having a cancer or a tumor, wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, and the method comprises: (a) comparing (i) the expression level of CD163 in a sample of the subject before receiving the therapy and (ii) the expression level of CD163 in a sample of the subject after receiving the therapy; and (b) increasing the dose or dosing frequency of the binding agent if the expression level of CD163 in (a-ii) is not reduced as compared to the expression level of CD163 in (a-i).
  • the present disclosure provides a method of treating a subject having a cancer or a tumor, comprising: (a) measuring (i) the expression level of CD163 in a sample of the subject before receiving a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, and (ii) the expression level of CD163 in a sample of the subject after receiving the therapy; (b) comparing the expression level of CD163 in (a-i) to the expression level of CD163 in (a-ii); and (c) increasing the dose or dosing frequency of the binding agent if the expression level of CD163 in (a-ii) is not reduced as compared to the expression level of CD163 in (a-i).
  • the sample is a peripheral blood sample or a tumor biopsy.
  • the sample comprises cells isolated from peripheral blood or a tumor biopsy of the subject.
  • the isolated cells are selected from the group consisting of monocytes, MDSCs, pan-myeloid cells, pan-dendritic cells, and combinations thereof.
  • the expression level of CD163 is a protein expression level of CD163.
  • the protein expression level is measured by flow cytometry, immunohistochemistry, Western Blot, or enzyme-linked immunosorbent assay (ELISA).
  • the expression level of CD163 is an mRNA expression level of CD163.
  • the mRNA expression level is measured by quantitative reverse- transcriptase PCR (RT-qPCR), microarray, Northern blot, or RNA sequencing.
  • RT-qPCR quantitative reverse- transcriptase PCR
  • the sample obtained from the subject after receiving the therapy is a sample obtained after the subject received one dose or multiple doses of the binding agent.
  • the sample obtained from the subject after receiving the therapy is a sample obtained from the subject between 15 days after the subject received a first dose of the binding agent and 15 days after the subject received a second dose of the binding agent.
  • the antigen-binding domain binds specifically to both human ILT2 and human ILT4 and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (VH-CDR1), a heavy chain complementarity- determining region 2 (VH-CDR2), and a heavy chain complementarity-determining region 3 (VH-CDR3) from the amino acid sequence of SEQ ID NO:144; and a light chain variable region (VL) comprising a light chain complementarity-determining region 1 (VL-CDR1), a light chain complementarity-determining region 2 (VL-CDR2), and a light chain complementarity- determining region 3 (VL-CDR3) from the amino acid sequence of SEQ ID NO:145.
  • VH heavy chain variable region
  • VH-CDR1 heavy chain complementarity-determining region 1
  • VH-CDR2 heavy chain complementarity-determining region 2
  • VH-CDR3 heavy chain complementarity-determining region 3
  • the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:115
  • the VH-CDR2 comprises the amino acid sequence of SEQ ID NO:112
  • the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:107
  • the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:89
  • the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:90
  • the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:91
  • the VH- CDR1 comprises the amino acid sequence of SEQ ID NO:111
  • the VH-CDR2 comprises the amino acid sequence of SEQ ID NO:112
  • the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:107
  • the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:89
  • the VL- CDR2 comprises the amino acid sequence of SEQ ID NO:90
  • the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:91
  • the VH has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:144, and/or the VL has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:145; or (b) the VH comprises the amino acid sequence of SEQ ID NO:144, and/or the VL comprises the amino acid sequence of SEQ ID NO:145.
  • the antigen-binding domain comprises (a) a heavy chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:156, and/or a light chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:157; or (b) a heavy chain comprising the amino acid sequence of SEQ ID NO:156, and/or a light chain comprising the amino acid sequence of SEQ ID NO:157.
  • the antigen-binding domain binds specifically to both human ILT2 and human ILT4 and comprises: (i) a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO:139; and a VL comprising a VL- CDR1, a VL-CDR2, and a VL-CDR3 from the amino acid sequence of SEQ ID NO:140; (ii) a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO:133; and a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from the amino acid sequence of SEQ ID NO:134; (iii) a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ
  • the VH comprises the VH-CDR1, the VH-CDR2, and the VH- CDR3 from the amino acid sequence of SEQ ID NO:139; and the VL comprises the VL-CDR1, the VL-CDR2, and the VL-CDR3 from the amino acid sequence of SEQ ID NO:140, and wherein (a) the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:95, the VH-CDR2 comprises the amino acid sequence of SEQ ID NO:87, the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:102, the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:103, the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:90, and the VL- CDR3 comprises the amino acid sequence of SEQ ID NO:91; (b) the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:86, the VH-CDR2 comprises the amino acid sequence of SEQ
  • the VH has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:139, and/or the VL has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:140; or the VH comprises the amino acid sequence of SEQ ID NO:139, and/or the VL comprises the amino acid sequence of SEQ ID NO:140, (b) the VH has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:133, and/or the VL has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:134; or the VH comprises the amino acid sequence of SEQ ID NO:133, and/or the VL comprises the amino acid sequence of SEQ ID NO:134, (c) the VH has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:143, and/or the VL has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:142; or the VH comprises the amino acid sequence of SEQ ID NO:143, and/
  • the antigen-binding domain comprises: (a) a heavy chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:148, and/or a light chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:149; (b) a heavy chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:152, and/or a light chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:153; (c) a heavy chain comprising the amino acid sequence of SEQ ID NO:148, and/or a light chain comprising the amino acid sequence of SEQ ID NO:149; or (d) a heavy chain comprising the amino acid sequence of SEQ ID NO:152, and/or a light chain comprising the amino acid sequence of SEQ ID NO:153.
  • the antigen-binding domain binds specifically to human ILT2 and comprises a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO:125; and a VL comprising a VL-CDR1, a VL-CDR2, and a VL- CDR3 from the amino acid sequence of SEQ ID NO:126.
  • the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:31
  • the VH-CDR2 comprises the amino acid sequence of SEQ ID NO:23
  • the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:24
  • the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:25
  • the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:26
  • the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:27
  • the VH- CDR1 comprises the amino acid sequence of SEQ ID NO:22
  • the VH-CDR2 comprises the amino acid sequence of SEQ ID NO:23
  • the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:24
  • the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:25
  • the VL- CDR2 comprises the amino acid sequence of SEQ ID NO:26
  • the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:
  • the VH has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:125, and/or the VL has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:126; or (b) the VH comprises the amino acid sequence of SEQ ID NO:125, and/or the VL comprises the amino acid sequence of SEQ ID NO:126.
  • the antigen-binding domain binds specifically to human ILT4 and comprises: (a) a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO:129; and a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from the amino acid sequence of SEQ ID NO:130; or (b) a VH comprising a VH- CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO:127; and a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from the amino acid sequence of SEQ ID NO:128.
  • the VH comprises the VH-CDR1, the VH-CDR2, and the VH-CDR3 from the amino acid sequence of SEQ ID NO:129; and the VL comprises the VL- CDR1, the VL-CDR2, and the VL-CDR3 from the amino acid sequence of SEQ ID NO:130, and wherein (i) the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:63, the VH- CDR2 comprises the amino acid sequence of SEQ ID NO:55, and the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:56,the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:57, the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:58, and the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:59; (ii) the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:54, the VH-CDR2 comprises the amino acid sequence of
  • the VH has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:129, and/or the VL has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:130;
  • the VH has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:127, and/or the VL has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:128;
  • the VH comprises the amino acid sequence of SEQ ID NO:129, and/or the VL comprises the amino acid sequence of SEQ ID NO:130; or
  • the VH comprises the amino acid sequence of SEQ ID NO:127, and/or the VL comprises the amino acid sequence of SEQ ID NO:128.
  • FIG. 1 depicts the expression of ILT2 and ILT4 in various immune cells assayed by flow cytometry.
  • Figure 2 depicts the inhibition of the interaction between ILT2 or ILT4 and MHC I molecules by anti-ILT antibodies.
  • Figure 3 depicts the binding of anti-ILT2/ILT4 antibodies to monocytes.
  • Figure 4A depicts the measurement by Raji reporter assay.
  • Figure 4B depicts the measurement by 721.221-HLA-G cell reporter assay.
  • Figure 5 depicts the inhibition of the interaction between cyno ILT2 and MHC I molecules by anti-ILT antibodies.
  • Figure 6 depicts the effect of anti-ILT antibodies on cytolytic activity of NKL cells.
  • Figure 7 depicts the effect of anti-ILT antibodies on activity of human primary NK cells.
  • Figure 8 depicts the effect of anti-ILT antibodies on cytolytic activity of primary NK cells.
  • Figure 9 depicts the effect of anti-ILT antibodies on cytolytic activity of primary NK cells in ADCC assay with classic MHC-I-expressing target cells.
  • Figure 10 depicts the effect of anti-ILT antibodies on MDSC activity in MLR assay.
  • Figures 11A-11C depict the effect of anti-ILT antibodies on MDSC activity in MLR assay.
  • Figure 11A depicts T cell proliferation.
  • Figure 11B depicts TNF- ⁇ secretion.
  • Figure 11C depicts GM-CSF secretion.
  • Figure 12 depicts the effect of anti-ILT antibodies on LPS-mediated stimulation of human PBMCs as assessed by cytokine production.
  • Figure 13 depicts the effect of anti-ILT antibodies on LPS-mediated stimulation of cyno PBMCs as assessed by cytokine production.
  • Figure 14 depicts the effect of anti-ILT antibodies on LPS-mediated stimulation of tolerized dendritic cells as assessed by cytokine production.
  • Figure 15 depicts the effect of anti-ILT antibodies on cytokine production from HMGB1-treated cells.
  • Figure 16 depicts the effect of anti-ILT antibodies on cytokine production from STING agonist-treated cells.
  • Figure 17 depicts the effect of anti-ILT antibodies suppression of T-cell mediated stimulation of myeloid cells.
  • Figure 18 depicts effect of anti-ILT antibodies on macrophage phagocytosis.
  • Figure 19 depicts the effect of anti-ILT antibodies on cytokine production by dendritic cells.
  • Figure 20 depicts the effect of anti-ILT antibodies on cytokine production from human blood cells.
  • Figure 21 depicts the effect of anti-ILT antibodies on cytokine production from cyno blood cells.
  • Figure 22 depicts the effect of anti-ILT antibodies on cytolytic activity of T cells.
  • Figure 23 depicts the effects of anti-ILT2/ILT4 antibodies on polarization of monocyte-derived macrophages by assaying expression markers via flow cytometry.
  • Figure 24 depicts the effect of Hz73D1.v1 (NGM707) on tumor growth in a CD34 humanized mouse melanoma model.
  • Figure 25 depicts the experimental design of a mouse melanoma xenograft model.
  • Figure 26 depicts the effect of ILT2 and ILT4 blockade individually on tumor growth.
  • Figure 27 depicts the additive effect of ILT2 and ILT4 blockade on monocyte LPS activation.
  • Figure 28 depicts the effect of Hz73D1.v1 (NGM707) in synergistically activating the immune system in vivo by blocking both ILT2 and ILT4 as shown by CX3CR1 MFI on Temra cells.
  • Figure 29 depicts the effect of Hz73D1.v1 (NGM707) in synergistically activating the immune system in vivo by blocking both ILT2 and ILT4 as shown by the interferon gamma secretion.
  • Figure 30 depicts the effect of Hz73D1.v1 (NGM707) in reversing a suppressive myeloid phenotype induced by cancer-associated fibroblasts.
  • Figure 31 depicts the objective responses and tumor lesion assessment of subjects treated with NGM707 with different doses. Each column represents one subject. EOT; end of therapy; PD: progressive disease; SD: stable disease; NCR/NPD: non-complete response/non- progressive disease; PR: partial response; AE: adverse events. Columns from top to bottom: 1-8, 1800 mg; 9-12, 1200 mg; 13-23, 600 mg; 24-27, 200 mg; 28-30, 60 mg, 31-32, 20 mg; 33-34, 6 mg.
  • Figures 32A and 32B depict the pharmacokinetic analysis.
  • Figure 32A depicts the NGM707 serum concentration in each subject after administration of a specific dose of NGM707 in three consecutive cycles of treatment.
  • Figure 32B depicts the mean NGM707 serum concentration from subjects administered with the same dose during the first cycle of treatment.
  • Figure 33 depicts the receptor occupancy measured in monocytes isolated from subjects treated with different doses of NGM707. Each line represents a subject.
  • Figures 34A and 34B depict the decreased CD163 expression on non-classical monocytes was associated with benefit from NGM707 monotherapy.
  • C1D1 cycle 1, day 1;
  • C1D15 cycle 1, day 15; SD: stable disease; PD: progressive disease.
  • Figure 34A depicts the raw expression level of CD163.
  • Figure 34B depicts the relative expression level of CD163 C1D15 to C1D1. SD or Better (circles ⁇ ); PD (diamonds, ⁇ ).
  • Figures 35A and 35B depict the immunohistochemical measurement of CD163 expression on patient biopsies.
  • Figure 35A depicts the immunohistochemical staining of CD163 on two colorectal cancer (CRC) patient biopsies collected before treatment (baseline) and after treatment with 600mg of NGM707 (collected on C1D20-C1D21).
  • CRC colorectal cancer
  • Pt 11002 and Pt 11004 are two CRC patients.
  • Figure 35B depicts the quantification of CD163+ staining from Figure 35A.
  • On-Txt on-treatment.11002 (circles ⁇ ), 11004 (triangles, ⁇ ).
  • Figure 36 depicts immunohistochemical measurement of CD163 in nonclassical monocytes from cultured PBMCs treated with NGM707 or anti-KLH control antibody.
  • Figures 37A and 37B depicts chemokine profile of partial responder on NGM707 monotherapy, 1800 mg.
  • Figure 37A shows the plasma concentration (pg/ml) of CCL3, CXCL9, CXCL10, and CXCL11 over the course of therapy.
  • Figure 37B presents the same data relative to the pretreatment (baseline) level of each individual chemokine.
  • Figure 38 depicts inflammatory immune status and tumor cell proliferation signature in patients treated with NGM707 monotherapy.
  • FIG 38 shows that T Cell Inflammation Signature (“TIS”) was significantly increased, and the proliferation signature significantly decreased, in patients showing stable disease (“SD”) or a better clinical outcome relative to those that showed progressive disease (“PD”) over the course of NGM707 monotherapy, as described in Example 24. 5.
  • TIS T Cell Inflammation Signature
  • SD stable disease
  • PD progressive disease
  • the present disclosure provides novel methods for using agents, including but not limited to polypeptides such as antibodies, that bind immunoglobulin-like transcript 2 (ILT2), immunoglobulin-like transcript 4 (ILT4), or both ILT2 and ILT4.
  • ILT2 immunoglobulin-like transcript 2
  • ILT4 immunoglobulin-like transcript 4
  • ILT-binding agents encompass ILT2-binding agents, ILT4-binding agents, and agents binding to both ILT2 and ILT4 (referred to as ILT2/ILT4-binding agents or ILT2/ILT4 dual binders).
  • ILT2/ILT4-binding agents referred to as ILT2/ILT4 dual binders.
  • LILRB family members are referred to by many names in art, the terms “ILT2” (also known as LILRB1) and “ILT4” (also known as LILRB2) are used in the present disclosure.
  • the ILT-binding agents include, but are not limited to, polypeptides, antibodies (including antigen-binding fragments thereof), scaffold proteins, and heterodimeric molecules.
  • ILT-binding agents include, but are not limited to, antagonists of ILT2 and/or ILT4 activity, inhibitors of ILT2 and/or ILT4 activity, and/or agents that inhibit ILT2 and/or ILT4 suppressive activity.
  • Related polypeptides, polynucleotides, vectors, compositions comprising the agents, cells comprising the related polynucleotides or vectors, and methods of making the agents are also provided.
  • the ILT-binding agent disclosed herein inhibits ILT2 and/or ILT4 activity.
  • the ILT-binding agent disclosed herein enhances an immune response.
  • the ILT-binding agent disclosed herein reverses suppression of an immune cell activity. 5.1.
  • binding agent refers to a molecule that binds a specific antigen or target (e.g., ILT2 and/or ILT4).
  • a binding agent may comprise a protein, peptide, nucleic acid, carbohydrate, lipid, or small molecular weight compound.
  • a binding agent comprises a full-length antibody.
  • a binding agent is an antigen-binding fragment of an antibody.
  • a binding agent comprises an alternative protein scaffold or artificial scaffold (e.g., a non-immunoglobulin backbone).
  • a binding agent is a fusion protein comprising an antigen-binding site.
  • a binding agent is a bispecific or multispecific molecule comprising at least one antigen-binding site.
  • antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to, an immunoglobulin molecule that recognizes and binds a target through at least one antigen-binding site, polyclonal antibodies, recombinant antibodies, monoclonal antibodies (including full length monoclonal antibodies), chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, multispecific antibodies, diabodies, tribodies, tetrabodies, single chain Fv (scFv) antibodies, and antibody fragments as long as they exhibit the desired antigen-binding activity.
  • the basic antibody structural unit comprises a tetramer.
  • Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function.
  • human light chains are classified as kappa and lambda light chains.
  • human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids.
  • the variable regions of each light/heavy chain pair form the antibody binding site.
  • an intact antibody has two binding sites.
  • the two binding sites are, in general, the same.
  • the variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), which are located within relatively conserved framework regions (FR).
  • CDRs complementarity determining regions
  • the CDRs are usually aligned by the framework regions, enabling binding to a specific epitope.
  • both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md. ; 5th ed.; NIH Publ. No.91-3242 (1991); Kabat (1978) Adv. Prot. Chem.32:1-75; Kabat, et al., (1977) J. Biol.
  • CDR or “CDRs” as used herein means complementarity determining region(s) in a immunoglobulin variable region, defined using the Kabat numbering system, unless otherwise indicated
  • Framework region or “FR” as used herein means the immunoglobulin variable regions excluding the CDR regions.
  • the term “intact antibody” or “full-length antibody” refers to an antibody having a structure substantially similar to a native antibody structure.
  • an antibody comprising two light chains each comprising a variable region and a light chain constant region (CL) and two heavy chains each comprising a variable region and at least heavy chain constant regions CH1, CH2, and CH3.
  • an intact antibody includes a hinge region (or a portion thereof) between the CH1 and CH2 regions.
  • antibody fragment or “antibody fragments” or “antigen binding fragment” as used herein refers to a molecule other than an intact antibody that comprises a portion of an antibody and generally an antigen-binding site. The antibody fragment retains the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions, e.g. all six CDRs.
  • antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv, single chain antibody molecules (e.g., scFv), sc(Fv)2, disulfide-linked scFv (dsscFv), diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies (e.g., camelid antibodies), and multispecific antibodies formed from antibody fragments.
  • Fab fragment antigen
  • Fab′ single chain antibody molecules
  • scFv single chain antibody molecules
  • sc(Fv)2 single chain antibody molecules
  • dsscFv disulfide-linked scFv
  • diabodies tribodies
  • tetrabodies minibodies
  • DVD dual variable domain antibodies
  • single variable domain antibodies e.g., camelid antibodies
  • multispecific antibodies formed from antibody fragments e.g., camelid antibodies
  • monoclonal antibody encompasses intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab′, F(ab′) 2 , Fv), single chain antibodies (e.g., scFv), fusion proteins comprising an antibody fragment, and any other modified immunoglobulin molecule comprising at least one antigen-binding site.
  • monoclonal antibody refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage library display, recombinant expression, and transgenic animals.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a first source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • humanized antibody refers to an antibody that comprises a human heavy chain variable region and a light chain variable region wherein the native CDR amino acid residues are replaced by residues from corresponding CDRs from a nonhuman antibody (e.g., mouse, rat, rabbit, or nonhuman primate), wherein the nonhuman antibody has the desired specificity, affinity, and/or activity.
  • one or more framework region amino acid residues of the human heavy chain or light chain variable regions are replaced by corresponding residues from the nonhuman antibody.
  • humanized antibodies can comprise amino acid residues that are not found in the human antibody or in the nonhuman antibody. In some embodiments, these modifications are made to further refine and/or optimize antibody characteristics.
  • the humanized antibody comprises at least a portion of a human immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc, and/or hinge region).
  • human antibody refers to an antibody that possesses an amino acid sequence that corresponds to an antibody produced by a human and/or an antibody that has been made using any of the techniques that are known to those of skill in the art for making human antibodies. These techniques include, but not limited to, phage display libraries, yeast display libraries, transgenic animals, recombinant protein production, and B-cell hybridoma technology.
  • epitopope and “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen or target capable of being recognized and bound by a particular antibody.
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of the protein.
  • Epitopes formed from contiguous amino acids also referred to as linear epitopes
  • epitopes formed by tertiary folding also referred to as conformational epitopes
  • An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation. Epitopes can be predicted using any one of a large number of software bioinformatic tools available on the internet.
  • X-ray crystallography may be used to characterize an epitope on a target protein by analyzing the amino acid residue interactions of an antigen/antibody complex.
  • the term “specifically binds” as used herein refers to an agent that interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to a particular antigen, epitope, protein, or target molecule than with alternative substances.
  • a binding agent that specifically binds an antigen can be identified, for example, by immunoassays, ELISAs, surface plasmon resonance (SPR), or other techniques known to those of skill in the art.
  • an agent that specifically binds an antigen can bind related antigens (e.g., rhesus ILT2 and/or cyno ILT2).
  • an agent that specifically binds an antigen e.g., human ILT2
  • can bind a second antigen e.g., human ILT4 and is referred to herein as a “dual binder”.
  • a binding agent that specifically binds an antigen can bind the target antigen at a higher affinity than its affinity for a different antigen.
  • the different antigen can be a related antigen.
  • a binding agent that specifically binds an antigen can bind the target antigen with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater, than its affinity for a different antigen.
  • a binding agent that specifically binds a particular antigen binds a different antigen at such a low affinity that binding cannot be detected using an assay described herein or otherwise known in the art.
  • affinity is measured using SPR technology in a Biacore system as described herein or as known to those of skill in the art.
  • polypeptide and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
  • polypeptides containing one or more analogs of an amino acid including but not limited to, unnatural amino acids, as well as other modifications known in the art.
  • polypeptide encompasses polypeptides as a single chain and polypeptides of two or more associated chains.
  • polynucleotide and nucleic acid and nucleic acid molecule are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art.
  • two nucleic acids or polypeptides of the disclosure are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 20-40, at least about 40-60, at least about 60-80 nucleotides or amino acid residues in length, or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 nucleotides or amino acid residues, such as at least about 80-100 nucleotides or amino acid residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, for example, (i) the coding region of a nucleotide sequence or (ii) an amino acid sequence.
  • amino acid substitution refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
  • basic side chains e.g.,
  • substitution of an alanine for a valine is considered to be a conservative substitution.
  • conservative substitutions in the sequences of polypeptides and/or antibodies do not abrogate the binding of the polypeptide or antibody to the target binding site.
  • Methods of identifying nucleotide and amino acid conservative substitutions that do not eliminate binding are well-known in the art.
  • the term “vector” as used herein means a construct that is capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.
  • isolated refers to a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature.
  • An “isolated” antibody is substantially free of material from the cellular source from which it is derived.
  • isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions are those that have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is isolated is substantially pure.
  • a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition can be isolated from a natural source (e.g., tissue) or from a source such as an engineered cell line.
  • substantially pure refers to material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
  • subject refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rabbits, rodents, and the like.
  • pharmaceutically acceptable refers to a substance approved or approvable by a regulatory agency or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, including humans.
  • pharmaceutically acceptable excipient, carrier, or adjuvant refers to an excipient, carrier, or adjuvant that can be administered to a subject, together with at least one therapeutic agent, and that is generally safe, non-toxic, and has no effect on the pharmacological activity of the therapeutic agent. In general, those of skill in the art and government agencies consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.
  • pharmaceutical formulation or “pharmaceutical composition” as used herein refers to a preparation that is in such form as to permit the biological activity of the agent to be effective.
  • a pharmaceutical formulation or composition generally comprises additional components, such as a pharmaceutically acceptable excipient, carrier, adjuvant, buffers, etc.
  • a pharmaceutically acceptable excipient such as a pharmaceutically acceptable excipient, carrier, adjuvant, buffers, etc.
  • therapeutically effective amount refers to the amount of an agent that is sufficient to reduce and/or ameliorate the severity and/or duration of (i) a disease, disorder or condition in a subject, and/or (ii) a symptom in a subject.
  • the term also encompasses an amount of an agent necessary for the (i) reduction or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction or amelioration of the recurrence, development, or onset of a given disease, disorder, or condition, and/or (iii) the improvement or enhancement of the prophylactic or therapeutic effect(s) of another agent or therapy (e.g., an agent other than the binding agents provided herein).
  • therapeutic effect refers to the effect and/or ability of an agent to reduce and/or ameliorate the severity and/or duration of (i) a disease, disorder, or condition in a subject, and/or (ii) a symptom in a subject.
  • the term also encompasses the ability of an agent to (i) reduce or ameliorate the advancement or progression of a given disease, disorder, or condition, (ii) reduce or ameliorate the recurrence, development, or onset of a given disease, disorder, or condition, and/or (iii) to improve or enhance the prophylactic or therapeutic effect(s) of another agent or therapy (e.g., an agent other than the binding agents provided herein).
  • another agent or therapy e.g., an agent other than the binding agents provided herein.
  • immune response refers to the partial or total inhibition of the development, recurrence, onset, or spread of a disease, disorder, or condition, or a symptom thereof in a subject.
  • immune response includes responses from both the innate immune system and the adaptive immune system. It includes both cell-mediated and/or humoral immune responses. It includes both T-cell and B-cell responses, as well as responses from other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, dendritic cells, etc.
  • NK natural killer
  • the term “receptor occupancy” or “RO” is a measure of the binding of a molecule to its target. Receptor occupancy assays measure the binding of a molecule to its receptor protein (or target) and provide quantitative data for the same.
  • the term “refractory” refers to a disease that does not respond to treatment, e.g., cancer. In embodiments, a refractory cancer may be resistant to treatment prior to or at the beginning of treatment. In other embodiments, refractory cancer may become refractory during treatment.
  • the term “relapsed” includes subjects who have received at least one prior treatment for the disease, e.g., cancer.
  • Kabat as used herein means an immunoglobulin alignment and numbering system pioneered by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.).
  • ILT-Binding Agents [00156] Amino acid (aa) sequences for human ILT2 (UniProtKB No. Q8NHL6), human ILT4 (UniProtKB No. Q8N423), rhesus macaque (“rhesus”) ILT2 (NCBI Ref No.
  • XP_028694980.1 house sequence has 98% identity to UniProtKB No. A0A2K5VN04
  • SEQ ID NO:1 SEQ ID NO:8, SEQ ID NO:15, and SEQ ID NO:166, respectively.
  • amino acid positions of ILT2 or ILT4 refer to the numbering of amino acid sequences including the signal sequence.
  • a genomic orthologue for human ILT2 is found in the monkey genome, however, no genomic orthologue for human ILT4 appears to exist. Expression patterns of the monkey ILT2 orthologue are comparable to the combined expression patterns of human ILT2 and human ILT4.
  • ILT2 is a single pass type I transmembrane protein with a predicted molecular weight of approximately 71 kDa.
  • ILT2 (human, rhesus, and cyno) is characterized by an extracellular domain comprising four Ig-like C2 type domains, a transmembrane domain, and a long cytoplasmic domain containing 4 ITIM domains (see, e.g., Borges et al., 1997, J. Immunol., 159:5192-5196).
  • the four Ig-like C2-type domains may be referred to herein as Domain 1 (D1), Domain 2 (D2), Domain 3 (D3), and Domain 4 (D4).
  • D1 is situated at the N-terminal portion of the protein, then D2, D3, with D4 situated closest to the transmembrane region.
  • human ILT2 is a protein of 650 amino acids (aa) - the signal sequence is aa 1-23, the extracellular domain is aa 24-461, the transmembrane region is aa 462- 482, and the cytoplasmic domain is aa 483-650.
  • D1 is aa 27- 115
  • D2 is aa 116-221
  • D3 is aa 222-312
  • D4 is aa 313-409
  • the “stem region” is aa 410-461.
  • ITIMs are aa 531-536, 560-565, 612-617, and 642-647.
  • Rhesus ILT2 is a protein of 639 amino acids (aa) - as compared to structural characterization of human ILT2 the signal sequence is aa 1-23, the extracellular domain is aa 24-460, the transmembrane region is aa 461-481, and the cytoplasmic domain is aa 482-639.
  • D1 is aa 27-114
  • D2 is aa 115-220
  • D3 is aa 221-311
  • D4 is aa 312-408
  • the “stem region” is aa 409-460.
  • ITIMs are aa 530-535, 559-564, 601- 606, and 631-636.
  • Cyno ILT2 is a protein of 651 amino acids (aa) - as compared to structural characterization of human ILT2 the signal sequence is aa 1-23, the extracellular domain is aa 24- 461, the transmembrane region is aa 462-482, and the cytoplasmic domain is aa 483-651.
  • D1 is aa 27-114
  • D2 is aa 115-220
  • D3 is aa 221-311
  • D4 is aa 312-408
  • the “stem region” is aa 409-461.
  • ITIMs are aa 531-536, 561-566, 613-618, and 643-648.
  • ILT2 is expressed (to varying degrees) on natural killer (NK) cells, monocytes, macrophages, eosinophils, basophils, dendritic cells (DCs), subset of T-cells, and B-cells.
  • NK natural killer
  • ILT2 Ligand known to interact with ILT2 include HLA class I molecules (e.g., HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G). ILT2 appears to bind more strongly with the “non-classical” MHC I molecule HLA-G than to classical HLA class I molecules.
  • ILT4 has a similar structure to ILT2. It is a single pass type I transmembrane protein with a predicted molecular weight of approximately 65 kDa.
  • ILT4 is characterized by an extracellular domain comprising four Ig-like C2 type domains, a transmembrane domain, and a long cytoplasmic domain containing 3 ITIM domains (see, e.g., Borges et al., 1997, J. Immunol., 159:5192-5196).
  • the four Ig-like C2-type domains may be referred to herein as D1, D2, D3, and D4.
  • D1 is situated at the N-terminal portion of the protein, then D2, D3, with D4 situated closest to the transmembrane region.
  • human ILT4 is a protein of 598 amino acids (aa) - the signal sequence is aa 1-21, the extracellular domain is aa 22-461, the transmembrane region is aa 462-482, and the cytoplasmic domain is aa 483-598.
  • D1 is aa 27-110
  • D2 is aa 111-229
  • D3 is aa 230-318
  • D4 is aa 330-419
  • the “stem region” is aa 420-461.
  • ITIMs are aa 531-536, 560-565, and 590-595.
  • ILT4 is expressed on myeloid cells such as monocytes, macrophages, dendritic cells, but not on lymphoid cells.
  • Ligands of ILT4 include HLA class I molecules, ANGPTL proteins, myelin inhibitors, and ⁇ -amyloid.
  • Domains of ILT2 or ILT4 e.g., human ILT2, rhesus ILT2, cyno ILT2, or human ILT4
  • the N-terminal amino acids and the C-terminal amino acids of any ILT2 or ILT4 domain or region may vary by 1, 2, 3, 4, 5, or more amino acid residues.
  • the present disclosure provides agents that bind ILT2, ILT4, or ILT2 and ILT4, i.e., ILT-binding agents.
  • Agents that bind both ILT2 and ILT4 may be referred to herein as “dual binders”.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds ILT2 or a fragment of ILT2.
  • a fragment of ILT2 comprises the extracellular domain of ILT2.
  • a fragment of ILT2 comprises one or more of the Ig-like C2 type domains (e.g., D1, D2, D3, and/or D4).
  • a fragment of ILT2 comprises D1 and D2. In some embodiments, a fragment of ILT2 comprises D2 and D3. In some embodiments, a fragment of ILT2 comprises D3 and D4. In some embodiments, a fragment of ILT2 comprises D1, D2, and D3. In some embodiments, a fragment of ILT2 comprises D2, D3, and D4. In some embodiments, a fragment of ILT2 comprises one or more of the Ig-like C2 type domains and the stem region. In some embodiments, a fragment of ILT2 comprises D4-stem, D3-D4-stem, or D2-D3-D4-stem.
  • the extracellular domain of human ILT2 comprises amino acids 24-461 of SEQ ID NO:1.
  • D1 of human ILT2 comprises amino acids 27- 115 of SEQ ID NO:1.
  • D2 of human ILT2 comprises amino acids 116- 221 of SEQ ID NO:1.
  • D3 of human ILT2 comprises amino acids 222- 312 of SEQ ID NO:1.
  • D4 of human ILT2 comprises amino acids 313- 409 of SEQ ID NO:1.
  • D1-D2 of human ILT2 comprises amino acids 27- 221 of SEQ ID NO:1.
  • D2-D3 of human ILT2 comprises amino acids 116-312 of SEQ ID NO:1. In some embodiments, D3-D4 of human ILT2 comprises amino acids 222-409 of SEQ ID NO:1. In some embodiments, D1-D2-D3 of human ILT2 comprises amino acids 27-312 of SEQ ID NO:1. In some embodiments, D2-D3-D4 of human ILT2 comprises amino acids 116-409 of SEQ ID NO:1. In some embodiments, D4-stem of human ILT2 comprises amino acids 313-461 of SEQ ID NO:1. In some embodiments, D3-D4-stem of human ILT2 comprises amino acids 222-461 of SEQ ID NO:1.
  • D2-D3- D4-stem of human ILT2 comprises amino acids 116-461 of SEQ ID NO:1.
  • a fragment of human ILT2 comprises the amino acid sequence of SEQ ID NO:3.
  • a fragment of human ILT2 comprises the amino acid sequence of SEQ ID NO:4.
  • a fragment of human ILT2 comprises the amino acid sequence of SEQ ID NO:5.
  • a fragment of human ILT2 comprises the amino acid sequence of SEQ ID NO:6.
  • a fragment of human ILT2 comprises the amino acid sequence of SEQ ID NO:7.
  • a fragment of human ILT2 comprises the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:5. In some embodiments, a fragment of human ILT2 comprises the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6. In some embodiments, a fragment of human ILT2 comprises the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7. In some embodiments, a fragment of human ILT2 comprises the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, a fragment of human ILT2 comprises the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.
  • the extracellular domain of rhesus ILT2 comprises amino acids 24-460 of SEQ ID NO:15.
  • D1 of rhesus ILT2 comprises amino acids 27- 114 of SEQ ID NO:15.
  • D2 of rhesus ILT2 comprises amino acids 115- 220 of SEQ ID NO:15.
  • D3 of rhesus ILT2 comprises amino acids 221- 311 of SEQ ID NO:15.
  • D4 of rhesus ILT2 comprises amino acids 312- 408 of SEQ ID NO:15.
  • D1-D2 of rhesus ILT2 comprises amino acids 27-220 of SEQ ID NO:15.
  • D2-D3 of rhesus ILT2 comprises amino acids 115-311 of SEQ ID NO:15.
  • D3-D4 of rhesus ILT2 comprises amino acids 221-408 of SEQ ID NO:15.
  • D1-D2-D3 of rhesus ILT2 comprises amino acids 27-311 of SEQ ID NO:15.
  • D2-D3-D4 of rhesus ILT2 comprises amino acids 115-408 of SEQ ID NO:15.
  • D4-stem of rhesus ILT2 comprises amino acids 312-460 of SEQ ID NO:15.
  • D3-D4-stem of rhesus ILT2 comprises amino acids 221-460 of SEQ ID NO:15.
  • D2-D3- D4-stem of rhesus ILT2 comprises amino acids 115-460 of SEQ ID NO:15.
  • a fragment of rhesus ILT2 comprises the amino acid sequence of SEQ ID NO:17.
  • a fragment of rhesus ILT2 comprises the amino acid sequence of SEQ ID NO:18.
  • a fragment of rhesus ILT2 comprises the amino acid sequence of SEQ ID NO:19. In some embodiments, a fragment of rhesus ILT2 comprises the amino acid sequence of SEQ ID NO:20. In some embodiments, a fragment of rhesus ILT2 comprises the amino acid sequence of SEQ ID NO:21. In some embodiments, a fragment of rhesus ILT2 comprises the amino acid sequences of SEQ ID NO:18 and SEQ ID NO:19. In some embodiments, a fragment of human ILT2 comprises the amino acid sequences of SEQ ID NO:19 and SEQ ID NO:20.
  • a fragment of human ILT2 comprises the amino acid sequences of SEQ ID NO:20 and SEQ ID NO:21. In some embodiments, a fragment of human ILT2 comprises the amino acid sequence of SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. In some embodiments, a fragment of human ILT2 comprises the amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21. [00164] In some embodiments, the extracellular domain of cyno ILT2 comprises amino acids 24-461 of SEQ ID NO:166. In some embodiments, D1 of cyno ILT2 comprises amino acids 27- 114 of SEQ ID NO:166.
  • D2 of cyno ILT2 comprises amino acids 115- 220 of SEQ ID NO:166.
  • D3 of cyno ILT2 comprises amino acids 221- 311 of SEQ ID NO:166.
  • D4 of cyno ILT2 comprises amino acids 312- 408 of SEQ ID NO:166.
  • D1-D2 of cyno ILT2 comprises amino acids 27-220 of SEQ ID NO:166.
  • D2-D3 of cyno ILT2 comprises amino acids 115-311 of SEQ ID NO:166.
  • D3-D4 of cyno ILT2 comprises amino acids 221-408 of SEQ ID NO:166.
  • D1-D2-D3 of cyno ILT2 comprises amino acids 27-311 of SEQ ID NO:166.
  • D2-D3-D4 of cyno ILT2 comprises amino acids 115-408 of SEQ ID NO:166.
  • D4-stem of cyno ILT2 comprises amino acids 312-461 of SEQ ID NO:166.
  • D3-D4-stem of cyno ILT2 comprises amino acids 221-461 of SEQ ID NO:166.
  • D2- D3-D4-stem of cyno ILT2 comprises amino acids 115-461 of SEQ ID NO:166.
  • a fragment of cyno ILT2 comprises the amino acid sequence of SEQ ID NO:168.
  • a fragment of cyno ILT2 comprises the amino acid sequence of SEQ ID NO:169.
  • a fragment of cyno ILT2 comprises the amino acid sequence of SEQ ID NO:170.
  • a fragment of cyno ILT2 comprises the amino acid sequence of SEQ ID NO:171.
  • a fragment of cyno ILT2 comprises the amino acid sequence of SEQ ID NO:172.
  • a fragment of cyno ILT2 comprises the amino acid sequences of SEQ ID NO:169 and SEQ ID NO:170. In some embodiments, a fragment of human ILT2 comprises the amino acid sequence of SEQ ID NO:170 and SEQ ID NO:171. In some embodiments, a fragment of human ILT2 comprises the amino acid sequences of SEQ ID NO:171 and SEQ ID NO:172. In some embodiments, a fragment of human ILT2 comprises the amino acid sequences of SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:171.
  • a fragment of human ILT2 comprises the amino acid sequences of SEQ ID NO:170, SEQ ID NO:171, and SEQ ID NO:172.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent disclosed herein binds a fragment of ILT2 (e.g., human ILT2, rhesus ILT2, and/or cyno ILT2).
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds within a specific region of ILT2.
  • the ILT2-binding agent or the ILT2/ILT4- binding agent binds within the extracellular domain of ILT2.
  • the ILT2- binding agent or the ILT2/ILT4-binding agent binds within the D1 domain of ILT2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds within the D2 domain of ILT2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds within the D3 domain of ILT2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds within the D4 domain of ILT2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds within the D4-stem region of ILT2.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds within the D1-D2 domains of ILT2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4- binding agent binds within the D2-D3 domains of ILT2. In some embodiments, the ILT2- binding agent or the ILT2/ILT4-binding agent binds within the D3-D4 domains of ILT2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds within the D1-D2-D3 domains of ILT2.
  • the ILT2-binding agent or the ILT2/ILT4- binding agent binds within the D2-D3-D4 domains of ILT2. In some embodiments, the ILT2- binding agent or the ILT2/ILT4-binding agent binds an epitope on ILT2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a conformational epitope on ILT2. In some embodiments, the ILT2-binding agent does not bind other human LILRB proteins (e.g., ILT3, ILT4, ILT5, or LILRB5).
  • human LILRB proteins e.g., ILT3, ILT4, ILT5, or LILRB5
  • the ILT2/ILT4-binding agent does not bind other human LILRB proteins (e.g., ILT3, ILT5, or LILRB5). In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent does not bind one or more of the human LILRA proteins (e.g., LILRA1, LILRA2, LILRA4, LILRA5, or LILRA6). In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent does not bind LILRA2, LILRA4, LILRA5, or LILRA6.
  • human LILRB proteins e.g., ILT3, ILT5, or LILRB5
  • the ILT2-binding agent or the ILT2/ILT4-binding agent does not bind one or more of the human LILRA proteins (e.g., LILRA1, LILRA2, LILRA4, LILRA5, or LILRA6). In some embodiments, the ILT2-binding agent or the ILT
  • the ILT2-binding agent or the ILT2/ILT4-binding agent disclosed herein binds human ILT2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds cyno ILT2 and/or rhesus ILT2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds human ILT2, cyno ILT2, and rhesus ILT2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:1.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:3. In some embodiments, the ILT2-binding agent or the ILT2/ILT4- binding agent binds a fragment comprising amino acids 24-461 of SEQ ID NO:1. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 27-115 of SEQ ID NO:1.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 116-221 of SEQ ID NO:1. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 222-312 of SEQ ID NO:1. In some embodiments, the ILT2- binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 313- 409 of SEQ ID NO:1. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 27-221 of SEQ ID NO:1.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 116-312 of SEQ ID NO:1. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 222-409 of SEQ ID NO:1. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:4. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:5. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:6.
  • the ILT2-binding agent or the ILT2/ILT4- binding agent binds SEQ ID NO:7.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent disclosed herein binds SEQ ID NO:15.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:16.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:17.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 24-460 of SEQ ID NO:15.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 27-114 of SEQ ID NO:15. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 115-220 of SEQ ID NO:15. In some embodiments, the ILT2-binding agent or the ILT2/ILT4- binding agent binds a fragment comprising amino acids 221-311 of SEQ ID NO:15. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 312-408 of SEQ ID NO:15.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 27-220 of SEQ ID NO:15. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 115-311 of SEQ ID NO:15. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 221-408 of SEQ ID NO:15. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:18.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:19. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:20. In some embodiments, the ILT2- binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:21. [00168] In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent disclosed herein binds SEQ ID NO:166. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:167.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:168. In some embodiments, the ILT2- binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 24-461 of SEQ ID NO:166. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 27-114 of SEQ ID NO:166. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 115-220 of SEQ ID NO:166.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 221-311 of SEQ ID NO:166. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 312-408 of SEQ ID NO:166. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 27-220 of SEQ ID NO:166. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 115-311 of SEQ ID NO:166.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 221-408 of SEQ ID NO:166. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:169. In some embodiments, the ILT2- binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:170. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:171. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:172.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:5.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:17.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:19. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:21.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:167. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:168. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:169. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:170.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:171. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:172. [00170] In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent disclosed herein binds an epitope comprising amino acids within SEQ ID NO:2. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:3.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:4. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:5. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:6. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:7.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:16 In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:17. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:18. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:19.
  • the ILT2- binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:20. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:21. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:167 In some embodiments, the ILT2-binding agent or the ILT2/ILT4- binding agent binds an epitope comprising amino acids within SEQ ID NO:168.
  • the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:169. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:170. In some embodiments, the ILT2-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:171. In some embodiments, the ILT2- binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:172.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds ILT4 or a fragment of ILT4.
  • a fragment of ILT4 comprises the extracellular domain of ILT4.
  • a fragment of ILT4 comprises one or more of the Ig-like C2 type domains (e.g., D1, D2, D3, and/or D4).
  • a fragment of ILT4 comprises D1 and D2.
  • a fragment of ILT4 comprises D2 and D3.
  • a fragment of ILT4 comprises D3 and D4.
  • a fragment of ILT4 comprises D1, D2, and D3.
  • a fragment of ILT4 comprises D2, D3, and D4. In some embodiments, a fragment of ILT4 comprises one or more of the Ig-like C2 type domains and the stem region. In some embodiments, a fragment of ILT4 comprises D4-stem, D3-D4-stem, or D2-D3-D4-stem. In some embodiments, the extracellular domain of human ILT4 comprises amino acids 22-461 of SEQ ID NO:8. In some embodiments, D1 of human ILT4 comprises amino acids 27-110 of SEQ ID NO:8. In some embodiments, D2 of human ILT4 comprises amino acids 111-229 of SEQ ID NO:8.
  • D3 of human ILT4 comprises amino acids 230-318 of SEQ ID NO:8. In some embodiments, D4 of human ILT4 comprises amino acids 330-419 of SEQ ID NO:8. In some embodiments, D1-D2 of human ILT4 comprises amino acids 27-229 of SEQ ID NO:8. In some embodiments, D2-D3 of human ILT4 comprises amino acids 111-318 of SEQ ID NO:8. In some embodiments, D3- D4 of human ILT4 comprises amino acids 230-419 of SEQ ID NO:8. In some embodiments, D1-D2-D3 of human ILT4 comprises amino acids 27-318 of SEQ ID NO:8.
  • D2-D3-D4 of human ILT4 comprises amino acids 111-419 of SEQ ID NO:8. In some embodiments, D4-stem of human ILT4 comprises amino acids 330-461 of SEQ ID NO:8. In some embodiments, D3-D4-stem of human ILT4 comprises amino acids 230-461 of SEQ ID NO:8. In some embodiments, D2-D3-D4-stem of human ILT4 comprises amino acids 111-461 of SEQ ID NO:8. [00172] In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds a fragment of ILT4.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds within a specific region of ILT4. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds within the extracellular domain of ILT4. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds within the D1 domain of ILT4. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds within the D2 domain of ILT4. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds within the D3 domain of ILT4.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds within the D4 domain of ILT4. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds within the D4-stem region of ILT4. In some embodiments, the ILT4-binding agent or the ILT2/ILT4- binding agent binds within the D1-D2 domains of ILT4. In some embodiments, the ILT4- binding agent or the ILT2/ILT4-binding agent binds within the D2-D3 domains of ILT4.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds within the D3-D4 domains of ILT4. In some embodiments, the ILT4-binding agent or the ILT2/ILT4- binding agent binds within the D1-D2-D3 domains of ILT4. In some embodiments, the ILT4- binding agent or the ILT2/ILT4-binding agent binds within the D2-D3-D4 domains of ILT4. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds an epitope on ILT4.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds a conformational epitope on ILT4.
  • the ILT4-binding agent does not bind other human LILRB proteins (e.g., ILT2, ILT3, ILT5, or LILRB5).
  • the ILT2/ILT4-binding agent does not bind other human LILRB proteins (e.g., ILT3, ILT5, or LILRB5).
  • the ILT4-binding agent or the ILT2/ILT4-binding agent does not bind one or more of the human LILRA proteins (e.g., LILRA1, LILRA2, LILRA4, LILRA5, or LILRA6). In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent does not bind LILRA2, LILRA4, LILRA5, or LILRA6. [00173] In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds human ILT4. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:8.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:9. In some embodiments, the ILT4-binding agent or the ILT2/ILT4- binding agent binds SEQ ID NO:10. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 22-461 of SEQ ID NO:8. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 27-110 of SEQ ID NO:8.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 111-229 of SEQ ID NO:8. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 230-318 of SEQ ID NO:8. In some embodiments, the ILT4- binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 330- 419 of SEQ ID NO:8. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 27-229 of SEQ ID NO:8.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 111-318 of SEQ ID NO:8. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds a fragment comprising amino acids 230-419 of SEQ ID NO:8. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:11. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:12.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:13. In some embodiments, the ILT4-binding agent or the ILT2/ILT4- binding agent binds SEQ ID NO:14. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:11 and SEQ ID NO:12. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:12 and SEQ ID NO:13.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:13 and SEQ ID NO:14. In some embodiments, the ILT4-binding agent or the ILT2/ILT4- binding agent binds SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. [00174] In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:9.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:11. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:12. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:13.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:14. [00175] In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:9. In some embodiments, the ILT4- binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:10. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:11.
  • the ILT4-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:12. In some embodiments, the ILT4-binding agent or the ILT2/ILT4- binding agent binds an epitope comprising amino acids within SEQ ID NO:13. In some embodiments, the ILT4-binding agent or the ILT2/ILT4-binding agent binds an epitope comprising amino acids within SEQ ID NO:14.
  • the ILT-binding agent binds human ILT2, human ILT4, or both human ILT2/ILT4, and has at least one or more of the following properties: (i) binding rhesus ILT2; (ii) binding cyno ILT2; (iii) not binding ILT3, ILT5, and LILRB5; (iv) not binding LILRA2, LILRA4, LILRA5, and LILRA6; (v) being an ILT2 antagonist; (vi) being an ILT4 antagonist, (vii) inhibiting ILT2 activity; (viii) inhibiting ILT4 activity; (ix) inhibiting ILT2 signaling in cells that express ILT2; (x) inhibiting ILT4 signaling in cells that express ILT4; (xi) inhibiting binding of ILT2 to MHC I molecules; (xii) inhibiting binding of ILT4 to MHC I molecules; (xiii) inhibiting ILT2-induced suppression of myeloid cells; (xiv) inhibit
  • the ILT-binding agent is an antibody. In some embodiments, the ILT2-binding agent is an antibody. In some embodiments, the ILT4-binding agent is an antibody. In some embodiments, the ILT2/ILT4-binding agent is an antibody. In some embodiments, the ILT-binding agent is an anti-ILT2 antibody. In some embodiments, the ILT- binding agent is an anti-ILT4 antibody. In some embodiments, the ILT-binding agent is an anti- ILT2/ILT4 antibody. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody.
  • the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody comprises an IgG heavy chain. In some embodiments, the antibody comprises an IgG1 heavy chain. In some embodiments, the antibody comprises an IgG2 heavy chain. In some embodiments, the antibody comprises an IgG4 heavy chain. In some embodiments, the antibody comprises a human IgG heavy chain.
  • the antibody comprises a human IgG1 heavy chain. In some embodiments, the antibody comprises a human IgG2 heavy chain. In some embodiments, the antibody comprises a human IgG4 heavy chain. In some embodiments, the antibody comprises a kappa light chain. In some embodiments, the antibody comprises a kappa light chain constant region. In some embodiments, the antibody comprises a human kappa light chain constant region. In some embodiments, the antibody comprises a lambda light chain. In some embodiments, the antibody comprises a lambda light chain constant region. In some embodiments, the antibody comprises a human lambda light chain constant region. In some embodiments, the antibody is an antibody fragment comprising at least one antigen-binding site.
  • the antibody is a scFv. In some embodiments, the antibody is a disulfide-linked scFv. In some embodiments, the antibody is a disulfide-linked sc(Fv) 2 . In some embodiments, the antibody is a Fab, Fab′, or a F(ab) 2 antibody. In some embodiments, the antibody is a diabody. In some embodiments, the antibody is a nanobody. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a bivalent antibody.
  • the antibody is a tetravalent antibody.
  • the antibody is isolated. In some embodiments, the antibody is substantially pure.
  • the ILT-binding agent e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4 binding agent
  • Polyclonal antibodies can be prepared by any method known to those of skill in the art.
  • polyclonal antibodies are produced by immunizing an animal (e.g., a rabbit, rat, mouse, goat, donkey) with an antigen of interest (e.g., a purified peptide fragment, a recombinant protein, or a fusion protein) using multiple subcutaneous or intraperitoneal injections.
  • an antigen of interest e.g., a purified peptide fragment, a recombinant protein, or a fusion protein
  • the antigen is conjugated to a carrier such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.
  • the antigen (with or without a carrier protein) is diluted in sterile saline and usually combined with an adjuvant (e.g., Complete or Incomplete Freund's Adjuvant) to form a stable emulsion.
  • an adjuvant e.g., Complete or Incomplete Freund's Adjuvant
  • polyclonal antibodies are recovered from the immunized animal (e.g., from blood or ascites).
  • the polyclonal antibodies are purified from serum or ascites according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and/or dialysis.
  • the ILT-binding agent e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4 binding agent
  • the ILT-binding agent is a monoclonal antibody.
  • Monoclonal antibodies can be prepared by any method known to those of skill in the art.
  • monoclonal antibodies are prepared using hybridoma methods known to one of skill in the art. For example, using a hybridoma method, a mouse, rat, rabbit, hamster, or other appropriate host animal, is immunized as described above.
  • lymphocytes are immunized in vitro.
  • the immunizing antigen is a human protein or a fragment thereof.
  • the immunizing antigen is a mouse protein or a fragment thereof. In some embodiments, the immunizing antigen is a rhesus protein or a fragment thereof. In some embodiments, the immunizing antigen is a cyno protein or a fragment thereof. In some embodiments, the immunizing antigen is a combination of two or more (e.g., 2, 3, 4) related proteins or fragments thereof.
  • lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. The hybridoma cells are selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process.
  • Hybridomas that produce monoclonal antibodies against a chosen antigen can be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay).
  • in vitro binding assays e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay.
  • the clones may be subcloned by limiting dilution techniques.
  • high-throughput methods are used to distribute single cell hybridoma cells into plates.
  • the hybridomas can be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal.
  • the monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.
  • monoclonal antibodies are made using recombinant DNA techniques as known to one skilled in the art.
  • the polynucleotides encoding an antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using standard techniques.
  • recombinant monoclonal antibodies are isolated from phage display libraries expressing variable domains or CDRs of a desired species. Screening of phage libraries can be accomplished by various techniques known in the art.
  • a monoclonal antibody is modified by using recombinant DNA technology to generate alternative antibodies.
  • the constant domains of the light chain and heavy chain of a mouse monoclonal antibody are substituted for constant regions of a human antibody to generate a chimeric antibody.
  • the constant regions are truncated or removed to generate a desired antibody fragment of a monoclonal antibody.
  • site-directed or high-density mutagenesis of the variable region(s) is used to optimize specificity and affinity of a monoclonal antibody.
  • the ILT-binding agent e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4 binding agent
  • the ILT-binding agent is a humanized antibody.
  • Various methods for generating humanized antibodies are known in the art.
  • a humanized antibody comprises one or more amino acid residues that have been introduced into it from a source that is non-human.
  • humanization is performed by substituting one or more non-human CDR sequences for the corresponding CDR sequences of a human antibody.
  • the humanized antibodies are constructed by substituting all six CDRs of a non-human antibody (e.g., a mouse antibody) for the corresponding CDRs of a human antibody.
  • the “best-fit” method is used where the sequence of the variable region of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable region sequences.
  • the human sequence that is most similar to that of the non-human (e.g., rodent) sequence is selected as the human variable region framework for the humanized antibody.
  • a particular variable region framework derived from a consensus sequence of all human antibodies of a particular subgroup of light or heavy chains is selected as the variable region framework.
  • the variable region framework sequence is derived from the consensus sequences of the most abundant human subclasses.
  • human germline genes are used as the source of the variable region framework sequences.
  • the ILT-binding agent e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4 binding agent
  • Human antibodies can be prepared using various techniques known in the art.
  • human antibodies are generated from immortalized human B lymphocytes immunized in vitro.
  • human antibodies are generated from lymphocytes isolated from an immunized individual.
  • cells that produce an antibody directed against a target antigen can be generated and isolated.
  • a human antibody is selected from a phage library, where that phage library expresses human antibodies.
  • phage display technology may be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable region gene repertoires from unimmunized human donors. Techniques for the generation and use of antibody phage libraries are well known in the art.
  • affinity maturation strategies known in the art, including but not limited to, chain shuffling and site-directed mutagenesis, may be employed to generate higher affinity human antibodies.
  • human antibodies are produced in transgenic mice that contain human immunoglobulin loci. Upon immunization these mice are capable of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • the ILT-binding agent e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4 binding agent
  • the ILT-binding agent is an antibody fragment.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an antibody and generally at least one antigen-binding site.
  • antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv, single chain antibody molecules (e.g., scFv), disulfide-linked scFv (dsscFv), nanobodies, diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies (e.g., camelid antibodies), and multispecific antibodies formed from antibody fragments.
  • the ILT-binding agent is a scFv antibody.
  • the scFv is a disulfide-linked scFv (dsscFv), which is a scFv comprising an engineered disulfide bond between the light chain variable region and heavy chain variable region of the scFv.
  • the disulfide bond increases stability of the scFv molecule.
  • the disulfide bond increases thermostability of the scFv molecule.
  • the ILT-binding agent is a Fv.
  • the ILT- binding agent is a Fab.
  • the ILT-binding agent is a F(ab′)2.
  • the ILT-binding agent is a F(ab′).
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody. The antibody fragments described herein can be produced using recombinant technologies known in the art (e.g., E.coli or phage expression).
  • the ILT-binding agent e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4 binding agent
  • the ILT-binding agent is a bispecific antibody. Bispecific antibodies are capable of recognizing and binding at least two different antigens or epitopes.
  • the different epitopes can either be within the same molecule (e.g., two epitopes on ILT2) or on different molecules (e.g., one epitope on ILT2 and a second epitope on a different target).
  • a bispecific antibody has enhanced potency as compared to an individual antibody or to a combination of more than one antibody.
  • a bispecific antibody has reduced toxicity as compared to an individual antibody or to a combination of more than one antibody. It is known to those of skill in the art that any therapeutic agent may have unique pharmacokinetics (PK) (e.g., circulating half-life).
  • PK pharmacokinetics
  • a bispecific antibody has the ability to synchronize the PK of two active binding agents wherein the two individual binding agents have different PK profiles.
  • a bispecific antibody has the ability to concentrate the actions of two agents in a common area (e.g., tissue) in a subject.
  • a bispecific antibody has the ability to concentrate the actions of two agents to a common target (e.g., a specific cell type).
  • a bispecific antibody has the ability to target the actions of two agents to more than one biological pathway or function.
  • a bispecific antibody has the ability to target two different cells and bring them closer together. [00194]
  • a bispecific antibody has decreased toxicity and/or side effects.
  • a bispecific antibody has decreased toxicity and/or side effects as compared to a mixture of the two individual antibodies or the antibodies as single agents. In some embodiments, a bispecific antibody has an increased therapeutic index. In some embodiments, a bispecific antibody has an increased therapeutic index as compared to a mixture of the two individual antibodies or the antibodies as single agents. [00195] Many techniques for making bispecific antibodies are known to those skilled in the art. In some embodiments, a bispecific antibody comprises heavy chain constant regions with modifications in the amino acids that are part of the interface between the two heavy chains. These modifications are made to enhance heterodimer formation and generally reduce or eliminate homodimer formation. In some embodiments, the bispecific antibody is generated using a knobs-into-holes (KIH) strategy.
  • KIH knobs-into-holes
  • the bispecific antibody comprises variant hinge regions incapable of forming disulfide linkages between identical heavy chains (e.g., reduce homodimer formation). In some embodiments, the bispecific antibody comprises heavy chains with changes in amino acids that result in altered electrostatic interactions. In some embodiments, the bispecific antibodies comprise heavy chains with changes in amino acids that result in altered hydrophobic/hydrophilic interactions. [00196] Bispecific antibodies can be intact antibodies or antibody fragments comprising antigen-binding sites. [00197] In some embodiments, the ILT-binding agent is an antibody that binds ILT2. In some embodiments, the anti-ILT antibody binds human ILT2. In some embodiments, the anti-ILT antibody binds cyno ILT2.
  • the anti-ILT antibody binds rhesus ILT2. In some embodiments, the anti-ILT antibody binds human ILT2 and cyno ILT2. In some embodiments, the anti-ILT antibody binds human ILT2 and rhesus ILT2. In some embodiments, the anti-ILT antibody binds human ILT2, rhesus ILT2, and cyno ILT2. In some embodiments, the anti-ILT2 antibody binds an ILT2 epitope. In some embodiments, the anti- ILT2 antibody binds an ILT2 epitope within the extracellular domain of human ILT2.
  • an anti-ILT2 antibody binds an ILT2 epitope within the extracellular domain of cyno ILT2. In some embodiments, the anti-ILT2 antibody binds an ILT2 epitope within the extracellular domain of rhesus ILT2. In some embodiments, the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 24- 461 of SEQ ID NO:1. In some embodiments, the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 27-115 of SEQ ID NO:1.
  • the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 116-221 of SEQ ID NO:1. In some embodiments, the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 222-312 of SEQ ID NO:1. In some embodiments, the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 313-409 of SEQ ID NO:1.
  • the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:3. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:4. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:5. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:6. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:7.
  • the anti- ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 24-460 of SEQ ID NO:15. In some embodiments, the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 27-114 of SEQ ID NO:15. In some embodiments, the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 115-220 of SEQ ID NO:15.
  • the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 221-311 of SEQ ID NO:15. In some embodiments, the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 312-408 of SEQ ID NO:15. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:17. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:18.
  • the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:19. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:20. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:21. In some embodiments, the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 24-461 of SEQ ID NO:166.
  • the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 27-114 of SEQ ID NO:166. In some embodiments, the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 115-220 of SEQ ID NO:166. In some embodiments, the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 221-311 of SEQ ID NO:166.
  • the anti-ILT2 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 312-408 of SEQ ID NO:166. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:168. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:169. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:170. In some embodiments, the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:171.
  • the anti-ILT2 antibody binds an epitope comprising amino acids within SEQ ID NO:172. In some embodiments, the epitope is a conformational epitope. In some embodiments, the epitope is a linear epitope.
  • the ILT-binding agent is an antibody that binds ILT4. In some embodiments, the anti-ILT antibody binds human ILT4. In some embodiments, the anti-ILT4 antibody binds an ILT4 epitope. In some embodiments, the anti-ILT4 antibody binds an ILT4 epitope within the extracellular domain of human ILT4.
  • the anti-ILT4 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 22-461 of SEQ ID NO:8. In some embodiments, the anti-ILT4 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 27- 110 of SEQ ID NO:8. In some embodiments, the anti-ILT4 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 111-229 of SEQ ID NO:8.
  • the anti-ILT4 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 230-318 of SEQ ID NO:8. In some embodiments, the anti-ILT4 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 330-419 of SEQ ID NO:8. In some embodiments, the anti-ILT4 antibody binds an epitope comprising amino acids within SEQ ID NO:9. In some embodiments, the anti-ILT4 antibody binds an epitope comprising amino acids within SEQ ID NO:10.
  • the anti-ILT4 antibody binds an epitope comprising amino acids within SEQ ID NO:11. In some embodiments, the anti-ILT4 antibody binds an epitope comprising amino acids within SEQ ID NO:12. In some embodiments, the anti-ILT4 antibody binds an epitope comprising amino acids within SEQ ID NO:13. In some embodiments, the anti-ILT4 antibody binds an epitope comprising amino acids within SEQ ID NO:14. In some embodiments, the epitope is a conformational epitope. In some embodiments, the epitope is a linear epitope. [00199] In some embodiments, the ILT-binding agent is an antibody that binds ILT2 and ILT4.
  • an antibody that binds ILT2 and ILT4 comprises at least one antigen-binding site that binds an epitope on both ILT2 and ILT4, in contrast to a bispecific antibody which would comprise one antigen-binding site that binds an epitope on ILT2 and a second antigen-binding site that binds a different epitope on ILT4.
  • the anti-ILT2/ILT4 antibody binds human ILT2 and human ILT4.
  • anti-ILT2/ILT4 antibody binds human ILT2, human ILT4, cyno ILT2, and rhesus ILT2.
  • the anti-ILT2/ILT4 antibody binds an ILT2 epitope and ILT4 epitope. In some embodiments, the anti-ILT2/ILT4 antibody binds an epitope within the extracellular domain of human ILT2 and an epitope within the extracellular domain of human ILT4, wherein the ILT2 epitope and the ILT4 epitope are the same or essentially the same.
  • the anti-ILT2/ILT4 antibody binds an ILT2 epitope within the extracellular domain of human ILT2, an ILT4 epitope within the extracellular domain of human ILT4, an ILT2 epitope within the extracellular domain of cyno ILT2, and an ILT2 epitope within the extracellular domain of rhesus ILT2.
  • the anti-ILT2/ILT4 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 24-461 of SEQ ID NO:1 and the same or essentially the same epitope within amino acids 22-461 of SEQ ID NO:8.
  • the anti-ILT2/ILT4 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 27-115 of SEQ ID NO:1 and the same or essentially the same epitope within amino acids 27-110 of SEQ ID NO:8. In some embodiments, the anti-ILT2/ILT4 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 116-221 of SEQ ID NO:1 and the same or essentially the same epitope within amino acids 111-229 of SEQ ID NO:8.
  • the anti-ILT2/ILT4 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 222-312 of SEQ ID NO:1 and the same or essentially the same epitope within amino acids 230-318 of SEQ ID NO:8. In some embodiments, the anti-ILT2/ILT4 antibody binds an epitope comprising at least one amino acid (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9) within amino acids 313-409 of SEQ ID NO:1 and the same or essentially the same epitope within amino acids 330-419 of SEQ ID NO:8. In some embodiments, the epitope is a conformational epitope.
  • the epitope is a linear epitope.
  • the ILT-binding agent is an anti-ILT2 antibody, an anti-ILT4 antibody, or an anti-ILT2/ILT4 antibody described herein.
  • the ILT- binding agent is a variant of an anti-ILT2 antibody, an anti-ILT4 antibody, or an anti-ILT2/ILT4 antibody described herein.
  • a variant of an anti-ILT antibody comprises one to thirty amino acid substitutions.
  • a variant of the anti-ILT antibody comprises one to twenty-five amino acid substitutions.
  • a variant of the anti-ILT antibody comprises one to twenty amino acid substitutions.
  • a variant of the anti-ILT antibody comprises one to fifteen amino acid substitutions. In some embodiments, a variant of the anti-ILT antibody comprises one to ten amino acid substitutions. In some embodiments, a variant of the anti-ILT antibody comprises one to five amino acid substitutions. In some embodiments, the variant of the anti-ILT antibody comprises one to three amino acid substitutions. In some embodiments, the amino acid substitution(s) is in a CDR of the antibody. In some embodiments, the amino acid substitution(s) is not in a CDR of the antibody. In some embodiments, the amino acid substitution(s) is in a framework region of the antibody. In some embodiments, the amino acid substitution(s) is a conservative amino acid substitution.
  • CDRs of an antibody are defined using a variety of methods/systems by those skilled in the art. These systems and/or definitions have been developed and refined over a number of years and include Kabat, Chothia, IMGT, AbM, and Contact.
  • the Kabat definition is based on sequence variability and is commonly used.
  • the Chothia definition is based on the location of the structural loop regions.
  • the IMGT system is based on sequence variability and location within the structure of the variable domain.
  • the AbM definition is a compromise between Kabat and Chothia.
  • the Contact definition is based on analyses of the available antibody crystal structures.
  • An Exemplary system is a combination of Kabat and Chothia.
  • the anti-ILT antibody described herein comprises the six CDRs of antibody 27F9, 47C8, 48A5, 47H6, Hz47H6.v2, 51A1, 64A12, Hz64A12, 73C4, 73D1, or Hz73D1.v1 based on the Kabat definition. In some embodiments, the anti-ILT antibody described herein comprises the six CDRs of antibody 27F9, 47C8, 48A5, 47H6, Hz47H6.v2, 51A1, 64A12, Hz64A12, 73C4, 73D1, or Hz73D1.v1 based on the Chothia definition.
  • the anti-ILT antibody described herein comprises the six CDRs of antibody 27F9, 47C8, 48A5, 47H6, Hz47H6.v2, 51A1, 64A12, Hz64A12, 73C4, 73D1, or Hz73D1.v1 based on the AbM definition. In some embodiments, the anti-ILT antibody described herein comprises the six CDRs of antibody 27F9, 47C8, 48A5, 47H6, Hz47H6.v2, 51A1, 64A12, Hz64A12, 73C4, 73D1, or Hz73D1.v1 based on the IMGT definition.
  • the anti-ILT antibody described herein comprises the six CDRs of antibody 27F9, 47C8, 48A5, 47H6, Hz47H6.v2, 51A1, 64A12, Hz64A12, 73C4, 73D1, or Hz73D1.v1 based on the Contact definition. In some embodiments, the anti-ILT antibody described herein comprises the six CDRs of antibody 27F9, 47C8, 48A5, 47H6, Hz47H6.v2, 51A1, 64A12, Hz64A12, 73C4, 73D1, or Hz73D1.v1 based on the Exemplary definition.
  • the ILT-binding agent is an anti-ILT antibody (e.g., an anti- ILT2 antibody, an anti-ILT4 antibody, or an anti-ILT2/ILT4 antibody) that comprises one, two, three, four, five, and/or six CDRs of any one of the antibodies described herein.
  • the anti-ILT2 antibody comprises (i) a heavy chain variable region (VH) comprising one, two, and/or three heavy chain variable region CDRs (VH-CDRs) from Table 1, and/or (ii) a light chain variable region (VL) comprising one, two, and/or three light chain variable region CDRs (VL-CDRs) from Table 1.
  • the anti-ILT4 antibody comprises (i) a VH comprising one, two, and/or three VH-CDRs from Table 2, and/or (ii) a light chain variable region comprising one, two, and/or three VL-CDRs from Table 2. In some embodiments, the anti-ILT4 antibody comprises (i) a VH comprising one, two, and/or three VH- CDRs from Table 3, and/or (ii) a VL comprising one, two, and/or three VL-CDRs from Table 3.
  • the anti-ILT2/ILT4 antibody comprises (i) a VH comprising one, two, and/or three VH-CDRs from Table 4A or Table 4B, and/or (ii) a VL comprising one, two, and/or three VL-CDRs from Table 4A or Table 4B. In some embodiments, the anti-ILT2/ILT4 antibody comprises (i) a VH comprising one, two, and/or three VH-CDRs from Table 5, and/or (ii) a VL comprising one, two, and/or three VL-CDRs from Table 5.
  • the anti-ILT2/ILT4 antibody comprises (i) a VH comprising one, two, and/or three VH-CDRs from Table 6A or Table 6B, and/or (ii) a VL comprising one, two, and/or three VL-CDRs from Table 6A or Table 6B. In some embodiments, the anti-ILT2/ILT4 antibody comprises (i) a VH comprising one, two, and/or three VH-CDRs from Table 7, and/or (ii) a VL comprising one, two, and/or three VL-CDRs from Table 7.
  • the anti-ILT2/ILT4 antibody comprises (i) a VH comprising one, two, and/or three VH-CDRs from Table 8A or Table 8B, and/or (ii) a VL comprising one, two, and/or three VL-CDRs from Table 8A or Table 8B.
  • the anti-ILT2 antibody comprises (i) a VH comprising three VH-CDRs from Table 1, and (ii) a VL comprising three VL-CDRs from Table 1.
  • the anti-ILT4 antibody comprises (i) a VH comprising three VH-CDRs from Table 2, and (ii) a VL comprising three VL-CDRs from Table 2.
  • the anti-ILT4 antibody comprises (i) a VH comprising three VH-CDRs from Table 3, and (ii) a VL comprising three VL-CDRs from Table 3.
  • the anti-ILT2/ILT4 antibody comprises (i) a VH comprising three VH-CDRs from Table 4A, and (ii) a VL comprising three VL-CDRs from Table 4A.
  • the anti-ILT2/ILT4 antibody comprises (i) a VH comprising three VH-CDRs from Table 4B, and (ii) a VL comprising three VL-CDRs from Table 4B.
  • the anti-ILT2/ILT4 antibody comprises (i) a VH comprising three VH- CDRs from Table 5, and (ii) a VL comprising three VL-CDRs from Table 5. In some embodiments, the anti-ILT2/ILT4 antibody comprises (i) a VH comprising three VH-CDRs from Table 6A, and (ii) a VL comprising three VL-CDRs from Table 6A. In some embodiments, the anti-ILT2/ILT4 antibody comprises (i) a VH comprising three VH-CDRs from Table 6B, and (ii) a VL comprising three VL-CDRs from Table 6B.
  • the anti-ILT2/ILT4 antibody comprises (i) a VH comprising three VH-CDRs from Table 7, and (ii) a VL comprising three VL-CDRs from Table 7. In some embodiments, the anti-ILT2/ILT4 antibody comprises (i) a VH comprising three VH-CDRs from Table 8A, and (ii) a VL comprising three VL-CDRs from Table 8B. In some embodiments, the anti- ILT2/ILT4 antibody comprises (i) a VH comprising three VH-CDRs from Table 8B, and (ii) a VL comprising three VL-CDRs from Table 8B.
  • the ILT-binding agent (e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4 binding agent) comprises a VH-CDR1, a VH-CDR2, and a VH- CDR3 and/or a VL-CDR1, a VL-CDR2, and a VL-CDR3 from an antibody described herein.
  • the ILT-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH- CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 from an antibody described herein.
  • the ILT-binding agent comprises: (a) a VH comprising a VH-CDR1, a VH- CDR2, and a VH-CDR3; and (b) a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from an antibody described herein.
  • the ILT-binding agent e.g., the anti-ILT2 antibody, the anti- ILT4 antibody, or the anti-ILT2/ILT4 antibody
  • a CDR comprises one amino acid substitution. In some embodiments, a CDR comprises two amino acid substitutions. In some embodiments, a CDR comprises three amino acid substitutions. In some embodiments, a CDR comprises four amino acid substitutions. In some embodiments, the CDR is a VH-CDR1. In some embodiments, the CDR is a VH-CDR2. In some embodiment, the CDR is a VH-CDR3. In some embodiments, the CDR is a VL-CDR1. In some embodiments, the CDR is a VL-CDR2. In some embodiments, the CDR is a VL-CDR3. In some embodiments, the substitutions are made as part of a humanization process.
  • the substitutions are made as part of a germline humanization process. In some embodiments, the substitutions are made as part of an affinity maturation process. In some embodiments, the substitutions are made as part of an optimization process.
  • the ILT-binding agent e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4 binding agent
  • the ILT-binding agent comprises one or more VH-CDRs or VL-CDRs that have been modified to reduce deamidation within the CDR sequence.
  • Deamidation is a chemical reaction in which an amide functional group in the side chain of the amino acids asparagine (Asn or N) or glutamine (Gln or Q) is removed or converted to another functional group.
  • asparagine is converted to aspartic acid or isoaspartic acid and glutamine is converted to glutamic acid or polyglutamic acid.
  • deamidation may change the structure, function, and/or stability of a polypeptide, potentially resulting in decreased biological activity.
  • the VH-CDR1, VH-CDR2, and/or VH-CDR3 of an antibody described herein is modified to reduce deamidation.
  • the VL- CDR1, VL-CDR2, and/or VL-CDR3 of an antibody described herein is modified to reduce deamidation.
  • the ILT-binding agent e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4 binding agent
  • the ILT-binding agent comprises one or more VH-CDRs or VL-CDRs that have been modified to reduce isomerization.
  • Isomerization is a chemical process by which a compound is transformed into any of its isomeric forms, i.e., forms with the same chemical composition but with different structure or configuration and, potentially with different physical and chemical properties.
  • the VH-CDR1, VH- CDR2, and/or VH-CDR3 of an antibody described herein is modified to reduce isomerization.
  • the VL-CDR1, VL-CDR2, and/or VL-CDR3 is modified to reduce isomerization.
  • the ILT-binding agent e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4 binding agent
  • the ILT-binding agent comprises one or more VH-CDRs or VL-CDRs that have been modified to reduce oxidation.
  • Oxidation is a chemical process by which an oxygen is added to an atom, for example, methionine is converted to methionine sulfoxide by addition of an oxygen to the sulfur atom. Oxidation of one or more amino acids can potentially affect the physical and chemical properties of a protein. Studies have shown that oxidation of methionine (Met or M) within a CDR has the potential to impact antibody binding and/or stability. In some embodiments, the VH-CDR1, VH-CDR2, and/or VH-CDR3 of an antibody described herein is modified to reduce oxidation (e.g., methionine oxidation).
  • the VL-CDR1, VL-CDR2, and/or VL-CDR3 of an antibody described herein is modified to reduce oxidation (e.g., methionine oxidation).
  • the ILT2-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and/or a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 27F9, a humanized version thereof, or variants thereof.
  • the ILT2-binding agent comprises a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from antibody 27F9.
  • the ILT2-binding agent comprises a VL comprising a VL-CDR1, a VL- CDR2, and a VL-CDR3 from antibody 27F9
  • the ILT2-binding agent comprises: (a) a VH comprising a VH-CDR1, a VH-CDR2, a VH-CDR3; and (b) a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 27F9.
  • the ILT2-binding agent is a humanized version of antibody 27F9.
  • the ILT2-binding agent is a variant of antibody 27F9 or humanized 27F9.
  • the ILT2-binding agent comprises: (a) a VH comprising a VH- CDR1 comprising the amino acid sequence GFSLTNYGVS (SEQ ID NO:22), a VH-CDR2 comprising the amino acid sequence IIWGDGSTNYHSALIS (SEQ ID NO:23), and a VH- CDR3 comprising the amino acid sequence PNWDTYAMDF (SEQ ID NO:24), and a VL comprising a VL-CDR1 comprising the amino acid sequence RASQDISNFLN (SEQ ID NO:25), a VL-CDR2 comprising the amino acid sequence CTSKLHS (SEQ ID NO:26), and a VL-CDR3 comprising the amino acid sequence QQGNTLPPT (SEQ ID NO:27); (b) a VH comprising a VH-CDR1 comprising the amino acid sequence GFSLTNY (SEQ ID NO:28), a VH-CDR2 comprising the amino acid sequence W
  • the ILT2-binding agent comprises: (a) a VH comprising a VH- CDR1 comprising the amino acid sequence GFSLTNYGVS (SEQ ID NO:22), a VH-CDR2 comprising the amino acid sequence IIWGDGSTNYHSALIS (SEQ ID NO:23), and a VH- CDR3 comprising the amino acid sequence PNWDTYAMDF (SEQ ID NO:24), and/or (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASQDISNFLN (SEQ ID NO:25), a VL-CDR2 comprising the amino acid sequence CTSKLHS (SEQ ID NO:26), and a VL-CDR3 comprising the amino acid sequence QQGNTLPPT (SEQ ID NO:27).
  • the ILT2-binding agent comprises a VH comprising VH-CDR1 comprising the amino acid sequence GFSLTNYGVS (SEQ ID NO:22), a VH-CDR2 comprising the amino acid sequence IIWGDGSTNYHSALIS (SEQ ID NO:23), and a VH-CDR3 comprising the amino acid sequence PNWDTYAMDF (SEQ ID NO:24).
  • the ILT2-binding agent comprises a VL comprising a VL-CDR1 comprising the amino acid sequence RASQDISNFLN (SEQ ID NO:25), a VL-CDR2 comprising the amino acid sequence CTSKLHS (SEQ ID NO:26), and a VL-CDR3 comprising the amino acid sequence QQGNTLPPT (SEQ ID NO:27).
  • the ILT2-binding agent comprises: (a) a VH comprising VH-CDR1 comprising the amino acid sequence GFSLTNYGVS (SEQ ID NO:22), a VH-CDR2 comprising the amino acid sequence IIWGDGSTNYHSALIS (SEQ ID NO:23), and a VH-CDR3 comprising the amino acid sequence PNWDTYAMDF (SEQ ID NO:24), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASQDISNFLN (SEQ ID NO:25), a VL-CDR2 comprising the amino acid sequence CTSKLHS (SEQ ID NO:26), and a VL-CDR3 comprising the amino acid sequence QQGNTLPPT (SEQ ID NO:27).
  • the anti-ILT2 binding agent comprises a VH comprising the amino acid sequence of VH-CDR1, VH-CDR2, and VH-CDR3 of antibody 27F9 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:125 and a VL comprising the amino acid sequence of VL-CDR1, VL-CDR2, and VL-CDR3 of antibody 27F9 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:126.
  • the ILT2-binding agent comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:125. In some embodiments, the ILT2-binding agent comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:126. In some embodiments, the ILT2-binding agent comprises a VH comprising an amino acid sequence of SEQ ID NO:125. In some embodiments, the ILT2-binding agent comprises a VL comprising an amino acid sequence of SEQ ID NO:126.
  • the ILT2-binding agent comprises a VH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:125 and a VL having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:126. In some embodiments, the ILT2-binding agent comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:125 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:126. In some embodiments, the ILT2-binding agent comprises a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:125 and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:126.
  • the ILT2-binding agent comprises a VH comprising an amino acid sequence of SEQ ID NO:125 and a VL comprising an amino acid sequence of SEQ ID NO:126.
  • the ILT4-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and/or a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 47C8, a humanized version thereof, or variants thereof.
  • the ILT4-binding agent comprises a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from antibody 47C8.
  • the ILT4-binding agent comprises a VL comprising a VL-CDR1, a VL- CDR2, and a VL-CDR3 from antibody 47C8
  • the ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1, a VH-CDR2, a VH-CDR3; and (b) a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 47C8.
  • the ILT4-binding agent is a humanized version of antibody 47C8.
  • the ILT4-binding agent is a variant of antibody 47C8 or humanized 47C8.
  • the ILT4-binding agent comprises: (a) a VH comprising a VH- CDR1 comprising the amino acid sequence GYSFTGYYMH (SEQ ID NO:38), a VH-CDR2 comprising the amino acid sequence RVYPNNGDTSYNQKFKV (SEQ ID NO:39), and a VH- CDR3 comprising the amino acid sequence GATVVESLFAY (SEQ ID NO:40), and a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDNYGNNFLH (SEQ ID NO:41), a VL-CDR2 comprising the amino acid sequence RTSNLES (SEQ ID NO:42), and a VL-CDR3 comprising the amino acid sequence QQSNEDPYT (SEQ ID NO:43); (b) a VH comprising a VH- CDR1 comprising the amino acid
  • the ILT4-binding agent comprises: (a) a VH comprising a VH- CDR1 comprising the amino acid sequence GYSFTGYYMH (SEQ ID NO:38), a VH-CDR2 comprising the amino acid sequence RVYPNNGDTSYNQKFKV (SEQ ID NO:39), and a VH- CDR3 comprising the amino acid sequence GATVVESLFAY (SEQ ID NO:40), and/or (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDNYGNNFLH (SEQ ID NO:41), a VL-CDR2 comprising the amino acid sequence RTSNLES (SEQ ID NO:42), and a VL-CDR3 comprising the amino acid sequence QQSNEDPYT (SEQ ID NO:43).
  • the ILT4-binding agent comprises a VH comprising a VH-CDR1 comprising the amino acid sequence GYSFTGYYMH (SEQ ID NO:38), a VH-CDR2 comprising the amino acid sequence RVYPNNGDTSYNQKFKV (SEQ ID NO:39), and a VH-CDR3 comprising the amino acid sequence GATVVESLFAY (SEQ ID NO:40).
  • the ILT4- binding agent comprises a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDNYGNNFLH (SEQ ID NO:41), a VL-CDR2 comprising the amino acid sequence RTSNLES (SEQ ID NO:42), and a VL-CDR3 comprising the amino acid sequence QQSNEDPYT (SEQ ID NO:43).
  • the ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYSFTGYYMH (SEQ ID NO:38), a VH-CDR2 comprising the amino acid sequence RVYPNNGDTSYNQKFKV (SEQ ID NO:39), and a VH-CDR3 comprising the amino acid sequence GATVVESLFAY (SEQ ID NO:40), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDNYGNNFLH (SEQ ID NO:41), a VL-CDR2 comprising the amino acid sequence RTSNLES (SEQ ID NO:42), and a VL-CDR3 comprising the amino acid sequence QQSNEDPYT (SEQ ID NO:43).
  • the anti-ILT4 binding agent comprises a VH comprising the amino acid sequence of VH-CDR1, VH-CDR2, and VH-CDR3 of antibody 47C8 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:127 and a VL comprising the amino acid sequence of VL-CDR1, VL-CDR2, and VL-CDR3 of antibody 47C8 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:128.
  • the ILT4-binding agent comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:127. In some embodiments, the ILT4-binding agent comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:128. In some embodiments, the ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:127.
  • the ILT4-binding agent comprises a VL comprising the amino acid sequence of SEQ ID NO:128.
  • the ILT4-binding agent comprises a VH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:127 and a VL having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:128.
  • the ILT4-binding agent comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:127 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:128.
  • the ILT4-binding agent comprises a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:127 and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:128. In some embodiments, the ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:127 and a VL comprising the amino acid sequence of SEQ ID NO:128.
  • the ILT4-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and/or a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 48A5, a humanized version thereof, or variants thereof.
  • the ILT4-binding agent comprises a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from antibody 48A5.
  • the ILT4-binding agent comprises a VL comprising a VL-CDR1, a VL- CDR2, and a VL-CDR3 from antibody 48A5
  • the ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1, a VH-CDR2, a VH-CDR3; and (b) a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 48A5.
  • the ILT4-binding agent is a humanized version of antibody 48A5.
  • the ILT4-binding agent is a variant of antibody 48A5 or humanized 48A5.
  • the ILT4-binding agent comprises: (a) a VH comprising a VH- CDR1 comprising the amino acid sequence GYTFTNYGMN (SEQ ID NO:54), a VH-CDR2 comprising the amino acid sequence WINTYIGEPIYADDFKG (SEQ ID NO:55), and a VH- CDR3 comprising the amino acid sequence RSDYDGYAMDY (SEQ ID NO:56), and a VL comprising a VL-CDR1 comprising the amino acid sequence KSSQSLLYSGNQKNYLA (SEQ ID NO:57), a VL-CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO:58), and a VL-CDR3 comprising the amino acid sequence QQHDSYPT (SEQ ID NO:59); (b) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTNY (SEQ ID NO:60), a VH-
  • the ILT4-binding agent comprises: (a) a VH comprising a VH- CDR1 comprising the amino acid sequence GYTFTNYGMN (SEQ ID NO:54), a VH-CDR2 comprising the amino acid sequence WINTYIGEPIYADDFKG (SEQ ID NO:55), and a VH- CDR3 comprising the amino acid sequence RSDYDGYAMDY (SEQ ID NO:56), and/or (b) a VL comprising a VL-CDR1 comprising the amino acid sequence KSSQSLLYSGNQKNYLA (SEQ ID NO:57), a VL-CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO:58), and a VL-CDR3 comprising the amino acid sequence QQHDSYPT (SEQ ID NO:59).
  • the ILT4-binding agent comprises a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTNYGMN (SEQ ID NO:54), a VH-CDR2 comprising the amino acid sequence WINTYIGEPIYADDFKG (SEQ ID NO:55), and a VH- CDR3 comprising the amino acid sequence RSDYDGYAMDY (SEQ ID NO:56).
  • the ILT4-binding agent comprises a VL comprising a VL-CDR1 comprising the amino acid sequence KSSQSLLYSGNQKNYLA (SEQ ID NO:57), a VL-CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO:58), and a VL-CDR3 comprising the amino acid sequence QQHDSYPT (SEQ ID NO:59).
  • the ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTNYGMN (SEQ ID NO:54), a VH-CDR2 comprising the amino acid sequence WINTYIGEPIYADDFKG (SEQ ID NO:55), and a VH-CDR3 comprising the amino acid sequence RSDYDGYAMDY (SEQ ID NO:56), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence KSSQSLLYSGNQKNYLA (SEQ ID NO:57), a VL-CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO:58), and a VL-CDR3 comprising the amino acid sequence QQHDSYPT (SEQ ID NO:59).
  • the anti-ILT4 binding agent comprises a VH comprising the amino acid sequence of VH-CDR1, VH-CDR2, and VH-CDR3 of antibody 48A5 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:129 and a VL comprising the amino acid sequence of VL-CDR1, VL-CDR2, and VL-CDR3 of antibody 48A5 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:130.
  • the ILT4-binding agent comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:129. In some embodiments, the ILT4-binding agent comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:130. In some embodiments, the ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:129.
  • the ILT4-binding agent comprises a VL comprising the amino acid sequence of SEQ ID NO:130.
  • the ILT4-binding agent comprises a VH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:129 and a VL having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:130.
  • the ILT4-binding agent comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:129 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:130.
  • the ILT4-binding agent comprises a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:129 and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:130. In some embodiments, the ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:129 and a VL comprising the amino acid sequence of SEQ ID NO:130.
  • the ILT2/ILT4-binding agent (e.g., a dual binder) comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and/or a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 47H6, a humanized version thereof (e.g., Hz47H6.v2), or variants thereof.
  • the ILT2/ILT4-binding agent comprises a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from antibody 47H6 or antibody Hz47H6.v2.
  • the ILT2/ILT4-binding agent comprises a VL comprising a VL-CDR1, a VL- CDR2, and a VL-CDR3 from antibody 47H6 or antibody Hz47H6.v2.
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1, a VH-CDR2, a VH- CDR3; and (b) a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 47H6.
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1, a VH-CDR2, a VH-CDR3; and (b) a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody Hz47H6.v2.
  • the ILT2/ILT4-binding agent is a humanized version of antibody 47H6.
  • the ILT2/ILT4-binding agent is a variant of antibody 47H6.
  • the ILT2/ILT4-binding agent is a variant of antibody Hz47H6.v2.
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH- CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71) or DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and a VL comprising a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75); (b) a VH comprising a VH-CDR1 comprising
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH- CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71) or DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and/or (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75).
  • the ILT2/ILT4- binding agent comprises a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72).
  • the ILT2/ILT4- binding agent comprises a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72).
  • the ILT2/ILT4- binding agent comprises a VL comprising a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75).
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75).
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75).
  • the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of VH-CDR1, VH-CDR2, and VH-CDR3 of antibody 47H6 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:131 and a VL comprising the amino acid sequence of VL-CDR1, VL-CDR2, and VL-CDR3 of antibody 47H6 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:132.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:131. In some embodiments, the ILT2/ILT4-binding agent comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:132. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:131.
  • the ILT2/ILT4-binding agent comprises a VL comprising the amino acid sequence of SEQ ID NO:132.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:131 and a VL having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:132.
  • an ILT2/ILT4-binding agent comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:131 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:132.
  • the ILT2/ILT4-binding agent comprises a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:131 and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:132. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:131 and a VL comprising the amino acid sequence of SEQ ID NO:132.
  • the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of VH-CDR1, VH-CDR2, and VH-CDR3 of antibody Hz47H6.v2 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:133 and a VL comprising the amino acid sequence of VL-CDR1, VL-CDR2, and VL-CDR3 of antibody Hz47H6.v2 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:134.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:133. In some embodiments, the ILT2/ILT4-binding agent comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:134. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:133.
  • the ILT2/ILT4-binding agent comprises a VL comprising the amino acid sequence of SEQ ID NO:134.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:133 and a VL having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:134.
  • the ILT2/ILT4-binding agent comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:133 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:134.
  • the ILT2/ILT4-binding agent comprises a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:133 and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:134. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:133 and a VL comprising the amino acid sequence of SEQ ID NO:134.
  • the ILT2/ILT4-binding agent comprises a heavy chain comprising a VH-CDR1 comprising GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71) or DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and a light chain comprising a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75), wherein the heavy chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least
  • the ILT2/ILT4-binding agent comprises a heavy chain comprising a VH- CDR1 comprising GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71) or DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and a light chain comprising a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75), wherein the heavy chain comprises at least 95% identity to the amino acid sequence of SEQ ID NO:148, and wherein the light chain comprises at least 95% identity
  • the ILT2/ILT4- binding agent comprises (a) a heavy chain comprising the amino acids of SEQ ID NO:148 and (b) a light chain comprising a VL-CDR1 comprising RASGNIHNYLA (SEQ ID NO:73), a VL- CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75), wherein the light chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:149.
  • the ILT2/ILT4-binding agent comprises (a) a heavy chain comprising the amino acids of SEQ ID NO:148 and (b) a light chain comprising a VL-CDR1 comprising RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75).
  • the ILT2/ILT4-binding agent comprises (a) a heavy chain comprising a VH-CDR1 comprising GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71) or DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), wherein the heavy chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:148, and (b) a light chain comprising the amino acid sequence of SEQ ID NO:149.
  • a heavy chain comprising a VH-CDR1 comprising GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising
  • the ILT2/ILT4-binding agent comprises (a) a heavy chain comprising a VH-CDR1 comprising GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71) or DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and (b) a light chain comprising the amino acid sequence of SEQ ID NO:149.
  • a heavy chain comprising a VH-CDR1 comprising GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71) or DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid
  • the ILT2/ILT4-binding agent is an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:148 and a light chain comprising the amino acid sequence of SEQ ID NO:149. [00238] In some embodiments, the ILT2/ILT4-binding agent comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:148. In some embodiments, the ILT2/ILT4-binding agent comprises a light chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:149.
  • the ILT2/ILT4-binding agent comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:148 and a light chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:149.
  • the ILT2/ILT4-binding agent comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:148.
  • the ILT2/ILT4-binding agent comprises a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:149.
  • the ILT2/ILT4-binding agent comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:148 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:149. In some embodiments, the ILT2/ILT4-binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:148. In some embodiments, the ILT2/ILT4-binding agent comprises a light chain comprising the amino acid sequence of SEQ ID NO:149. In some embodiments, the ILT2/ILT4-binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:148 and a light chain comprising the amino acid sequence of SEQ ID NO:149.
  • the ILT2/ILT4-binding agent is an antibody that comprises a heavy chain of SEQ ID NO:148 and/or a light chain of SEQ ID NO:149. In some embodiments, the ILT2/ILT4- binding agent is an antibody that comprises a heavy chain of SEQ ID NO:148. In some embodiments, the ILT2/ILT4-binding agent is an antibody that comprises a light chain of SEQ ID NO:149. In some embodiments, the ILT2/ILT4-binding agent is an antibody that comprises a heavy chain of SEQ ID NO:148 and a light chain of SEQ ID NO:149.
  • the ILT2/ILT4-binding agent (e.g., a dual binder) comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and/or a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 51A1, a humanized version thereof, or variants thereof.
  • the ILT2/ILT4-binding agent comprises a VH comprising a VH-CDR1, a VH-CDR2, and a VH- CDR3 from antibody 51A1.
  • the ILT2/ILT4-binding agent comprises a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 51A1.
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1, a VH-CDR2, a VH-CDR3; and (b) a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 51A1.
  • the ILT2/ILT4-binding agent is a humanized version of antibody 51A1.
  • the ILT2/ILT4-binding agent is a variant of antibody 51A1 or humanized 51A1.
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH- CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYGTMYYYAMDY (SEQ ID NO:88), and a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91); (b)
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH- CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYGTMYYYAMDY (SEQ ID NO:88), and/or (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises a VH comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYGTMYYYAMDY (SEQ ID NO:88).
  • the ILT2/ILT4- binding agent comprises a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYGTMYYYAMDY (SEQ ID NO:88), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of VH-CDR1, VH-CDR2, and VH-CDR3 of antibody 51A1 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:135 and a VL comprising the amino acid sequence of VL-CDR1, VL-CDR2, and VL-CDR3 of antibody 51A1 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:136.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:135. In some embodiments, the ILT2/ILT4-binding agent comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:136. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:135.
  • the ILT2/ILT4-binding agent comprises a VL comprising the amino acid sequence of SEQ ID NO:136.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:135 and a VL having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:136.
  • the ILT2/ILT4-binding agent comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:135 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:136.
  • the ILT2/ILT4-binding agent comprises a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:135 and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:136. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:135 and a VL comprising the amino acid sequence of SEQ ID NO:136.
  • the ILT2/ILT4-binding agent (e.g., a dual binder) comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and/or a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 64A12, a humanized version thereof (e.g., Hz64A12), or variants thereof.
  • the ILT2/ILT4-binding agent comprises a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from antibody 64A12 or Hz64A12.
  • the ILT2/ILT4-binding agent comprises a VL comprising a VL-CDR1, a VL-CDR2, and a VL- CDR3 from antibody 64A12 or Hz64A12.
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1, a VH-CDR2, a VH-CDR3; and (b) a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 64A12 or Hz64A12.
  • the ILT2/ILT4-binding agent is a humanized version of antibody 64A12.
  • the ILT2/ILT4-binding agent is a variant of antibody 64A12. In some embodiments, the ILT2/ILT4-binding agent is a variant of antibody Hz64A12. [00246] In some embodiments, the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH- CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYDTMYYYAMDY (SEQ ID NO:102), and a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFIY (SEQ ID NO:103), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH- CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYDTMYYYAMDY (SEQ ID NO:102), and/or (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFIY (SEQ ID NO:103), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises a VH comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYDTMYYYAMDY (SEQ ID NO:102).
  • the ILT2/ILT4-binding agent comprises a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFIY (SEQ ID NO:103), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYDTMYYYAMDY (SEQ ID NO:102), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFIY (SEQ ID NO:103), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of VH-CDR1, VH-CDR2, and VH-CDR3 of antibody 64A12 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:137 and a VL comprising the amino acid sequence of VL-CDR1, VL-CDR2, and VL- CDR3 of antibody 64A12 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:138.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:137. In some embodiments, the ILT2/ILT4-binding agent comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:138. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:137.
  • the ILT2/ILT4-binding agent comprises a VL comprising the amino acid sequence of SEQ ID NO:138.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:137 and a VL having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:138.
  • the ILT2/ILT4-binding agent comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:137 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:138.
  • the ILT2/ILT4-binding agent comprises a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:137 and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:138. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:137 and a VL comprising the amino acid sequence of SEQ ID NO:138.
  • the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of VH-CDR1, VH-CDR2, and VH-CDR3 of antibody Hz64A12 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:139 and a VL comprising the amino acid sequence of VL-CDR1, VL-CDR2, and VL- CDR3 of antibody Hz64A12 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:140.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:139. In some embodiments, the ILT2/ILT4-binding agent comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:140. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:139.
  • the ILT2/ILT4-binding agent comprises a VL comprising the amino acid sequence of SEQ ID NO:140.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:139 and a VL having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:140.
  • the ILT2/ILT4-binding agent comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:139 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:140.
  • the ILT2/ILT4-binding agent comprises a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:139 and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:140. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:139 and a VL comprising the amino acid sequence of SEQ ID NO:140.
  • the ILT2/ILT4-binding agent comprises a heavy chain comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYDTMYYYAMDY (SEQ ID NO:102), and a light chain comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFIY (SEQ ID NO:103), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91), wherein the heavy chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least
  • the ILT2/ILT4-binding agent comprises a heavy chain comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYDTMYYYAMDY (SEQ ID NO:102), and a light chain comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFIY (SEQ ID NO:103), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91), wherein the heavy chain comprises at least 95% identity to the amino acid sequence of SEQ ID NO:152, and wherein the light chain comprises at least 95% identity to the amino acid sequence of
  • the ILT2/ILT4-binding agent comprises (a) a heavy chain comprising the amino acids of SEQ ID NO:152 and (b) a light chain comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFIY (SEQ ID NO:103), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91), wherein the light chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:153.
  • the ILT2/ILT4-binding agent comprises (a) a heavy chain comprising the amino acids of SEQ ID NO:152 and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFIY (SEQ ID NO:103), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises (a) a heavy chain comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYDTMYYYAMDY (SEQ ID NO:102), wherein the heavy chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:152, and (b) a light chain comprising the amino acid sequence of SEQ ID NO:153.
  • the ILT2/ILT4-binding agent comprises (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYDTMYYYAMDY (SEQ ID NO:102), and (b) a light chain comprising the amino acid sequence of SEQ ID NO:153.
  • the ILT2/ILT4-binding agent is an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:152 and a light chain comprising the amino acid sequence of SEQ ID NO:153. [00255] In some embodiments, the ILT2/ILT4-binding agent comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:152. In some embodiments, the ILT2/ILT4-binding agent comprises a light chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:153.
  • the ILT2/ILT4-binding agent comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:152 and a light chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:153.
  • the ILT2/ILT4-binding agent comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:152.
  • the ILT2/ILT4-binding agent comprises a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:153.
  • the ILT2/ILT4-binding agent comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:152 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:153. In some embodiments, the ILT2/ILT4-binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:152. In some embodiments, the ILT2/ILT4-binding agent comprises a light chain comprising the amino acid sequence of SEQ ID NO:153. In some embodiments, the ILT2/ILT4-binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:152 and a light chain comprising the amino acid sequence of SEQ ID NO:153.
  • the ILT2/ILT4-binding agent is an antibody that comprises a heavy chain of SEQ ID NO:152 and/or a light chain of SEQ ID NO:153. In some embodiments, the ILT2/ILT4- binding agent is an antibody that comprises a heavy chain of SEQ ID NO:152. In some embodiments, the ILT2/ILT4-binding agent is an antibody that comprises a light chain of SEQ ID NO:153. In some embodiments, the ILT2/ILT4-binding agent is an antibody that comprises a heavy chain of SEQ ID NO:152 and a light chain of SEQ ID NO:153.
  • the ILT2/ILT4-binding agent (e.g., a dual binder) comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and/or a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 73C4, a humanized version thereof, or variants thereof.
  • the ILT2/ILT4-binding agent comprises a VH comprising a VH-CDR1, a VH-CDR2, and a VH- CDR3 from antibody 73C4.
  • the ILT2/ILT4-binding agent comprises a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 73C4.
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1, a VH-CDR2, a VH-CDR3; and (b) a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 73C4.
  • the ILT2/ILT4-binding agent is a humanized version of antibody 73C4.
  • the ILT2/ILT4-binding agent is a variant of antibody 73C4 or humanized 73C4.
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH- CDR2 comprising the amino acid sequence NVNPNNGGTSYNQKFKG (SEQ ID NO:106), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH- CDR2 comprising the amino acid sequence NVNPNNGGTSYNQKFKG (SEQ ID NO:106), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and/or (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence NVNPNNGGTSYNQKFKG (SEQ ID NO:106), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107).
  • the ILT2/ILT4- binding agent comprises a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence NVNPNNGGTSYNQKFKG (SEQ ID NO:106), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of VH-CDR1, VH-CDR2, and VH-CDR3 of antibody 73C4 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:141 and a VL comprising the amino acid sequence of VL-CDR1, VL-CDR2, and VL-CDR3 of antibody 73C4 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:142.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:141. In some embodiments, the ILT2/ILT4-binding agent comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:142. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:141.
  • the ILT2/ILT4-binding agent comprises a VL comprising the amino acid sequence of SEQ ID NO:142.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:141 and a VL having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:142.
  • the ILT2/ILT4-binding agent comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:141 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:142.
  • the ILT2/ILT4-binding agent comprises a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:141 and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:142. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:141 and a VL comprising the amino acid sequence of SEQ ID NO:142.
  • the ILT2/ILT4-binding agent (e.g., a dual binder) comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and/or a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 73D1, a humanized version thereof (e.g., Hz73D1), or variants thereof.
  • the ILT2/ILT4-binding agent comprises a VH comprising a VH-CDR1, a VH- CDR2, and a VH-CDR3 from antibody 73D1 or antibody Hz73D1.v1.
  • the ILT2/ILT4-binding agent comprises a VL comprising a VL-CDR1, a VL-CDR2, and a VL- CDR3 from antibody 73D1 or antibody Hz73D1.v1.
  • the ILT2/ILT4- binding agent comprises: (a) a VH comprising a VH-CDR1, a VH-CDR2, a VH-CDR3; and (b) a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 73D1 or antibody Hz73D1.v1.
  • the ILT2/ILT4-binding agent is a humanized version of antibody 73D1.
  • the ILT2/ILT4-binding agent is a variant of antibody 73D1. In some embodiments, the ILT2/ILT4-binding agent is a variant of antibody Hz73D1.v1. [00263] In some embodiments, the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYIN (SEQ ID NO:111), a VH- CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYIN (SEQ ID NO:111), a VH- CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and/or (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYIN (SEQ ID NO:111), a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107).
  • the ILT2/ILT4- binding agent comprises a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYIN (SEQ ID NO:111), a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYIN (SEQ ID NO:111), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; a VL-CDR2 comprising the amino acid sequence FA
  • the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of VH-CDR1, VH-CDR2, and VH-CDR3 of antibody 73D1 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:143 and a VL comprising the amino acid sequence of VL-CDR1, VL-CDR2, and VL-CDR3 of antibody 73D1 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:142.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:143. In some embodiments, the ILT2/ILT4-binding agent comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:142. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:143.
  • the ILT2/ILT4-binding agent comprises a VL comprising the amino acid sequence of SEQ ID NO:142.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:143 and a VL having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:142.
  • the ILT2/ILT4-binding agent comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:143 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:142.
  • the ILT2/ILT4-binding agent comprises a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:143 and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:142. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:143 and a VL comprising the amino acid sequence of SEQ ID NO:142.
  • the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of VH-CDR1, VH-CDR2, and VH-CDR3 of antibody Hz73D1.v1 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:144 and a VL comprising the amino acid sequence of VL-CDR1, VL-CDR2, and VL-CDR3 of antibody Hz73D1.v1 and which has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:145.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:144. In some embodiments, the ILT2/ILT4-binding agent comprises a VL having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:145. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:144.
  • the ILT2/ILT4-binding agent comprises a VL comprising the amino acid sequence of SEQ ID NO:145.
  • the ILT2/ILT4-binding agent comprises a VH having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:144 and a VL having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:145.
  • the ILT2/ILT4-binding agent comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:144 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:145.
  • the ILT2/ILT4-binding agent comprises a VH having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:144 and a VL having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:145. In some embodiments, the ILT2/ILT4-binding agent comprises a VH comprising the amino acid sequence of SEQ ID NO:144 and a VL comprising the amino acid sequence of SEQ ID NO:145.
  • the ILT2/ILT4-binding agent comprises a VH comprising a VH-CDR1 comprising GYTFTDYYIN (SEQ ID NO:111), a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and a VL comprising a VL- CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL- CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91), wherein the VH comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% identity to the amino acid
  • the ILT2/ILT4-binding agent comprises a VH comprising a VH-CDR1 comprising GYTFTDYYIN (SEQ ID NO:111), a VH- CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91), wherein the VH comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% identity to the amino acid sequence of SEQ ID NO:111
  • the ILT2/ILT4-binding agent comprises a heavy chain comprising a VH-CDR1 comprising GYTFTDYYIN (SEQ ID NO:111), a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH- CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and a light chain comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91), wherein the heavy chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%
  • the ILT2/ILT4-binding agent comprises a heavy chain comprising a VH-CDR1 comprising GYTFTDYYIN (SEQ ID NO:111), a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and a light chain comprising a VL- CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL- CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91), wherein the heavy chain comprises at least 95% identity to the amino acid sequence of SEQ ID NO:156, and wherein the light chain comprises at least 95% identity to the amino acid sequence of SEQ ID NO:156
  • the ILT2/ILT4-binding agent comprises (a) a heavy chain comprising the amino acids of SEQ ID NO:156 and (b) a light chain comprising a VL-CDR1 comprising RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91), wherein the light chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:157.
  • the ILT2/ILT4-binding agent comprises (a) a heavy chain comprising the amino acids of SEQ ID NO:156 and (b) a light chain comprising a VL-CDR1 comprising RASESVDYYGNSFMY (SEQ ID NO:89), a VL- CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises: (a) a heavy chain comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYIN (SEQ ID NO:111), a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), wherein the heavy chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:156, and (b) a light chain comprising the amino acid sequence of SEQ ID NO:157.
  • the ILT2/ILT4-binding agent comprises: (a) a heavy chain comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYIN (SEQ ID NO:111), a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and (b) a light chain comprising the amino acid sequence of SEQ ID NO:157.
  • the ILT2/ILT4-binding agent is an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:156 and a light chain comprising the amino acid sequence of SEQ ID NO:157.
  • the ILT2/ILT4-binding agent comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:156.
  • the ILT2/ILT4-binding agent comprises a light chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:157.
  • the ILT2/ILT4-binding agent comprises a heavy chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:156 and a light chain having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:157.
  • the ILT2/ILT4-binding agent comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:156.
  • the ILT2/ILT4-binding agent comprises a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:157.
  • the ILT2/ILT4-binding agent comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:156 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:157. In some embodiments, the ILT2/ILT4-binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:156. In some embodiments, the ILT2/ILT4-binding agent comprises a light chain comprising the amino acid sequence of SEQ ID NO:157. In some embodiments, the ILT2/ILT4-binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:156 and a light chain comprising the amino acid sequence of SEQ ID NO:157.
  • the ILT2/ILT4-binding agent is an antibody that comprises a heavy chain of SEQ ID NO:156 and/or a light chain of SEQ ID NO:157. In some embodiments, the ILT2/ILT4- binding agent is an antibody that comprises a heavy chain of SEQ ID NO:156. In some embodiments, the ILT2/ILT4-binding agent is an antibody that comprises a light chain of SEQ ID NO:157. In some embodiments, the ILT2/ILT4-binding agent is an antibody that comprises a heavy chain of SEQ ID NO:156 and a light chain of SEQ ID NO:157. [00275] In some embodiments, the ILT2/ILT4-binding agent is antibody 73D1.
  • the ILT2/ILT4-binding agent is antibody Hz73D1.v1.
  • an agent competes with one or more of the antibodies described herein for binding to ILT2, ILT4, or both ILT2 and ILT4.
  • an agent that competes with one or more of the antibodies described herein is an antibody.
  • an agent binds the same epitope as one of the antibodies described herein.
  • an agent binds an epitope overlapping with an epitope bound by one of the antibodies described herein.
  • an agent competes for binding to human ILT2, ILT4, or both ILT2 and ILT4 with a reference antibody, wherein the reference antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYIN (SEQ ID NO:111), a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90
  • an agent competes for binding to human ILT2 with a reference antibody
  • the reference antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYIN (SEQ ID NO:111), a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • an agent competes for binding to human ILT2, ILT4, or both ILT2 and ILT4 with a reference antibody
  • the reference antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71) or DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and (b) a VL comprising a VL- CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (
  • an agent competes for binding to human ILT4 with a reference antibody, wherein the reference antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71) or DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75).
  • the reference antibody comprises: (a) a VH
  • the ILT-binding agent (e.g., the ILT2-binding agent, the ILT4, binding agent, or the ILT2/ILT4 binding agent) described herein comprises an antibody in which at least one or more of the constant regions of the antibody has been modified or deleted.
  • an antibody comprises one or more modifications to one or more of the three heavy chain constant regions (CH1, CH2 or CH3) and/or to the light chain constant region (CL).
  • an antibody comprises one or more modifications to the hinge region.
  • the heavy chain constant region of the modified antibody comprises at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody comprises more than one human constant region.
  • modifications to the constant region comprise additions, deletions, or substitutions of one or more amino acids in one or more regions.
  • one or more regions are partially or entirely deleted from the constant regions of a modified antibody.
  • the entire CH2 domain has been removed from an antibody ( ⁇ CH2 constructs).
  • one or more regions are partially or entirely deleted from the hinge region of a modified antibody.
  • a deleted constant region is replaced by a short amino acid spacer that provides some of the molecular flexibility typically imparted by the absent constant region.
  • a deleted hinge region is replaced by a short amino acid spacer that provides some of the molecular flexibility typically imparted by the absent hinge region.
  • a modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody. In some embodiments, a modified antibody comprises a peptide spacer inserted between the hinge region and modified CH2 and/or CH3 domains.
  • the constant region(s) of an antibody mediates several effector functions and these effector functions can vary depending on the isotype of the antibody. For example, binding of the C1 component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can be involved in autoimmune hypersensitivity.
  • the Fc region of an antibody can bind a Fc receptor (FcR) on the surface of a cell.
  • Fc receptor Fc receptor
  • IgG gamma receptors
  • IgE epsilon receptors
  • IgA alpha receptors
  • IgM mu receptors
  • Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.
  • ADCC antibody-dependent cell cytotoxicity
  • the ILT-binding agent comprises a variant Fc region.
  • the amino acid sequences of the Fc region of human IgG1, IgG2, IgG3, and IgG4 are known to those of ordinary skill in the art (e.g., a representative human IgG1 region is SEQ ID NO:158).
  • Fc regions with amino acid variations have been identified in native antibodies.
  • a variant Fc region is engineered with substitutions at specific amino acid positions as compared to a native Fc region.
  • variant Fc regions are well-known in the art and include, but are not limited to, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, and SEQ ID NO:163.
  • a modified antibody provides for altered effector functions that, in turn, affect the biological profile of the antibody.
  • the deletion or inactivation (through point mutations or other means) of a constant region reduces Fc receptor binding of a modified antibody as it circulates.
  • constant region modifications increase the serum half-life of an antibody.
  • constant region modifications reduce the serum half-life of an antibody.
  • constant region modifications decrease or remove ADCC and/or complement-dependent cytotoxicity (CDC) of an antibody.
  • specific amino acid substitutions in a human IgG1 Fc region with corresponding IgG2 or IgG4 residues reduce effector functions (e.g., ADCC and CDC) in a modified antibody.
  • a modified antibody does not have one or more effector functions.
  • a modified antibody does not have any detectable effector functions (e.g., “effectorless” antibodies).
  • a modified antibody has no ADCC activity and/or no CDC activity.
  • a modified antibody does not bind an Fc receptor and/or complement factors.
  • a modified antibody has no effector function(s).
  • constant region modifications increase or enhance ADCC and/or CDC of an antibody.
  • the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties.
  • the constant region is modified to add/substitute one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites.
  • Modifications to the constant region of antibodies described herein may be made using well-known biochemical or molecular engineering techniques.
  • antibody variants are prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide.
  • the present disclosure further embraces additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate biological properties of the antibody, including but not limited to, specificity, thermostability, expression level, effector function(s), glycosylation, immunogenicity, and/or solubility.
  • amino acid changes may alter post- translational processes of an antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.
  • Variations may be generated by the substitution, deletion, or insertion of one or more nucleotides into a polynucleotide encoding the antibody or polypeptide that results in a change in an amino acid or the amino acid sequence as compared with the native antibody or polypeptide sequence.
  • amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine (i.e., conservative amino acid replacements).
  • the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule.
  • variations in the amino acid sequence that are biologically useful and/or relevant are determined by systematically making insertions, deletions, or substitutions in the sequence and testing the resulting variant proteins for activity as compared to the parental antibody.
  • variants may include addition of amino acid residues at the amino- and/or carboxyl-terminal end of the antibody or polypeptide.
  • a variant comprises an N-terminal methionyl residue.
  • the variant comprises an additional polypeptide/protein to create a fusion protein.
  • a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g., a fluorescent tag, a fluorescent protein, or an enzyme).
  • a cysteine residue not involved in maintaining the proper conformation of an antibody is substituted or deleted to modulate the antibody’s characteristics, for example, to improve oxidative stability and/or prevent aberrant disulfide crosslinking.
  • an antibody of the present disclosure is “deimmunized”.
  • the deimmunization of antibodies generally consists of introducing specific amino acid mutations (e.g., substitutions, deletions, additions) that result in removal of T-cell epitopes (known or predicted) without significantly reducing the binding affinity or other desired activities of the antibody.
  • the variant antibodies or polypeptides described herein may be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.
  • the ILT-binding agent (e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4-binding agent) described herein is chemically modified.
  • the ILT-binding agent is (i) an anti-ILT2 antibody, (ii) an anti-ILT4 antibody, or (iii) an anti-ILT2/ILT4 antibody that is chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications may be carried out by known techniques.
  • the ILT-binding agent is the antibody fragment (e.g., scFv, Fv, Fab, F(ab′)2, or F(ab′)), wherein the antibody fragment is attached (either directly or indirectly) to a half-life extending moiety including, but not limited to, polyethylene glycol (PEG), a PEG mimetic, XTEN®, serum albumin, polysialic acid, N-(2-hydroxypropyl)methacrylamide, or dextran.
  • PEG polyethylene glycol
  • PEG mimetic e.g., a PEG mimetic
  • XTEN® e.g., XTEN®
  • serum albumin e.g., polysialic acid
  • N-(2-hydroxypropyl)methacrylamide e.g., N-(2-hydroxypropyl)methacrylamide
  • a non- immunoglobulin-based binding agent is an agent that competes with (i) an anti-ILT2 antibody, (ii) an anti-ILT4 antibody, and/or (iii) an anti-ILT2/ILT4 antibody described herein in a competitive binding assay.
  • alternative ILT-binding agents comprise a scaffold protein.
  • scaffold proteins can be assigned to one of three groups based on the architecture of their backbone (1) scaffolds consisting of ⁇ -helices; (2) small scaffolds with few secondary structures or an irregular architecture of ⁇ -helices and ⁇ -sheets; and (3) scaffolds consisting of predominantly ⁇ -sheets.
  • Scaffold proteins include, but are not limited to, anticalins, which are based upon the lipocalin scaffold; adnectins, which are based on the 10 th domain of human fibronectin type 3; affibodies, which are based on the B-domain in the Ig- binding region of Staphylococcus aureus protein A; darpins, which are based on ankyrin repeat domain proteins; fynomers, which are based on the SH3 domain of the human Fyn protein kinase; affitins, which are based on Sac7d from Sulfolobus acidocaldarius; affilins, which are based on human ⁇ -B-crystallin or human ubiquitin; avimers, which are based on the A-domains of membrane receptor proteins; knottins (cysteine knot miniproteins), which are based upon a stable 30-amino acid anti-parallel ⁇ -strand protein fold; and Kunitz domain inhibitor
  • the ILT2-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 shown in Table 1.
  • the ILT2-binding agent comprises an engineered scaffold protein comprising a VH-CDR1 comprising the amino acid sequence GFSLTNYGVS (SEQ ID NO:22), a VH-CDR2 comprising the amino acid sequence IIWGDGSTNYHSALIS (SEQ ID NO:23), a VH-CDR3 comprising the amino acid sequence PNWDTYAMDF (SEQ ID NO:24), a VL-CDR1 comprising the amino acid sequence RASQDISNFLN (SEQ ID NO:25), a VL-CDR2 comprising the amino acid sequence CTSKLHS (SEQ ID NO:26), and a VL-CDR3 comprising the amino acid sequence QQGNTLPPT (SEQ ID NO:27).
  • a VH-CDR1 comprising the amino acid sequence GFSLTNYGVS (SEQ ID NO:22), a VH-CDR2 comprising the amino acid sequence IIWGDGSTNYHSALIS (SEQ ID NO:23), a VH-CDR3 compris
  • the ILT2-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL- CDR1, a VL-CDR2, and a VL-CDR3 from antibody 27F9.
  • the ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 shown in Table 2.
  • the ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1 comprising the amino acid sequence GYSFTGYYMH (SEQ ID NO:38), a VH-CDR2 comprising the amino acid sequence RVYPNNGDTSYNQKFKV (SEQ ID NO:39), a VH-CDR3 comprising the amino acid sequence GATVVESLFAY (SEQ ID NO:40), a VL-CDR1 comprising the amino acid sequence RASESVDNYGNNFLH (SEQ ID NO:41), a VL-CDR2 comprising the amino acid sequence RTSNLES (SEQ ID NO:42), and a VL-CDR3 comprising the amino acid sequence QQSNEDPYT (SEQ ID NO:43).
  • the ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL- CDR1, a VL-CDR2, and a VL-CDR3 from antibody 47C8.
  • the ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 shown in Table 3.
  • the ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1 comprising the amino acid sequence GYTFTNYGMN (SEQ ID NO:54), a VH-CDR2 comprising the amino acid sequence WINTYIGEPIYADDFKG (SEQ ID NO:55), a VH-CDR3 comprising the amino acid sequence RSDYDGYAMDY (SEQ ID NO:56), a VL-CDR1 comprising the amino acid sequence KSSQSLLYSGNQKNYLA (SEQ ID NO:57), a VL-CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO:58), and a VL-CDR3 comprising the amino acid sequence QQHDSYPT (SEQ ID NO:59).
  • the ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL- CDR1, a VL-CDR2, and a VL-CDR3 from antibody 48A5.
  • the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 shown in Table 4A or Table 4B.
  • the ILT2/ILT4- binding agent comprises an engineered scaffold protein comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71) or DFNPNNAGTTYNQKFEG (SEQ ID NO:118), a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75).
  • a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN
  • VH-CDR2 comprising the amino acid sequence DFN
  • the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 47H6 or antibody Hz47H6.v2.
  • the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 shown in Table 5.
  • the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), a VH-CDR3 comprising the amino acid sequence DGIYYYGTMYYYAMDY (SEQ ID NO:88), a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH- CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 51A1.
  • the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 shown in Table 6A or Table 6B.
  • the ILT2/ILT4- binding agent comprises an engineered scaffold protein comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), a VH-CDR3 comprising the amino acid sequence DGIYYYDTMYYYAMDY (SEQ ID NO:102), a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFIY (SEQ ID NO:103), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 64A12. [00298] In some embodiments, the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 shown in Table 7.
  • the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence NVNPNNGGTSYNQKFKG (SEQ ID NO:106), a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH- CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 73C4. [00299] In some embodiments, the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 shown in Table 8A or Table 8B.
  • the ILT2/ILT4- binding agent comprises an engineered scaffold protein comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYIN (SEQ ID NO:111), a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the ILT2/ILT4-binding agent comprises an engineered scaffold protein comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 from antibody 73D1 or antibody Hz73D1.v1.
  • a composition comprises an ILT-binding agent described herein.
  • a composition comprises an ILT2-binding agent described herein.
  • a composition comprises an ILT4-binding agent described herein.
  • a composition comprises an ILT2/ILT4-binding agent described herein.
  • a composition comprises an anti-ILT2 antibody described herein. In some embodiments, a composition comprises a monoclonal anti-ILT2 antibody described herein. In some embodiments, a composition comprises an anti-ILT4 antibody described herein. In some embodiments, a composition comprises a monoclonal anti-ILT4 antibody described herein. In some embodiments, a composition comprises an anti-ILT2/ILT4 antibody described herein. In some embodiments, a composition comprises a monoclonal anti-ILT2/ILT4 antibody described herein.
  • a composition comprises an antibody selected from the group consisting of: antibody 27F9, antibody 47C8, antibody 48A5, antibody 47H6, antibody 51A1, antibody 64A12, antibody 73C4, or antibody 73D1, or humanized versions thereof.
  • a pharmaceutical composition comprises an ILT-binding agent described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises an ILT2-binding agent described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises an ILT4-binding agent described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises an ILT2/ILT4-binding agent described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises an anti-ILT2 antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, a composition comprises a monoclonal anti-ILT2 antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises an anti-ILT4 antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, a composition comprises a monoclonal anti-ILT4 antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises an anti-ILT2/ILT4 antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, a composition comprises a monoclonal anti-ILT2/ILT4 antibody described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises an antibody selected from the group consisting of: antibody 27F9, antibody 47C8, antibody 48A5, antibody 47H6, antibody 51A1, antibody 64A12, antibody 73C4, or antibody 73D1, or humanized versions thereof and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises antibody Hz73D1.v1 and a pharmaceutically acceptable carrier.
  • the ILT-binding agent is isolated. In some embodiments, the ILT-binding agent is substantially pure.
  • antigen-antibody interactions are non-covalent and reversible, formed by a combination of hydrogen bonds, hydrophobic interactions, and electrostatic and van der Waals forces.
  • K D is the ratio of an antibody dissociation rate (k off ) (how quickly it dissociates from its antigen) to the antibody association rate (k on ) (how quickly it binds to its antigen).
  • KD values are determined by measuring the kon and koff rates of a specific antibody/antigen interaction and then using a ratio of these values to calculate the K D value. K D values may be used to evaluate and rank order the strength of individual antibody/antigen interactions.
  • affinity is measured using SPR technology (e.g., using a Biacore system).
  • Avidity gives a measure of the overall strength of an antibody-antigen complex. It is dependent on three major parameters: (i) affinity of the antibody for the target, (ii) valency of both the antibody and antigen, and (iii) structural arrangement of the parts that interact.
  • the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a dissociation constant (KD) of 1 ⁇ M or less, 100 nM or less, 40 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. In some embodiments, the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a KD of about 20 nM or less.
  • KD dissociation constant
  • the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a K D of 10 nM or less. In some embodiments, the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a K D of 5 nM or less. In some embodiments, the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a KD of 3 nM or less. In some embodiments, the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a KD of 2 nM or less.
  • the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a KD of 1 nM or less. In some embodiments, the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a KD of 0.5 nM or less. In some embodiments, the ILT- binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a K D of 0.1 nM or less. In some embodiments, the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a K D of 50 pM or less.
  • the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a K D of 25 pM or less. In some embodiments, the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a K D of 10 pM or less. In some embodiments, the ILT- binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a K D of 1 pM or less. In some embodiments, the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a K D of 0.01 nM to 2.5 nM.
  • the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a KD of 0.1 nM to 5 nM. In some embodiments, the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a K D of 1 nM to 5 nM. In some embodiments, the dissociation constant of the binding agent for ILT2 and/or ILT4 is the dissociation constant determined using an ILT protein (e.g., ILT2 or ILT4) immobilized on a Biacore chip and the binding agent flowed over the chip.
  • an ILT protein e.g., ILT2 or ILT4
  • the dissociation constant of the binding agent for ILT2 and/or ILT4 is the dissociation constant determined using the binding agent captured by an anti-human IgG antibody on a Biacore chip and soluble ILT2 or ILT4 flowed over the chip.
  • the ILT-binding agent binds ILT2, ILT4, or both ILT2 and ILT4 with a half maximal effective concentration (EC50) of 1 ⁇ M or less, 100 nM or less, 40 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less.
  • the ILT-binding agent binds human ILT2, ILT4, or both ILT2 and ILT4 with an EC 50 of 1 ⁇ M or less, 100 nM or less, 40 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In some embodiments, the ILT-binding agent binds cyno or rhesus ILT2 with an EC50 of 40 nM or less, 20 nM or less, 10 nM or less, 1 nM or less or 0.1 nM or less.
  • the ILT-binding agent binds ILT2 and/or ILT4 with an EC50 of 0.1 nM to about 3 nM, 0.1 nM to 2 nM, 0.1 nM to 1 nM, 0.5 nM to 3 nM, 0.5 nM to 2 nM, or 0.5 nM to 1 nM.
  • the ILT-binding agent binds human ILT2 and human ILT4 and has at least one or more of the following properties: (i) binding rhesus ILT2/ILT4; (ii) binding cyno ILT2/4; (iii) not binding ILT3, ILT5, and LILRB5; (iv) not binding LILRA2, LILRA4, LILRA5, and LILRA6; (v) being an ILT2 antagonist; (vi) being an ILT4 antagonist, (vii) inhibits ILT2 activity; (viii) inhibiting ILT4 activity; (ix) inhibiting ILT2 signaling in cells that express ILT2; (x) inhibiting ILT4 signaling in cells that express ILT4; (xi) inhibiting binding of ILT2 to MHC I molecules; (xii) inhibiting binding of ILT4 to MHC I molecules; (xiii) inhibiting ILT2-induced suppression of myeloid cells; (xiv) inhibiting ILT
  • the ILT-binding agents (e.g., ILT2-binding agents, ILT4-binding agents, or ILT2/ILT4-binding agents) described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide functional variants thereof.
  • a DNA sequence encoding a polypeptide of interest is constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized.
  • oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated.
  • the individual oligonucleotides typically contain 5′ or 3′ overhangs for complementary assembly.
  • the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or expression of a biologically active polypeptide in a suitable host.
  • recombinant expression vectors are used to amplify and express DNA encoding the ILT-binding agents described herein.
  • recombinant expression vectors can be replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of the ILT-binding agent, such as an anti-ILT2/ILT4 antibody, or antigen-binding fragment thereof, operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral, or insect genes.
  • a transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters and/or enhancers, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences.
  • Regulatory elements can include an operator sequence to control transcription.
  • the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated.
  • DNA regions are “operatively linked” when they are functionally related to each other.
  • DNA for a signal peptide is operatively linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
  • structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • a polypeptide may include an N-terminal methionine residue.
  • This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • the choice of an expression control sequence and an expression vector generally depends upon the choice of host. A wide variety of expression host/vector combinations can be employed.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E.
  • the ILT-binding agent e.g., the ILT2-binding agent, the ILT4- binding agent, or the ILT2/ILT4-binding agent
  • the ILT-binding agent of the present disclosure is expressed from one or more vectors.
  • a VH is expressed by one vector and a VL is expressed by a second vector.
  • a VH and a VL are expressed by one vector.
  • a vector encodes a VH of an ILT-binding agent described herein.
  • a vector encodes a VL of an ILT-binding agent described herein. In some embodiments, a vector encodes a VH and a VL of an ILT-binding agent described herein. In some embodiments, a heavy chain polypeptide is expressed by one vector and a light chain polypeptide is expressed by a second vector. In some embodiments, a heavy chain polypeptide and a light chain polypeptide are expressed by one vector. In some embodiments, a vector encodes a heavy chain polypeptide of an ILT-binding agent described herein. In some embodiments, a vector encodes a light chain polypeptide of an ILT-binding agent described herein.
  • a vector encodes a heavy chain polypeptide and a light chain polypeptide of an ILT-binding agent described herein.
  • Suitable host cells for expression of the ILT-binding agent e.g., ILT2-binding agents, ILT4-binding agents, or ILT2/ILT4-binding agents
  • ILT2 or ILT4 protein or fragment thereof to use as an antigen or immunogen include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters.
  • Prokaryotes include gram- negative or gram-positive organisms, for example E. coli or Bacillus.
  • Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Cell-free translation systems may also be employed. Appropriate cloning vectors and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well known in the art. [00313] Various mammalian culture systems may be used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells may be desirable because these proteins are generally correctly folded, appropriately modified, and biologically functional.
  • suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney- derived), L-929 (murine fibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney-derived) cell lines and variants thereof.
  • Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5′ or 3′ flanking non-transcribed sequences, and 5′ or 3′ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5′ or 3′ flanking non-transcribed sequences, and 5′ or 3′ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • expression of recombinant proteins in insect cell culture systems e.g., baculovirus
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
  • the present disclosure provides cells comprising the ILT-binding agents described herein.
  • the cells produce the ILT-binding agents described herein.
  • the cells produce an antibody.
  • the cells produce an antibody that binds human ILT2.
  • the cells produce an antibody that binds human ILT4.
  • the cells produce an antibody that binds human ILT2 and ILT4.
  • the cells produce an antibody that binds human ILT2 and cyno ILT2.
  • the cells produce an anti-ILT2 antibody designated 27F9.
  • the cells produce an anti-ILT4 antibody designated 47C8.
  • the cells produce an anti-ILT4 antibody designated 48A5. In some embodiments, the cells produce an anti-ILT2/ILT4 antibody designated 47H6. In some embodiments, the cells produce a humanized anti-ILT2/ILT4 antibody designated Hz47H6.v2. In some embodiments, the cells produce an anti-ILT2/ILT4 antibody designated 51A1. In some embodiments, the cells produce an anti-ILT2/ILT4 antibody designated 64A12. In some embodiments, the cells produce a humanized anti-ILT2/ILT4 antibody designated Hz64A12. In some embodiments, the cells produce an anti-ILT2/ILT4 antibody designated 73C4. In some embodiments, the cells produce an anti-ILT2/ILT4 antibody designated 73D1.
  • the cells produce a humanized anti-ILT2/ILT4 antibody designated Hz73D1.v1.
  • the cell is a prokaryotic cell.
  • the cell is an eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is a hybridoma cell.
  • Proteins produced by a host cell can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • Affinity tags such as hexahistidine (His6; SEQ ID NO:173), maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column.
  • Affinity chromatography methods used for purifying immunoglobulins can include, but are not limited to, Protein A, Protein G, and Protein L chromatography. Isolated proteins can be physically characterized using techniques that include, but are not limited to, proteolysis, size exclusion chromatography (SEC), mass spectrometry (MS), nuclear magnetic resonance (NMR), isoelectric focusing (IEF), high performance liquid chromatography (HPLC), and x-ray crystallography.
  • the purity of isolated proteins can be determined using techniques known to those of skill in the art, including but not limited to, SDS-PAGE, SEC, capillary gel electrophoresis, IEF, and capillary isoelectric focusing (cIEF).
  • supernatants from expression systems that secrete recombinant protein into culture media are first concentrated using a commercially available protein concentration filter, for example, an Amicon® or Millipore Pellicon® ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix.
  • an anion exchange resin is employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification.
  • a cation exchange step is employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • a hydroxyapatite media is employed, including but not limited to, ceramic hydroxyapatite (CHT).
  • CHT ceramic hydroxyapatite
  • one or more reverse-phase HPLC steps employing hydrophobic RP- HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, are employed to further purify a recombinant protein.
  • hydrophobic interaction chromatography is used to separate recombinant proteins based on their hydrophobicity.
  • HIC is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions than some other techniques. Some or all of the foregoing purification steps, in various combinations, can be employed to provide a homogeneous recombinant protein.
  • ILT-binding agents e.g., ILT2-binding agents, ILT4-binding agents, or ILT2/ILT4- binding agents
  • ILT2/ILT4- binding agents may be analyzed for their physical/chemical properties and/or biological activities by various assays known in the art.
  • an anti- ILT2 antibody is tested for its ability to bind ILT2 (e.g., human ILT2 and/or cyno/rhesus ILT2).
  • an anti-ILT4 antibody is tested for its ability to bind ILT4 (e.g., human ILT4).
  • an anti-ILT2/ILT4 antibody is tested for its ability to bind ILT2 and ILT4 (e.g., human ILT2, human ILT4, and cyno/rhesus ILT2).
  • Binding assays include, but are not limited to, SPR (e.g., Biacore), ELISA, and FACS.
  • an anti-ILT2 antibody is tested for its ability to inhibit, reduce, or block ILT2 binding to MHC class I antigens.
  • an anti-ILT4 antibody is tested for its ability to inhibit, reduce, or block ILT4 binding to MHC class I antigens.
  • an anti-ILT2/ILT4 antibody is tested for its ability to inhibit, reduce, or block ILT2 and ILT4 binding to MHC class I antigens.
  • antibodies may be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency.
  • monoclonal antibodies generated against ILT2, ILT4, or ILT2 and ILT4 are grouped based upon the epitope each individual antibody recognizes, a process known as “epitope binning”.
  • epitope binning Generally, antibodies are tested in a pairwise combinatorial manner and antibodies that compete with each other are grouped together into bins. For example, in a premix binning assay, a first antibody is immobilized on a surface and a premixed solution of a second antibody and antigen is flowed over the immobilized first antibody. In tandem, the antigen is immobilized on a surface and the two antibodies are flowed over the immobilized antigen and compete to bind.
  • an epitope bin comprises at least one antibody from the group consisting of: 27F9, 47C8, 48A5, 47H6, 51A1, 64A12, 73C4, and 73D1.
  • an epitope bin comprises at least antibodies 27F9 and 73D1. In some embodiments, an epitope bin comprises at least antibodies 27F9, 73C4, and 73D1. In some embodiments, an epitope bin comprises at least antibodies 48A5 and 47H6. [00321] Epitope mapping is the process of identifying the binding site, or epitope on a target protein/antigen where an antibody (or other binding agent) binds. A variety of methods are known in the art for mapping epitopes on target proteins.
  • mutagenesis including but not limited to, shotgun mutagenesis, site-directed mutagenesis, and alanine scanning
  • domain or fragment scanning e.g., peptide scanning (e.g., Pepscan technology)
  • display methods including but not limited to, phage display, microbial display, and ribosome/mRNA display
  • methods involving proteolysis and mass spectroscopy e.g., methods involving amide hydrogen/deuterium exchange
  • structural determination including but not limited to, x-ray crystallography and NMR.
  • purified anti-ILT antibodies are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, differential scanning fluorimetry (DSF), nanoDSF, capillary isoelectric focusing (cIEF), ion exchange chromatography, and papain digestion.
  • assays including, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, differential scanning fluorimetry (DSF), nanoDSF, capillary isoelectric focusing (cIEF), ion exchange chromatography, and papain digestion.
  • in vitro assays that characterize immune cell function include, but are not limited to, cell activation assays (e.g., cell proliferation assays), cytotoxic T-cell (CTL) assays, T-cell suppression assays, MDSC assays, natural killer (NK) cell assays, mixed lymphocyte reaction (MLR) assays, cytokine/chemokine production assays, FcR binding assays, phagocytosis assays, and cell migration assays.
  • cell activation assays e.g., cell proliferation assays
  • CTL cytotoxic T-cell
  • T-cell suppression assays e.g., TTL
  • MDSC natural killer cell assays
  • MLR mixed lymphocyte reaction
  • cytokine/chemokine production assays e/chemokine production assays
  • FcR binding assays e.g., phagocytosis assays
  • phagocytosis assays e.g.
  • “Affect or affecting ILT activity” may include, for example, inhibiting, reducing, blocking, antagonizing, suppressing, and/or interfering with ILT2 activity, ILT4 activity, or ILT2 and ILT4 activity.
  • ILT2 and ILT4 generally act as negative regulator/inhibitory molecules, in some embodiments, inhibiting, reducing, blocking, antagonizing, suppressing, and/or interfering with ILT2 and/or ILT4 activity results in a blockade of ILT2-induced and/or ILT4-induced suppression of a biological function.
  • ILT2 is expressed on myeloid cells, such as monocytes, macrophages, dendritic cells (DCs), and APCs, as well as NK cells, B-cells, and CD8+ T-cells (CTLs).
  • myeloid cells such as monocytes, macrophages, dendritic cells (DCs), and APCs, as well as NK cells, B-cells, and CD8+ T-cells (CTLs).
  • ILT2 activity or ILT2 signaling activity includes, but is not limited to, suppression of myeloid cells, suppression of myeloid cell activity, suppression of tumor-associated myeloid cells, suppression of NK cells, and suppression of cytolytic T-cells (CTLs).
  • CTLs cytolytic T-cells
  • inhibiting, reducing, blocking, antagonizing, suppressing, and/or interfering with ILT2 activity results in a release of ILT2-induced suppression of an activation signal.
  • the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody disclosed herein inhibits ILT2 signaling.
  • the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody inhibits ILT2 signaling thereby reversing an ILT2-induced suppressive effect. In some embodiments, the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody inhibits an ILT2-induced extinction signal. In some embodiments, the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody increases myeloid cell activity. In some embodiments, the anti-ILT2 antibody or the anti- ILT2/ILT4 antibody increases APC activity. In some embodiments, the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody increases macrophage activity.
  • the anti- ILT2 antibody or the anti-ILT2/ILT4 antibody increases macrophage phagocytosis. In some embodiments, the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody increases NK cell activity. In some embodiments, the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody increases CTL activity. [00325] ILT4 is expressed on myeloid cells, such as monocytes, macrophages, dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), and APCs, as well as neutrophils and eosinophils.
  • myeloid cells such as monocytes, macrophages, dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), and APCs, as well as neutrophils and eosinophils.
  • ILT4 activity or ILT4 signaling activity includes, but is not limited to, suppression of myeloid cells, suppression of myeloid cell activity, and suppression of tumor-associated myeloid cells.
  • inhibiting, reducing, blocking, antagonizing, suppressing, and/or interfering with ILT4 activity results in a release of ILT4-induced suppression of an activation signal.
  • the anti-ILT4 antibody or the anti-ILT2/ILT4 antibody inhibits ILT4 signaling.
  • the anti-ILT4 antibody or the anti-ILT2/ILT4 antibody inhibits ILT4 signaling thereby reversing an ILT4-induced suppressive effect.
  • the anti-ILT4 antibody or the anti-ILT2/ILT4 antibody inhibits an ILT4-induced extinction signal.
  • the anti-ILT4 antibody or the anti-ILT2/ILT4 antibody increases myeloid cell activity.
  • the anti-ILT4 antibody or the anti- ILT2/ILT4 antibody increases macrophage activity.
  • the anti-ILT4 antibody or the anti-ILT2/ILT4 antibody decreases MDSCs.
  • the anti- ILT4 antibody or the anti-ILT2/ILT4 antibody decreases suppression by MDSCs.
  • the anti-ILT4 antibody or the anti-ILT2/ILT4 antibody induces a switch of MDSCs to activated macrophages.
  • the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway.
  • the anti-ILT2 antibody or the anti- ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and activates myeloid cells.
  • the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and activates APCs.
  • the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and activates dendritic cells. In some embodiments, the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and activates primary dendritic cells. In some embodiments, the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and increases NK cell activity. In some embodiments, the anti-ILT2 antibody or the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and increases CTL activity.
  • the anti-ILT4 antibody or the anti-ILT2/ILT4 antibody disrupts the ILT4 signaling pathway. In some embodiments, the anti-ILT4 antibody or the anti- ILT2/ILT4 antibody disrupts the ILT4 signaling pathway and activates myeloid cells. In some embodiments, the anti-ILT4 antibody or the anti-ILT2/ILT4 antibody disrupts the ILT4 signaling pathway and activates APCs. In some embodiments, the anti-ILT4 antibody or the anti-ILT2/ILT4 antibody disrupts the ILT4 signaling pathway and activates dendritic cells.
  • the anti-ILT4 antibody or the anti-ILT2/ILT4 antibody disrupts the ILT4 signaling pathway and activates primary dendritic cells.
  • the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and the ILT4 signaling pathway.
  • the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and the ILT4 signaling pathway and activates myeloid cells.
  • the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and the ILT4 signaling pathway and activates APCs.
  • the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and the ILT4 signaling pathway and activates dendritic cells. In some embodiments, the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and the ILT4 signaling pathway and activates primary dendritic cells. In some embodiments, the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and the ILT4 signaling pathway and increases myeloid cell activity. In some embodiments, the anti- ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and the ILT4 signaling pathway and increases NK cell activity.
  • the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and the ILT4 signaling pathway and increases CTL activity. In some embodiments, the anti-ILT2/ILT4 antibody disrupts the ILT2 signaling pathway and the ILT4 signaling pathway and decreases MDSC activity. [00329] In some embodiments, the terms “inhibiting”, “reducing”, “blocking”, “antagonizing”, “suppressing”, and “interfering” are relative to levels and/or activity in the absence of treatment with the ILT-binding agent.
  • the terms “inhibiting”, “reducing”, “blocking”, “antagonizing”, “suppressing”, and “interfering” are relative to levels and/or activity prior to treatment with the ILT-binding agent.
  • the terms “activating”, “promoting”, “increasing”, and “enhancing” are relative to levels and/or activity in the absence of treatment with the ILT- binding agent.
  • the terms “activating”, “promoting”, “increasing”, and “enhancing” are relative to levels and/or activity prior to treatment with the ILT-binding agent.
  • an anti-ILT2 antibody that inhibits ILT2 activity is antibody 27F9.
  • an anti-ILT4 antibody that inhibits ILT4 activity is antibody 47C8. In some embodiments, an anti-ILT4 antibody that inhibits ILT4 activity is antibody 48A5. In some embodiments, an anti-ILT2/ILT4 antibody that inhibits ILT2 and ILT4 activity is antibody 47H6. In some embodiments, an anti-ILT2/ILT4 antibody that inhibits ILT2 and ILT4 activity is antibody Hz47H6.v2. In some embodiments, an anti-ILT2/ILT4 antibody that inhibits ILT2 and ILT4 activity is antibody 51A1. In some embodiments, an anti-ILT2/ILT4 antibody that inhibits ILT2 and ILT4 activity is antibody 64A12.
  • an anti- ILT2/ILT4 antibody that inhibits ILT2 and ILT4 activity is antibody Hz64A12. In some embodiments, an anti-ILT2/ILT4 antibody that inhibits ILT2 and ILT4 activity is antibody 73C4. In some embodiments, an anti-ILT2/ILT4 antibody that inhibits ILT2 and ILT4 activity is antibody 73D1. In some embodiments, an anti-ILT2/ILT4 antibody that inhibits ILT2 and ILT4 activity is antibody Hz73D1.v1 [00332]
  • the present disclosure also provides conjugates comprising an anti-ILT2, an anti-ILT4, or an anti-ILT2/ILT4 antibody described herein. In some embodiments, the antibody is attached to a second molecule.
  • the antibody is conjugated to a cytotoxic agent or moiety. In some embodiments, the antibody is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate).
  • the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamycin/doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines (PBDs), or other intercalating agents.
  • the cytotoxic agent is a microtubule inhibitor including, but not limited to, auristatins, maytansinoids (e.g., DM1 and DM4), and tubulysins.
  • the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tric
  • an antibody is conjugated to one or more small molecule toxins, such as calicheamicins, maytansinoids, trichothenes, and CC1065.
  • a derivative of any one of these toxins may be used as long as the derivative retains the cytotoxic activity of the parent molecule.
  • Conjugates comprising an anti-ILT antibody (e.g., an ILT2 antibody, an ILT4 antibody, or an ILT2/ILT4 antibody) described herein may be made using any suitable method known in the art.
  • conjugates are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyridyidi
  • an anti-ILT antibody e.g., an anti-ILT2 antibody, an anti-ILT4 antibody, or an anti-ILT2/ILT4 antibody
  • a detectable substance or molecule that allows the antibody to be used for diagnosis and/or detection.
  • a labeled anti-ILT antibody is used to monitor immune cells in a tumor or in the microenvironment of a tumor.
  • a labeled anti-ILT antibody is used to monitor immune cells in a tumor or in the microenvironment of a tumor after treatment.
  • a detectable substance can include but is not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine(s); fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), and phycoerythrin; bioluminescent materials, such as luciferase; radioactive materials, such as 212 Bi, 14 C, 57 Co, 51 Cr, 67 Cu, 18 F, 68 Ga, 67 Ga, 153 Gd, 159 Gd, 68 Ge, 3 H, 166 Ho, 131 I, 125 I, 123 I, 121 I,
  • an anti-ILT antibody e.g., an anti-ILT2 antibody, an anti-ILT4 antibody, or an anti-ILT2/ILT4 antibody
  • an immunoassay is known to those of skill in the art and include, but are not limited to, ELISA, SPR (e.g., Biacore), FACS, and immunohistochemistry (IHC).
  • an anti- ILT antibody described herein is used on a tissue sample or a tumor sample.
  • An anti-ILT antibody (e.g., an anti-ILT2 antibody, an anti-ILT4 antibody, or an anti- ILT2/ILT4 antibody) described herein can also be conjugated to a second antibody to form an antibody heteroconjugate.
  • An anti-ILT antibody (e.g., an anti-ILT2 antibody, an anti-ILT4 antibody, or an anti- ILT2/ILT4 antibody) as described herein may be attached to a solid support.
  • Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • immobilized anti-ILT antibodies are used in immunoassays.
  • polynucleotides comprising polynucleotides that encode a polypeptide (e.g., an ILT-binding agent) described herein.
  • polynucleotides that encode a polypeptide encompasses a polynucleotide that includes only coding sequences for the polypeptide as well as a polynucleotide that includes additional coding and/or non-coding sequences.
  • the polynucleotides of the disclosure can be in the form of RNA or in the form of DNA.
  • DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.
  • the polynucleotide comprises a polynucleotide encoding a VH and/or a VL of an ILT2-binding agent described herein. In some embodiments, the polynucleotide comprises a polynucleotide encoding a VH of an ILT2-binding agent described herein.
  • the polynucleotide comprises a polynucleotide encoding a VL of an ILT2-binding agent described herein. In some embodiments, the polynucleotide comprises a polynucleotide encoding a VH of an ILT2-binding agent described herein and a polynucleotide encoding a VL of the ILT2-binding agent. In some embodiments, the polynucleotide comprises a polynucleotide encoding a VH and/or a VL of an ILT4-binding agent described herein.
  • the polynucleotide comprises a polynucleotide encoding a VL of an ILT4- binding agent described herein. In some embodiments, the polynucleotide comprises a polynucleotide encoding a VH of an ILT4-binding agent described herein. In some embodiments, the polynucleotide comprises a polynucleotide encoding a VH of an ILT4-binding agent described herein and a polynucleotide encoding a VL of the ILT4-binding agent.
  • the polynucleotide comprises a polynucleotide encoding a VH and/or a VL of an ILT2/ILT4-binding agent described herein. In some embodiments, the polynucleotide comprises a polynucleotide encoding a VH of an ILT2/ILT4-binding agent described herein. In some embodiments, the polynucleotide comprises a polynucleotide encoding a VL of an ILT2/ILT4-binding agent described herein.
  • the polynucleotide comprises a polynucleotide encoding a VH of an ILT2/ILT4-binding agent described herein and a polynucleotide encoding a VL of the ILT2/ILT4-binding agent.
  • the polynucleotide comprises polynucleotide encoding a heavy chain and/or a light chain of an ILT2-binding agent described herein.
  • the polynucleotide comprises a polynucleotide encoding a heavy chain of an ILT2-binding agent described herein.
  • the polynucleotide comprises a polynucleotide encoding a light chain of an ILT2-binding agent described herein. In some embodiments, the polynucleotide comprises a polynucleotide encoding a heavy chain of an ILT2-binding agent described herein and a polynucleotide encoding a light chain of the ILT2-binding agent. In some embodiments, the polynucleotide comprises a polynucleotide encoding a heavy chain and/or a light chain of an ILT4-binding agent described herein.
  • the polynucleotide comprises a polynucleotide encoding a heavy chain of an ILT4-binding agent described herein. In some embodiments, the polynucleotide comprises a polynucleotide encoding a light chain of an ILT4-binding agent described herein. In some embodiments, the polynucleotide comprises a polynucleotide encoding a heavy chain of an ILT4-binding agent described herein and a polynucleotide encoding a light chain of the ILT4-binding agent.
  • the polynucleotide comprises a polynucleotide encoding a heavy chain and/or a light chain of an ILT2/ILT4-binding agent described herein. In some embodiments, the polynucleotide comprises a polynucleotide encoding a heavy chain of an ILT2/ILT4-binding agent described herein. In some embodiments, the polynucleotide comprises a polynucleotide encoding a light chain of an ILT2/ILT4-binding agent described herein.
  • the polynucleotide comprises a polynucleotide encoding a heavy chain of an ILT2/ILT4-binding agent described herein and a polynucleotide encoding a light chain of the ILT2/ILT4-binding agent.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs:125-145.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:125.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:126. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:127. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:128. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:129.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:130. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:131. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:132. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:133.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:134. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:135. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:136. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:137.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:138. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:139. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:140. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:141.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:142. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:143. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:144. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:145.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs:146-157. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:146. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:147.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:148. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:149. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:150. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:151.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:152. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:153. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:154. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:155.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:156. In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:157. [00343] In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising more than one amino acid sequence selected from the group consisting of: SEQ ID NOs:125-145.
  • the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:125 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:126. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:127 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:128.
  • the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:129 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:130. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:131 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:132.
  • the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:133 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:134. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:135 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:136.
  • the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:137 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:138. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:139 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:140.
  • the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:141 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:142. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:143 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:142.
  • the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:144 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:145. [00344] In some embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising more than one amino acid sequence selected from the group consisting of: SEQ ID NOs:146-157.
  • the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:146 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:147. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:148 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:149.
  • the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:150 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:151. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:152 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:153.
  • the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:154 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:155. In some embodiments, the polynucleotide comprises a polynucleotide encoding (i) a polypeptide comprising an amino acid sequence of SEQ ID NO:156 and (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO:157.
  • the present disclosure also provides variants of the polynucleotides described herein, wherein a variant encodes, for example, a fragment, an analog, and/or a derivative of a polypeptide.
  • the present disclosure provides a polynucleotide comprising a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polynucleotide encoding a polypeptide described herein.
  • the polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, at least 97%, at least 98%, or at least 99% identical to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NOs:125-157.
  • a polynucleotide that comprises a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NOs:125-157.
  • the hybridization is under conditions of high stringency as is known to those skilled in the art.
  • the phrase “a polynucleotide having a nucleotide sequence at least 95% identical to a polynucleotide sequence” is intended to mean that the nucleotide sequence of the polynucleotide is identical to a reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. It is understood by those of skill in the art that an appropriate calculation would be made for other “% identical” statements, for example, 90% identical or 85% identical.
  • the mutations of the reference sequence can occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variant contains alterations that produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, the polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code).
  • the polynucleotide variant comprises one or more mutated codons comprising one or more (e.g., 1, 2, or 3) substitutions to the codon that change the amino acid encoded by that codon.
  • Methods for introducing one or more substitutions into a codon are known in the art, including but not limited to, PCR mutagenesis and site-directed mutagenesis.
  • Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
  • the polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence. [00349] In some embodiments, the polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, the polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, the polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, the polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
  • the polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
  • the polynucleotide comprises the coding sequence for a polypeptide fused in the same reading frame to a polynucleotide that aids in expression and secretion of a polypeptide from a host cell.
  • the polynucleotide that aids in expression and secretion is a leader sequence that functions as a secretory sequence for controlling transport of a polypeptide.
  • the polypeptide has a leader sequence cleaved by the host cell to form a “mature” form of the polypeptide.
  • the polynucleotide comprises the coding sequence for a polypeptide fused in the same reading frame to a marker or tag sequence.
  • the marker sequence is a hexa-histidine tag (HIS-tag; SEQ ID NO:173) that allows for efficient purification of the polypeptide fused to the marker.
  • the marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host is used.
  • the marker sequence is a FLAGTM tag.
  • the marker may be used in conjunction with other markers or tags.
  • the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure. [00353] Vectors and cells comprising each and every one of the polynucleotides described herein are also provided.
  • the vector comprises a polynucleotide molecule encoding an ILT-binding agent (e.g., an ILT2-binding agent, an ILT4-binding agent, or an ILT2/ILT4-binding agent) described herein. In some embodiments, the vector comprises a polynucleotide molecule encoding a polypeptide that is part of an ILT-binding agent described herein.
  • an ILT-binding agent e.g., an ILT2-binding agent, an ILT4-binding agent, or an ILT2/ILT4-binding agent
  • the cell comprises a vector comprising a polynucleotide molecule encoding an ILT-binding agent described herein. In some embodiments, the cell comprises a vector comprising a polynucleotide molecule encoding a polypeptide that is part of an ILT- binding agent described herein. In some embodiments, the cell comprises a polynucleotide molecule encoding an ILT-binding agent described herein. In some embodiments, the cell comprises one or more polynucleotides encoding an ILT-binding agent described herein. In some embodiments, the cell comprises a single polynucleotide encoding an ILT-binding agent described herein.
  • the cell comprises a first polynucleotide encoding a VH of an ILT-binding agent described herein and a second polynucleotide encoding a VL of an ILT-binding agent described herein. In some embodiments, the cell comprises a polynucleotide encoding a VH and a VL of an ILT-binding agent described herein. In some embodiments, the cell comprises a first polynucleotide encoding a heavy chain of an ILT-binding agent described herein and a second polynucleotide encoding a light chain of an ILT-binding agent described herein.
  • the cell comprises a polynucleotide encoding a heavy chain and a light chain of an ILT-binding agent described herein. In some embodiments, the cell comprises one or more vectors encoding an ILT-binding agent described herein. In some embodiments, the cell comprises a vector encoding an ILT-binding agent described herein. In some embodiments, the cell comprises a first vector encoding a VH of an ILT-binding agent described herein and a second vector encoding a VL of an ILT-binding agent described herein. In some embodiments, the cell comprises a single vector encoding a VH and a VL of an ILT-binding agent described herein.
  • the cell comprises a first vector encoding a heavy chain of an ILT-binding agent described herein and a second vector encoding a light chain of an ILT-binding agent described herein. In some embodiments, the cell comprises a single vector encoding a heavy chain and a light chain of an ILT-binding agent described herein. 5.4. Methods of Making Binding Agents [00354] The disclosure provides methods for making the ILT-binding agents (e.g., ILT2- binding agents, ILT4-binding agents, or ILT2/ILT4-binding agents) described herein.
  • ILT-binding agents e.g., ILT2- binding agents, ILT4-binding agents, or ILT2/ILT4-binding agents
  • the method comprises providing a cell comprising one or more polynucleotides encoding a heavy chain and/or light chain of an ILT-binding agent described herein, culturing the cell under conditions that permit the expression of the binding agent, and isolating the binding agent. In some embodiments, the method further comprises purifying the binding agent. In some embodiments, the method further comprises formulating the binding gent as a pharmaceutical composition. [00355] In some embodiments, the cell comprises one or more polynucleotides encoding the heavy chain and the light chain of an ILT-binding agent described herein.
  • the cell comprises a first polynucleotide encoding the heavy chain of the ILT- binding agent and a second polynucleotide encoding the light chain of the ILT-binding agent.
  • a cell comprises a polynucleotide encoding the heavy chain and the light chain of the ILT-binding agent described herein.
  • the polynucleotide encoding the ILT-binding agent described herein is transiently transfected into a cell.
  • the polynucleotide encoding an ILT-binding agent described herein is stably transfected into a cell.
  • the cell comprises one or more vectors encoding the VH and the VL of an ILT-binding agent described herein. In some embodiments, the cell comprises a first vector encoding the VH of an ILT-binding agent and a second vector encoding the VL of an ILT-binding agent. In other embodiments, a cell comprises a vector encoding the VH and the VL of an ILT-binding agent. In some embodiments, the cell comprises one or more vectors encoding the heavy chain and the light chain of an ILT-binding agent described herein.
  • the cell comprises a first vector encoding the heavy chain of an ILT-binding agent and a second vector encoding the light chain of an ILT-binding agent.
  • a cell comprises a vector encoding the heavy chain and the light chain of an ILT-binding agent described herein.
  • an ILT-binding agent e.g., the ILT2-binding agent, an ILT4- binding gent, or an ILT2/ILT4-binding agent
  • the method involves providing a cell comprising a polynucleotide encoding the fragment of the anti-ILT antibody, incubating the cell under conditions that permit the expression of the antibody fragment, and isolating the antibody fragment.
  • the cell comprises a polynucleotide encoding an antibody fragment described herein.
  • the cell comprises a vector encoding an antibody fragment described herein.
  • the method comprises purifying the antibody fragment.
  • the antibody fragment is a Fab, Fab′, F(ab′)2, Fv, scFv, (scFv)2, single chain antibody, dual variable region antibody, diabody, or nanobody.
  • the ILT-binding agent is a scFv and the method involves providing a cell comprising the scFv, incubating the cell under conditions that permit the expression of the scFv, and isolating the scFv.
  • the cell comprises a vector described herein encoding the scFv.
  • the cell comprises a polynucleotide described herein encoding the scFv.
  • the method comprises purifying the scFv.
  • the cell used to make an ILT-binding agent is a bacterial cell.
  • the cell used to make an ILT-binding agent is a yeast cell.
  • the cell used to make an ILT-binding agent is a mammalian cell.
  • the cell used to make an ILT-binding agent is a CHO cell.
  • the cell used to make an ILT-binding agent is a HEK-293 cell. 5.5.
  • the ILT-binding agents are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as treatment of cancer.
  • the therapeutic treatment methods comprise immunotherapy for cancer.
  • an ILT-binding agent described herein is useful for activating, promoting, increasing, and/or enhancing an immune response to cancer or cancer cells.
  • an ILT-binding agent described herein is useful for activating, promoting, increasing, and/or enhancing an immune response to a tumor or tumor cells.
  • the methods of use may be in vitro, ex vivo, or in vivo methods.
  • the present disclosure provides methods of disrupting, inhibiting, or blocking the binding of ILT2 to one or more MHC I molecules.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 to one or more MHC I molecules comprises contacting cells with an ILT2-binding agent described herein.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 to one or more MHC I molecules comprises contacting cells with an ILT2-binding agent described herein, wherein the method results in disrupting, inhibiting, or blocking MHC I-induced ILT2 activity.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 to one or more MHC I molecules comprises contacting cells with an ILT2-binding agent described herein, wherein the method results in disrupting, inhibiting, or blocking ILT2-induced suppression of myeloid cells.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 to one or more MHC I molecules comprises contacting cells with an ILT2- binding agent described herein, wherein the method results in disrupting, inhibiting, or blocking of ILT2-induced suppression of myeloid cell activity.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 to one or more MHC I molecules restores FcR signaling activity in myeloid cells.
  • the myeloid cell is a monocyte. In some embodiments, the myeloid cell is a macrophage. In some embodiments, the myeloid cell is a dendritic cell. In some embodiments, the myeloid cell is an APC. In some embodiments, the method of disrupting, inhibiting, or blocking the binding of ILT2 to one or more MHC I molecules comprises contacting cells with an ILT2-binding agent described herein, wherein the method results in increasing NK cell activity. In some embodiments, the method of disrupting, inhibiting, or blocking the binding of ILT2 to one or more MHC I molecules comprises contacting cells with an ILT2-binding agent described herein, wherein the method results in increasing CTL activity.
  • the MHC I molecule is a classical MHC I molecule. In some embodiments of the methods described herein, the MHC I molecule is a non-classical MHC I molecule. In some embodiments of the methods described herein, the MHC I molecule is HLA-A, HLA-B, HLA-C, HLA-E, and/or HLA-G. [00362]
  • the present disclosure provides methods of disrupting, inhibiting, or blocking the binding of ILT4 to one or more MHC I molecules. In some embodiments, the method of disrupting, inhibiting, or blocking the binding of ILT4 to one or more MHC I molecules comprises contacting cells with an ILT4-binding agent described herein.
  • the method of disrupting, inhibiting, or blocking the binding of ILT4 to one or more MHC I molecules comprises contacting cells with an ILT4-binding agent described herein, wherein the method results in disrupting, inhibiting, or blocking MHC I-induced ILT4 activity. In some embodiments, the method of disrupting, inhibiting, or blocking the binding of ILT4 to one or more MHC I molecules comprises contacting cells with an ILT4-binding agent described herein, wherein the method results in disrupting, inhibiting, or blocking ILT4-induced suppression of myeloid cells.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 to one or more MHC I molecules comprises contacting cells with an ILT4- binding agent described herein, wherein the method results in disrupting, inhibiting, or blocking of ILT4-induced suppression of myeloid cell activity.
  • the method of disrupting, inhibiting, or blocking the binding of ILT4 to one or more MHC I molecules restores FcR signaling activity in myeloid cells.
  • the myeloid cell is a monocyte.
  • the myeloid cell is a macrophage.
  • the myeloid cell is a dendritic cell.
  • the myeloid cell is an APC.
  • the MHC I molecule is a classical MHC I molecule. In some embodiments of the methods described herein, the MHC I molecule is a non- classical MHC I molecule. In some embodiments of the methods described herein, the MHC I molecule is HLA-A, HLA-B, HLA-C, HLA-E, and/or HLA-G. [00363] The present disclosure provides methods of disrupting, inhibiting, or blocking the binding of ILT2 and ILT4 to one or more MHC I molecules.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 and ILT4 to one or more MHC I molecules comprises contacting cells with an ILT2/ILT4-binding agent described herein. In some embodiments, the method of disrupting, inhibiting, or blocking the binding of ILT2 and ILT4 to one or more MHC I molecules comprises contacting cells with an ILT2/ILT4-binding agent described herein, wherein the method results in disrupting, inhibiting, or blocking MHC I- induced ILT2 and/or ILT4 activity.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 and ILT4 to one or more MHC I molecules comprises contacting cells with an ILT2/ILT4-binding agent described herein, wherein the method results in disrupting, inhibiting, or blocking ILT2-induced and/or ILT4-induced suppression of myeloid cells.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 and ILT4 to one or more MHC I molecules comprises contacting cells with an ILT2/ILT4- binding agent described herein, wherein the method results in disrupting, inhibiting, or blocking of ILT2-induced and/or ILT4-induced suppression of myeloid cell activity.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 and ILT4 to one or more MHC I molecules restores FcR signaling activity in myeloid cells.
  • the myeloid cell is a monocyte.
  • the myeloid cell is a macrophage.
  • the myeloid cell is a dendritic cell.
  • the myeloid cell is an APC.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 and ILT4 to one or more MHC I molecules comprises contacting cells with an ILT2/ILT4-binding agent described herein, wherein the method results in increasing NK cell activity.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 and ILT4 to one or more MHC I molecules comprises contacting cells with an ILT2/ILT4-binding agent described herein, wherein the method results in increasing CTL activity.
  • the present disclosure provides methods of disrupting, inhibiting, or blocking the binding of ILT2 and/or ILT4 to MHC I molecules in a subject.
  • the method of disrupting, inhibiting, or blocking the binding of ILT2 and/or ILT4 to MHC I molecules in a subject comprises administering to the subject an effective amount of an ILT2/ILT4-binding agent described herein.
  • the method of disrupting, inhibiting, or blocking MHC I-induced ILT2 and/or ILT4 activity in a subject comprises administering to the subject an effective amount of an ILT2/ILT4-binding agent described herein. In some embodiments, the method of disrupting, inhibiting, or blocking ILT2-induced and/or ILT4-induced suppression of myeloid cells in a subject comprises administering to the subject an effective amount of an ILT2/ILT4-binding agent described herein. In some embodiments, the method of disrupting, inhibiting, or blocking ILT2-induced and/or ILT4- induced suppression of myeloid cell activity in a subject comprises administering to the subject an effective amount of an ILT2/ILT4-binding agent described herein.
  • the method of disrupting, inhibiting, or blocking ILT2-induced and/or ILT4-induced suppression of antigen-presenting cell activity in a subject restores FcR activity in myeloid cells.
  • the myeloid cell is a monocyte.
  • the myeloid cell is a macrophage.
  • the myeloid cell is a dendritic cell.
  • the myeloid cell is an APC.
  • the disclosure provides methods for increasing an immune response in a subject using an ILT2/ILT4-binding agent described herein. In some embodiments, the disclosure provides methods for enhancing an immune response in a subject using an ILT2/ILT4-binding agent described herein. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises stimulating myeloid cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises stimulating monocytes. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises stimulating macrophages. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises stimulating dendritic cells.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises stimulating APCs. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing cell-mediated immunity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing effector T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises enhancing NK cell activity.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of Tregs. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of MDSCs.
  • the immune response is a result of antigenic stimulation. In some embodiments, the antigenic stimulation is a tumor cell. In some embodiments, the antigenic stimulation is cancer.
  • the disclosure also provides methods of disrupting and/or inhibiting ILT2 and/or ILT4 signaling in a cell comprising contacting the cell with an effective amount of an ILT2-binding agent, an ILT4-binding agent, or an ILT2/ILT4-binding agent described herein.
  • the method of disrupting and/or inhibiting ILT2 signaling in a cell comprises contacting the cell with an effective amount of antibody 27F9, antibody 47H6, antibody 51A1, antibody 64A12, antibody 73C4, or antibody 73D1, or a humanized version thereof.
  • the method of disrupting and/or inhibiting ILT4 signaling in a cell comprises contacting the cell with an effective amount of antibody 47C8, antibody 48A5, antibody 47H6, antibody 51A1, antibody 64A12, antibody 73C4, or antibody 73D1, or a humanized version thereof.
  • the method of disrupting and/or inhibiting ILT2 signaling and ILT4 signaling in a cell comprises contacting the cell with an effective amount of antibody 47H6, antibody 51A1, antibody 64A12, antibody 73C4, or antibody 73D1, or a humanized version thereof.
  • the method of disrupting and/or inhibiting ILT2 signaling and ILT4 in a cell comprises contacting the cell with an effective amount of antibody Hz47H6.v2. In some embodiments, the method of disrupting and/or inhibiting ILT2 signaling and ILT4 in a cell comprises contacting the cell with an effective amount of antibody Hz64A12. In some embodiments, the method of disrupting and/or inhibiting ILT2 signaling and ILT4 in a cell comprises contacting the cell with an effective amount of antibody Hz73D1.v1. In some embodiments, the disclosure provides use of an ILT2-binding agent described herein in the manufacture or preparation of a medicament for disrupting and/or inhibiting ILT2 signaling in a cell.
  • the disclosure provides use of an ILT4-binding agent described herein in the manufacture or preparation of a medicament for disrupting and/or inhibiting ILT4 signaling in a cell.
  • the disclosure provides use of an ILT2/ILT4-binding agent described herein in the manufacture or preparation of a medicament for disrupting and/or inhibiting ILT2 signaling and ILT4 signaling in a cell.
  • the cell is a myeloid cell.
  • the cell is a monocyte.
  • the cell is a macrophage.
  • the cell is a dendritic cell.
  • the cell is an antigen-presenting cell.
  • the cells is a NK cell.
  • the cell is a CTL.
  • the method is an in vivo method wherein the step of contacting the cell with the agent comprises administering a therapeutically effective amount of an ILT-binding agent to a subject.
  • the method is an in vitro or ex vivo method.
  • the present disclosure also provides methods for inhibiting growth of a tumor using an ILT2-binding agent, an ILT4-binding agent, or an ILT2/ILT4-binding agent described herein.
  • the method of inhibiting growth of a tumor comprises using an ILT2- binding agent described herein.
  • the method of inhibiting growth of a tumor comprises using antibody 27F9 or a humanized version thereof.
  • the method of inhibiting growth of a tumor comprises using an ILT4-binding agent described herein. In some embodiments, the method of inhibiting growth of a tumor comprises using antibody 47C8 or antibody 48A5, or a humanized version thereof. In some embodiments, the method of inhibiting growth of a tumor comprises using an ILT2/ILT4-binding agent described herein. In some embodiments, the method of inhibiting growth of a tumor comprises using antibody 47H6, antibody 51A1, antibody 64A12, antibody 73C4, or antibody 73D1, or humanized versions thereof. In some embodiments, the method of inhibiting growth of a tumor comprises using antibody Hz47H6.v2. In some embodiments, the method of inhibiting growth of a tumor comprises using antibody Hz64A12.
  • the method of inhibiting growth of a tumor comprises using antibody Hz73D1.v1.
  • the method of inhibiting growth of a tumor comprises contacting a cell mixture with an ILT-binding agent in vitro.
  • an immortalized cell line or a cancer cell line mixed with immune cells e.g., a myeloid cell
  • an immortalized cell line or a cancer cell line mixed with immune cells e.g., a myeloid cell
  • tumor cells are isolated from a patient sample such as, for example, a tissue biopsy, pleural effusion, or blood sample, mixed with immune cells (e.g., myeloid cells), and cultured in medium to which is added a test agent that binds ILT2 and/or ILT4.
  • the disclosure provides use of an ILT2-binding agent described herein in the manufacture or preparation of a medicament for inhibiting growth of a tumor or a tumor cell. In some embodiments, the disclosure provides use of an ILT4-binding agent described herein in the manufacture or preparation of a medicament for inhibiting growth of a tumor or a tumor cell. In some embodiments, the disclosure provides use of an ILT2/ILT4- binding agent described herein in the manufacture or preparation of a medicament for inhibiting growth of a tumor or a tumor cell. In some embodiments, the ILT2-binding agent increases, promotes, and/or enhances the activity of effector immune cells.
  • the ILT2-binding agent inhibits tumor cell growth by increasing, promoting, and/or enhancing the activity of effector immune cells. In some embodiments, an ILT4-binding agent increases, promotes, and/or enhances the activity of effector immune cells. In some embodiments, an ILT4-binding agent inhibits tumor cell growth by increasing, promoting, and/or enhancing the activity of effector immune cells. In some embodiments, an ILT2/ILT4-binding agent increases, promotes, and/or enhances the activity of effector immune cells. In some embodiments, an ILT2/ILT4-binding agent inhibits tumor cell growth by increasing, promoting, and/or enhancing the activity of effector immune cells.
  • the method of inhibiting tumor growth comprises contacting the tumor and/or tumor microenvironment with an ILT-binding agent (e.g., an ILT2-binding agent, an ILT4-binding agent, or an ILT2/ILT4-binding agent) described herein in vivo.
  • an ILT-binding agent e.g., an ILT2-binding agent, an ILT4-binding agent, or an ILT2/ILT4-binding agent
  • contacting a tumor and/or tumor microenvironment with an ILT-binding agent described herein is undertaken in an animal model.
  • a test agent e.g., an ILT2/ILT4-binding agent
  • the ILT2/ILT4-binding agent increases, promotes, and/or enhances the activity of immune cells in the mice.
  • the ILT2/ILT4-binding agent inhibits tumor growth. In some embodiments, the ILT2/ILT4-binding agent causes a tumor to regress. In some embodiments, the ILT2/ILT4-binding agent is administered at the same time or shortly after introduction of tumor cells into the animal to prevent tumor growth (“preventative model”). In some embodiments, the ILT2/ILT4-binding agent is administered after tumors have grown to a specified size or have become “established” for treatment (“therapeutic model”).
  • the ILT2/ILT4-binding agent is administered to a transgenic animal (e.g., a transgenic mouse) that expresses human ILT2 and/or ILT4, wherein the transgenic animal has a tumor derived from human cells.
  • the method of inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of an ILT-binding agent (e.g., an ILT2-binding agent, an ILT4-binding agent, or an ILT2/ILT4-binding agent) described herein.
  • the method of inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of an ILT2/ILT4-binding agent described herein.
  • the method of increasing or enhancing an immune response to a tumor or tumor cells in a subject comprises administering to the subject a therapeutically effective amount of an ILT2/ILT4-binding agent described herein.
  • the method of activating or enhancing a persistent or long-term immune response to a tumor or tumor cells in a subject comprises administering to the subject a therapeutically effective amount of an ILT2/ILT4- binding agent described herein.
  • the method of inhibiting tumor relapse or tumor regrowth in a subject comprises administering to the subject a therapeutically effective amount of an ILT2/ILT4-binding agent described herein.
  • the method of inducing a persistent or long-term immunity that inhibits tumor relapse or tumor regrowth in a subject comprises administering to the subject a therapeutically effective amount of an ILT2/ILT4-binding agent described herein.
  • the tumor is a solid tumor.
  • the tumor is a pancreatic tumor, a breast tumor, a lung tumor, a non-small cell lung tumor, a small cell lung tumor, a squamous cell carcinoma, a head and neck tumor, an esophageal cancer, a colorectal tumor, a prostate tumor, a skin tumor, a melanoma tumor, a stomach tumor, a gastric tumor, an intestinal tumor, an ovarian tumor, a cervical tumor, an uterine tumor, an endometrial tumor, a bladder tumor, a brain tumor, an esophageal tumor, a liver tumor, a kidney tumor, a renal cell carcinoma, a hepatocellular carcinoma, a biliary duct cancer, a thyroid tumor, a mesothelioma, a glioblastoma, or a testicular tumor.
  • the tumor is a hepatocellular carcinoma, a small cell lung tumor, a thyroid tumor, a mesothelioma, a glioblastoma, a renal cell carcinoma, a non-small cell lung tumor, a melanoma (skin cutaneous and uveal), a pancreatic ductal adenocarcinoma, a gastric tumor, a squamous cell carcinoma of the head and neck, a biliary duct tumor, a breast tumor, a triple-negative breast tumor, an ovarian tumor, a cervical tumor, an endocervical tumor, a colorectal tumor, an esophageal tumor, a gastroesophageal junction adenocarcinoma, a squamous cell carcinoma of head and neck, a bladder tumor, an uterine tumor, a cholangiocarcinoma, or a prostate tumor.
  • a pancreatic ductal adenocarcinoma
  • the tumor is a pancreatic tumor (e.g, a pancreatic ductal adenocarcinoma).
  • the tumor is a non-small cell lung tumor.
  • the tumor is a renal cell carcinoma (RCC).
  • the subject has a tumor or the subject had a tumor that was at least partially removed.
  • the subject is a human.
  • the disclosure provides use of an ILT2-binding agent, an ILT4- binding agent, or an ILT2/ILT4-binding agent described herein in the manufacture or preparation of a medicament for inhibiting growth of a tumor or tumor cell.
  • the method of inhibiting growth of a tumor comprises administering to a subject a therapeutically effective amount of antibody 27F9, antibody 47C8, antibody 48A5, antibody 47H6, antibody 51A1, antibody 64A12, antibody 73C4, or antibody 73D1, or humanized versions thereof.
  • the method of inhibiting growth of a tumor comprises administering to a subject a therapeutically effective amount of antibody Hz47H6.v2.
  • the method of inhibiting growth of a tumor comprises administering to a subject a therapeutically effective amount of antibody Hz64A12.
  • the method of inhibiting growth of a tumor comprises administering to a subject a therapeutically effective amount of antibody Hz73D1.v1.
  • the subject is a human.
  • the present disclosure provides methods of treating cancer.
  • the method of treating cancer comprises administering to a subject a therapeutically effective amount of an ILT2-binding agent described herein.
  • the method of treating cancer comprises administering to a subject a therapeutically effective amount of an ILT4-binding agent described herein.
  • the method of treating cancer comprises administering to a subject a therapeutically effective amount of an ILT2/ILT4-binding agent described herein.
  • the ILT2/ILT4-binding agent binds ILT2 and/or ILT4 and inhibits or reduces growth of the cancer.
  • the ILT2/ILT4- binding agent binds human ILT2-expressing cells and/or ILT4-expressing cells, enhances an immune response to a cancer, and inhibits or reduces growth of the cancer. In some embodiments, the ILT2/ILT4-binding agent binds human ILT2-expressing cells and/or ILT4- expressing cells, activates myeloid cells, enhances an immune response to a cancer, and inhibits or reduces growth of the cancer. In some embodiments, the subject is a human. In some embodiments, the subject has a cancerous tumor. In some embodiments, the subject has had the cancer at least partially removed.
  • the disclosure provides use of an ILT2-binding agent described herein in the manufacture or preparation of a medicament for the treatment of cancer. In some embodiments, the disclosure provides use of an ILT4-binding agent described herein in the manufacture or preparation of a medicament for the treatment of cancer. In some embodiments, the disclosure provides use of an ILT2/ILT4-binding agent described herein in the manufacture or preparation of a medicament for the treatment of cancer.
  • the cancer is pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, non-small cell lung cancer (NSCLC), head and neck cancer, colorectal cancer, prostate cancer, skin cancer, melanoma, stomach cancer, gastric cancer, intestinal cancer, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, bladder cancer, brain cancer, esophageal cancer, liver cancer, kidney cancer, renal cell carcinoma (RCC), or testicular cancer.
  • the cancer is pancreatic cancer.
  • the cancer is breast cancer (e.g., triple-negative breast cancer).
  • the cancer is non-small cell lung cancer.
  • the cancer is renal cell carcinoma.
  • the method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of antibody 27F9 or a humanized version thereof.
  • the method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of antibody 47C8 or antibody 48A5, or a humanized version thereof.
  • the method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of antibody 47H6, antibody 51A1, antibody 64A12, antibody 73C4, or antibody 73D1, or a humanized version thereof.
  • the method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of antibody Hz47H6.v2. In some embodiments, the method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of antibody Hz64A12. In some embodiments, the method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of antibody Hz73D1.v1.
  • the cancer is hepatocellular carcinoma, small cell lung cancer, thyroid cancer, mesothelioma, glioblastoma, renal cell carcinoma, non-small cell lung cancer, melanoma (skin cutaneous and uveal), pancreatic ductal adenocarcinoma, gastric cancer, squamous cell carcinoma of the head and neck, biliary duct cancer, breast cancer, triple-negative breast cancer, ovarian cancer, cervical cancer, endocervical cancer, colorectal cancer, esophageal cancer, gastroesophageal junction adenocarcinoma, squamous cell carcinoma of head and neck, bladder cancer, uterine cancer, cholangiocarcinoma, or prostate cancer.
  • the cancer is hepatocellular carcinoma. In certain embodiments, the cancer is small cell lung cancer. In certain embodiments, the cancer is thyroid cancer. [00376] In some embodiments, the tumor comprises a microsatellite instability – high (MSI-H) tumor. In some embodiments, the tumor comprises a deficient mismatch repair (dMMR) positive tumor.
  • MSI-H microsatellite instability – high
  • dMMR deficient mismatch repair
  • the tumor is a small cell lung tumor, thyroid tumor, pancreatic tumor, a breast tumor, a lung tumor, a non-small cell lung tumor, a head and neck tumor, a colorectal tumor, a prostate tumor, a skin tumor, a melanoma, a gastric tumor, a colorectal tumor, an ovarian tumor, a cervical tumor, a uterine tumor, an endometrial tumor, an endocervical tumor, a bladder tumor, a brain tumor, an esophageal tumor, a liver tumor, a kidney tumor, a renal tumor, mesothelioma, glioblastoma, a biliary duct tumor, or a testicular tumor.
  • the tumor is a hepatocellular carcinoma, a small cell lung tumor, a thyroid tumor, a mesothelioma, a glioblastoma, a renal cell carcinoma, a non-small cell lung tumor, a melanoma (skin cutaneous and uveal), a pancreatic ductal adenocarcinoma, a gastric tumor, a squamous cell carcinoma of the head and neck, a biliary duct tumor, a breast tumor, a triple-negative breast tumor, an ovarian tumor, a cervical tumor, an endocervical tumor, a colorectal tumor, an esophageal tumor, a gastroesophageal junction adenocarcinoma, a squamous cell carcinoma of head and neck, a bladder tumor, an uterine tumor, a cholangiocarcinoma, or a prostate tumor.
  • a pancreatic ductal adenocarcinoma
  • the tumor is a liver tumor. In certain embodiments, the tumor is a small cell lung tumor. In certain embodiments, the tumor is a thyroid tumor. [00377] Provided herein is use of the binding agent or antibody of the present disclosure for treatment of cancer.
  • the cancer is hepatocellular carcinoma, small cell lung cancer, thyroid cancer, mesothelioma, glioblastoma, renal cell carcinoma, non-small cell lung cancer, melanoma (skin cutaneous and uveal), pancreatic ductal adenocarcinoma, gastric cancer, squamous cell carcinoma of the head and neck, biliary duct cancer, breast cancer, triple- negative breast cancer, ovarian cancer, cervical cancer, endocervical cancer, colorectal cancer, esophageal cancer, gastroesophageal junction adenocarcinoma, squamous cell carcinoma of head and neck, bladder cancer, uterine cancer, cholangiocarcinoma, or prostate cancer.
  • the binding agent or antibody of the present disclosure for treatment of hepatocellular cancer.
  • use of the binding agent or antibody of the present disclosure for treatment of small cell lung cancer.
  • use of the binding agent or antibody of the present disclosure for treatment of thyroid cancer.
  • use of the binding agent or antibody of the present disclosure for treatment of a microsatellite instability – high (MSI-H) tumor.
  • use of the binding agent or antibody of the present disclosure for treatment of a deficient mismatch repair (dMMR) positive tumor.
  • dMMR deficient mismatch repair
  • the cancer is hepatocellular carcinoma, small cell lung cancer, thyroid cancer, mesothelioma, glioblastoma, renal cell carcinoma, non-small cell lung cancer, melanoma (skin cutaneous and uveal), pancreatic ductal adenocarcinoma, gastric cancer, squamous cell carcinoma of the head and neck, biliary duct cancer, breast cancer, triple-negative breast cancer, ovarian cancer, cervical cancer, endocervical cancer, colorectal cancer, esophageal cancer, gastroesophageal junction adenocarcinoma, squamous cell carcinoma of head and neck, bladder cancer, uterine cancer, cholangiocarcinoma, or prostate cancer.
  • the binding agent or antibody of the present disclosure in the manufacture of a medicament for treatment of hepatocellular cancer.
  • use of the binding agent or antibody of the present disclosure in the manufacture of a medicament for treatment of small cell lung cancer.
  • use of the binding agent or antibody of the present disclosure in the manufacture of a medicament for treatment of thyroid cancer.
  • use of the binding agent or antibody of the present disclosure in the manufacture of a medicament for treatment of a microsatellite instability – high (MSI-H) tumor.
  • MSI-H microsatellite instability – high
  • dMMR deficient mismatch repair
  • the disclosure provides methods of activating myeloid cells in the tumor microenvironment.
  • the method of activating myeloid cells in the tumor microenvironment in a subject with a tumor comprises administering to the subject a therapeutically effective amount of an ILT2-binding agent, an ILT4-binding agent, or an ILT2/ILT4-binding agent described herein.
  • the method of activating myeloid cells in the tumor microenvironment in a subject with a tumor comprises administering to the subject a therapeutically effective amount of an ILT2/ILT4-binding agent described herein.
  • the myeloid cells are primary dendritic cells.
  • the myeloid cells are monocytes.
  • the myeloid cells are macrophages. In some embodiments, the myeloid cells are APCs. [00380] In some embodiments, the disclosure provides methods of activating NK cells in the tumor microenvironment. In some embodiments, the method of activating NK cells in the tumor microenvironment in a subject with a tumor comprises administering to the subject a therapeutically effective amount of an ILT2-binding agent or an ILT2/ILT4-binding agent described herein. In some embodiments, the method of activating NK cells in the tumor microenvironment in a subject with a tumor comprises administering to the subject a therapeutically effective amount of an ILT2-binding agent described herein.
  • the method of activating NK cells in the tumor microenvironment in a subject with a tumor comprises administering to the subject a therapeutically effective amount of an ILT2/ILT4-binding agent described herein.
  • the disclosure provides methods of activating CTLs in the tumor microenvironment.
  • the method of activating CTLs in the tumor microenvironment in a subject with a tumor comprises administering to the subject a therapeutically effective amount of an ILT2-binding agent or an ILT2/ILT4-binding agent described herein.
  • the method of activating CTLs in the tumor microenvironment in a subject with a tumor comprises administering to the subject a therapeutically effective amount of an ILT2-binding agent described herein. In some embodiments, the method of activating CTLs in the tumor microenvironment in a subject with a tumor comprises administering to the subject a therapeutically effective amount of an ILT2/ILT4-binding agent described herein.
  • the ILT2-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 of antibody 27F9.
  • the ILT2-binding agent is an anti-ILT2 antibody.
  • the anti-ILT2 antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GFSLTNYGVS (SEQ ID NO:22), a VH-CDR2 comprising the amino acid sequence IIWGDGSTNYHSALIS (SEQ ID NO:23), and a VH-CDR3 comprising the amino acid sequence PNWDTYAMDF (SEQ ID NO:24), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASQDISNFLN (SEQ ID NO:25), a VL-CDR2 comprising the amino acid sequence CTSKLHS (SEQ ID NO:26), and a VL-CDR3 comprising the amino acid sequence QQGNTLPPT (SEQ ID NO:27).
  • the anti-ILT2 antibody comprises: (a) a VH of SEQ ID NO:125 and (b) a VL of SEQ ID NO:126.
  • the anti-ILT2 antibody is antibody 27F9.
  • the anti-ILT2 antibody is a humanized version of 27F9.
  • the ILT4-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 of antibody 47C8.
  • an ILT4-binding agent is an anti-ILT4 antibody.
  • the anti-ILT4 antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYSFTGYYMH (SEQ ID NO:38), a VH-CDR2 comprising the amino acid sequence RVYPNNGDTSYNQKFKV (SEQ ID NO:39), and a VH-CDR3 comprising the amino acid sequence GATVVESLFAY (SEQ ID NO:40), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDNYGNNFLH (SEQ ID NO:41), a VL-CDR2 comprising the amino acid sequence RTSNLES (SEQ ID NO:42), and a VL-CDR3 comprising the amino acid sequence QQSNEDPYT (SEQ ID NO:43.
  • the anti-ILT4 antibody comprises: (a) a VH of SEQ ID NO:127 and (b) a VL of SEQ ID NO:128.
  • the anti-ILT4 antibody is antibody 47C8.
  • the anti-ILT4 antibody is a humanized version of antibody 47C8.
  • the ILT4-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 of antibody 48A5.
  • an ILT4-binding agent is an anti-ILT4 antibody.
  • the anti-ILT4 antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTNYGMN (SEQ ID NO:54), a VH-CDR2 comprising the amino acid sequence WINTYIGEPIYADDFKG (SEQ ID NO:55), and a VH-CDR3 comprising the amino acid sequence RSDYDGYAMDY (SEQ ID NO:56), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence KSSQSLLYSGNQKNYLA (SEQ ID NO:57), a VL-CDR2 comprising the amino acid sequence WASTRES (SEQ ID NO:58), and a VL-CDR3 comprising the amino acid sequence QQHDSYPT (SEQ ID NO:59).
  • the anti-ILT4 antibody comprises: (a) a VH of SEQ ID NO:129 and (b) a VL of SEQ ID NO:130.
  • the anti-ILT4 antibody is antibody 48A5.
  • the anti- ILT4 antibody is a humanized version of antibody 48A5.
  • the ILT2/ILT4-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 of antibody 47H6 or antibody Hz47H6.v2.
  • an ILT2/ILT4-binding agent is an anti-ILT2/ILT4 antibody.
  • the anti- ILT2/ILT4 antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71) or DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75).
  • the anti- ILT2/ILT4 antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNGGTTYNQKFEG (SEQ ID NO:71), and a VH-CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75).
  • the anti-ILT2/ILT4 antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence DFNPNNAGTTYNQKFEG (SEQ ID NO:118), and a VH- CDR3 comprising the amino acid sequence GRFYYGSLYSFDY (SEQ ID NO:72), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASGNIHNYLA (SEQ ID NO:73), a VL-CDR2 comprising the amino acid sequence NAKTLAD (SEQ ID NO:74), and a VL-CDR3 comprising the amino acid sequence QHFWTSIT (SEQ ID NO:75).
  • the anti-ILT2/ILT4 antibody comprises: (a) a VH of SEQ ID NO:131 and (b) a VL of SEQ ID NO:132. In some embodiments of the methods described herein, the anti-ILT2/ILT4 antibody comprises: (a) a VH of SEQ ID NO:133 and (b) a VL of SEQ ID NO:134. In some embodiments of the methods described herein, the anti- ILT2/ILT4 antibody comprises: (a) a heavy chain of SEQ ID NO:148 and (b) a light chain of SEQ ID NO:149. In some embodiments of the methods described herein, the anti-ILT2/ILT4 antibody is antibody 47H6.
  • the anti- ILT2/ILT4 antibody is a humanized version of antibody 47H6. In some embodiments of the methods described herein, the anti-ILT2/ILT4 antibody is antibody Hz47H6.v2. [00390] In some embodiments of the methods described herein, the ILT2/ILT4-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 of antibody 51A1 or a humanized version of antibody 51A1.
  • an ILT2/ILT4-binding agent is an anti-ILT2/ILT4 antibody.
  • the anti- ILT2/ILT4 antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYGTMYYYAMDY (SEQ ID NO:88), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPW
  • the anti-ILT2/ILT4 antibody comprises: (a) a VH of SEQ ID NO:135 and (b) a VL of SEQ ID NO:136.
  • the anti-ILT2/ILT4 antibody is antibody 51A1.
  • the anti-ILT2/ILT4 antibody is a humanized version of antibody 51A1.
  • the ILT2/ILT4-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 of antibody 64A12, a humanized version of antibody 64A12, or antibody Hz64A12.
  • an ILT2/ILT4-binding agent is an anti-ILT2/ILT4 antibody.
  • the anti- ILT2/ILT4 antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GFTFNTYAMH (SEQ ID NO:86), a VH-CDR2 comprising the amino acid sequence RIRSKSSNYATYYADSVKD (SEQ ID NO:87), and a VH-CDR3 comprising the amino acid sequence DGIYYYDTMYYYAMDY (SEQ ID NO:102), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFIY (SEQ ID NO:103), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the anti-ILT2/ILT4 antibody comprises: (a) a VH of SEQ ID NO:137 and (b) a VL of SEQ ID NO:138. In some embodiments of the methods described herein, the anti-ILT2/ILT4 antibody comprises: (a) a VH of SEQ ID NO:139 and (b) a VL of SEQ ID NO:140. In some embodiments of the methods described herein, the anti-ILT2/ILT4 antibody comprises: (a) a heavy chain of SEQ ID NO:152 and (b) a light chain of SEQ ID NO:153. In some embodiments of the methods described herein, the anti-ILT2/ILT4 antibody is antibody 64A12.
  • the anti-ILT2/ILT4 antibody is a humanized version of antibody 64A12. In some embodiments of the methods described herein, the anti-ILT2/ILT4 antibody is antibody Hz64A12. [00394] In some embodiments of the methods described herein, the ILT2/ILT4-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 of antibody 73C4 or a humanized version of antibody 73C4.
  • an ILT2/ILT4-binding agent is an anti-ILT2/ILT4 antibody.
  • the anti- ILT2/ILT4 antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYMN (SEQ ID NO:70), a VH-CDR2 comprising the amino acid sequence NVNPNNGGTSYNQKFKG (SEQ ID NO:106), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNED
  • the anti-ILT2/ILT4 antibody comprises: (a) a VH of SEQ ID NO:141 and (b) a VL of SEQ ID NO:142.
  • the anti-ILT2/ILT4 antibody is antibody 73C4.
  • the anti-ILT2/ILT4 antibody is a humanized version of antibody 73C4.
  • the ILT2/ILT4-binding agent comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3 and a VL-CDR1, a VL-CDR2, and a VL-CDR3 of antibody 73D1, a humanized version of antibody 73D1, or antibody Hz73D1.v1.
  • an ILT2/ILT4-binding agent is an anti-ILT2/ILT4 antibody.
  • the anti- ILT2/ILT4 antibody comprises: (a) a VH comprising a VH-CDR1 comprising the amino acid sequence GYTFTDYYIN (SEQ ID NO:111), a VH-CDR2 comprising the amino acid sequence NVNPNDGGTTYNQKFKG (SEQ ID NO:112), and a VH-CDR3 comprising the amino acid sequence REIYFYGTIYYYAMDY (SEQ ID NO:107), and (b) a VL comprising a VL-CDR1 comprising the amino acid sequence RASESVDYYGNSFMY (SEQ ID NO:89), a VL-CDR2 comprising the amino acid sequence FASNLES (SEQ ID NO:90), and a VL-CDR3 comprising the amino acid sequence QQNNEDPWT (SEQ ID NO:91).
  • the anti-ILT2/ILT4 antibody comprises: (a) a VH of SEQ ID NO:143 and (b) a VL of SEQ ID NO:142. In some embodiments of the methods described herein, the anti-ILT2/ILT4 antibody comprises: (a) a VH of SEQ ID NO:144 and (b) a VL of SEQ ID NO:145. In some embodiments of the methods described herein, the anti-ILT2/ILT4 antibody comprises: (a) a heavy chain of SEQ ID NO:156 and (b) a light chain of SEQ ID NO:157. In some embodiments of the methods described herein, the anti-ILT2/ILT4 antibody is antibody 73D1.
  • the anti-ILT2/ILT4 antibody is a humanized version of antibody 73D1. In some embodiments of the methods described herein, the anti-ILT2/ILT4 antibody is antibody Hz73D1.v1.
  • an ILT-binding agent e.g., an ILT2-binding agent, an ILT4-binding agent, or an ILT2/ILT4-binding agent
  • the appropriate dosage of an ILT-binding agent of the present disclosure depends on the disorder or disease to be treated, the severity and course of the disorder or disease, the responsiveness of the disorder or disease, whether the agent is administered for therapeutic or preventative purposes, previous therapy, the patient's clinical history, and so on.
  • an ILT-binding agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is affected or a diminution of the disease state is achieved.
  • the binding agent is administered at a dose of about 1 mg, about 3 mg, about 5 mg, about 6 mg, about 20 mg, about 60 mg, about 200 mg, about 600 mg, about 1200 mg, about 1500 mg, about 1800 mg, about 2100 mg, or about 2400 mg.
  • the binding agent is administered at a dose of between about 50 mg and about 2000 mg.
  • the binding agent is administered at a dose of between about 60 mg and about 1800 mg, between about 60 mg and about 1500 mg, between about 60 mg and about 1200 mg, between about 60 mg and about 900 mg, between about 60 mg and about 800 mg, between about 60 mg and about 600 mg, between about 60 mg and about 400 mg, between about 60 mg and about 200 mg, between about 200 mg and about 1800 mg, between about 200 mg and about 1500 mg, between about 200 mg and about 1200 mg, between about 200 mg and about 900 mg, between about 200 mg and about 800 mg, between about 200 mg and about 600 mg, between about 200 mg and about 400 mg, between about 400 mg and about 1800 mg, between about 400 mg and about 1500 mg, between about 400 mg and about 1200 mg, between about 400 mg and about 900 mg, between about 400 mg and about 800 mg, between about 400 mg and about 600 mg, between about 600 mg and about 1800 mg, between about 600 mg and about 1500 mg, between about 400 mg and about 1200 mg, between about 400 mg and about 900 mg, between about 400 mg and about 800 mg, between about
  • the binding agent is administered at a dose of between about 400 mg and about 700 mg, between about 500 mg and about 800 mg, or between about 500 mg and about 700 mg. In some embodiments of the method or use, the binding agent is administered at a dose of about 600 mg. In some embodiments of the method or use, the binding agent is administered at a dose of between about 1000 mg and about 1300 mg, between about 1100 mg and about 1400 mg, or between about 1100 mg and about 1300 mg. In some embodiments of the method or use, the binding agent is administered at a dose of about 1200 mg.
  • the binding agent is administered at a dose of about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, or about 1800 mg.
  • the dose is a flat dose.
  • the binding agent is administered at a dose of between about 1 mg/kg and about 100 mg/kg of body weight.
  • the binding agent is administered at a dose of between about 1 mg/kg and about 30 mg/kg, between about 5 mg/kg and about 30 mg/kg, between about 10 mg/kg and about 30 mg/kg, between about 15 mg/kg and about 30 mg/kg, between about 20 mg/kg and about 30 mg/kg, between about 25 mg/kg and about 30 mg/kg, between about 1 mg/kg and about 25 mg/kg, between about 5 mg/kg and about 25 mg/kg, between about 10 mg/kg and about 25 mg/kg, between about 15 mg/kg and about 25 mg/kg, between about 20 mg/kg and about 25 mg/kg, between about 1 mg/kg and about 20 mg/kg, between about 5 mg/kg and about 20 mg/kg, between about 10 mg/kg and about 20 mg/kg, between about 15 mg/kg and about 20 mg/kg, between about 1 mg/kg and about 15 mg/kg, between about 5 mg/kg and about 15 mg/kg, between about 10 mg/kg and about 15 mg/kg, between about 10 mg/kg and about 15 mg
  • the binding agent is administered at a dose of between about 5 mg/kg and about 15 mg/kg of body weight. In some embodiments of the method or use, the binding agent is administered at a dose of about 10 mg/kg of body weight. In some embodiments of the method or use, the binding agent is administered at a dose of between about 15 mg/kg and about 25 mg/kg of body weight. In some embodiments of the method or use, the binding agent is administered at a dose of about 20 mg/kg of body weight.
  • the binding agent is administered at a dose of about 10 mg/kg, about 12 mg/kg, about 14 mg/kg, about 16 mg/kg, about 18 mg/kg, about 20 mg/kg, about 22 mg/kg, about 24 mg/kg, about 26 mg/kg, about 28 mg/kg, or about 30 mg/kg.
  • the dose is a body weight-based dose.
  • the binding agent is administered once or sequentially.
  • the binding agent is administered once every week (e.g., every 5-9, 6-9, 7-9, 5-8, 5-7, 6-8, 6-7, 7-8 days, or every 7 days), every 2 weeks (e.g., every 2-16, 13-16, 14-16, 12-15, 12-14, 13-15, 13-14, or 14-15 days, or every 14 days), every 3 weeks (e.g., every 19-23, 20-23, 21-23, 19-22, 19-21, 20-22, 20-21, 21-22 days, or every 21 days), every 4 weeks (e.g., every 26-30, 27-30, 28-30, 26-29, 26-28, 27-29, 27-28, 28-29 days, or every 28 days), every 5 weeks (e.g., every 33-37, 34-37, 35-37, 34-36, 34-36, 35- 37, 36-37, 35-36 days, or every 35 days), or every 6 weeks (e.g., every 40-44, 40-43
  • the binding agent is administered once every 3 weeks. In some embodiments, the binding agent is administered once every 21 days. In some embodiments, the binding agent is administered once every 6 weeks. In some embodiments, the binding agent is administered once every 42 days. [00404] In some embodiments of the method or use, the binding agent is administered over about 30 minutes, about 60 minutes, or about 90 minutes. In some embodiments of the method or use, the binding agent is administered over about 30 minutes at a dose of ⁇ 600 mg (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg).
  • ⁇ 600 mg e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg.
  • the binding agent is administered over about 60 minutes at a dose of ⁇ 600 mg (e.g., about 600 mg, about 800 mg, about 1000 mg, about 1200 mg, about 1400 mg, about 1600 mg, and about 1800 mg). In some embodiments of the method or use, the binding agent is administered over about 90 minutes at a dose of ⁇ 1800 mg.
  • the dose comprises a starting dose. In some embodiments of the method or use, the dose is modified relative to the starting dose. In some embodiments of the method or use, the dose is modified to be about two thirds of the starting dose. In some embodiments of the method or use, the dose is modified to be about one half of the starting dose.
  • the dose is modified to be about one third of the starting dose.
  • the binding agent is administered intravenously, subcutaneously, or intra-peritoneally. In some embodiments of the method or use, the binding agent is administered intravenously. [00407] In some embodiments of the method or use, the binding agent is administered intravenously at a dose of about 600 mg. In some embodiments of the method or use, the binding agent is administered intravenously at a dose of about 1200 mg. In some embodiments of the method or use, the binding agent is administered intravenously at a dose of about 600 mg, once every 3 weeks (e.g., every 21 days).
  • the binding agent is administered intravenously at a dose of about 1200 mg, once every 3 weeks (e.g., every 21 days). In some embodiments of the method or use, the binding agent is administered intravenously at a dose of about 600 mg over about 30 minutes, once every 3 weeks (e.g., every 21 days). In some embodiments of the method or use, the binding agent is administered intravenously at a dose of about 1200 mg over about 60 minutes, once every 3 weeks (e.g., every 21 days). [00408] In some embodiments of the method or use, the binding agent is administered intravenously at a dose of between about 200 mg and about 1800 mg, once every 6 weeks (e.g., every 42 days).
  • a method for determining receptor occupancy comprising the steps of: a. collecting a first sample (e.g., a blood sample or a tissue biopsy) from a subject treated with the binding agent or antibody of the present disclosure; b. contacting the first sample with a detection agent, wherein optionally the detection agent comprises an antibody or antigen binding fragment thereof, that binds at least one molecule of the binding agent or antibody of the present disclosure; c. collecting a second sample (e.g., a blood sample or a tissue biopsy) from the subject; d. contacting the second sample with the binding agent or antibody of the present disclosure ex vivo; e.
  • a first sample e.g., a blood sample or a tissue biopsy
  • the receptor occupancy is determined between about 20 days and about 30 days after treating the subject with the binding agent or antibody. In some embodiments, the receptor occupancy is determined between 7 days and 21 days after treating the subject with the binding agent or antibody. In some embodiments, the receptor occupancy is determined 7 days, 14 days or 21 days after treating the subject with the binding agent or antibody. [00411] In some embodiments, provided herein is a method for determining receptor occupancy, said method comprising the steps of: a.
  • a sample e.g., a blood sample or a tissue biopsy
  • a first detection agent wherein the detection agent comprises the binding agent or antibody of the present disclosure
  • a second detection agent wherein the second detection agent comprises an antibody or antigen binding fragment thereof, that binds at least one molecule of the binding agent or antibody of the present disclosure and the second detection agent does not compete with the first detection agent
  • determining receptor occupancy by comparing the amount of first detection agent to the amount of second detection agent.
  • the receptor occupancy is determined between about 20 days and about 30 days after treating the subject with the binding agent or antibody. In some embodiments, the receptor occupancy is determined between 7 days and 21 days after treating the subject with the binding agent or antibody. In some embodiments, the receptor occupancy is determined 7 days, 14 days or 21 days after treating the subject with the binding agent or antibody. [00413] In certain embodiments, the sample is a peripheral blood sample. In certain embodiments, the sample is a tissue biopsy. In certain embodiments, the sample is a tumor biopsy.
  • the sample is labeled with markers for certain cell types (e.g., immune cells, e.g., monocytes, myeloid derived suppressor cells (MDSCs), pan-myeloid cells, CD33+ myeloid cells).
  • the sample is co-stained with competitive and non-competitive binding agents that bind to IL2 and/or IL4 to determine the receptor occupancy.
  • the subject is treated with a single dose of the binding agent or antibody at between about 1 mg/kg and about 100 mg/kg.
  • the subject is treated with a single dose of the binding agent or antibody at between about 1 mg/kg and about 30 mg/kg, between about 5 mg/kg and about 30 mg/kg, between about 10 mg/kg and about 30 mg/kg, between about 15 mg/kg and about 30 mg/kg, between about 20 mg/kg and about 30 mg/kg, between about 25 mg/kg and about 30 mg/kg, between about 1 mg/kg and about 25 mg/kg, between about 5 mg/kg and about 25 mg/kg, between about 10 mg/kg and about 25 mg/kg, between about 15 mg/kg and about 25 mg/kg, between about 20 mg/kg and about 25 mg/kg, between about 1 mg/kg and about 20 mg/kg, between about 5 mg/kg and about 20 mg/kg, between about 10 mg/kg and about 20 mg/kg, between about 15 mg/kg and about 20 mg/kg between about 1 mg/kg and about 15 mg/kg, between about 5 mg/kg and about 15 mg/kg, between about 10 mg/kg and about 15 mg/kg, between about 10 mg/
  • the subject is treated with a single dose of the binding agent or antibody at about 10 mg/kg. In some embodiments, the subject is treated with a single dose of the binding agent or antibody at about 20 mg/kg. In some embodiments, the subject is treated with a single dose of the binding agent or antibody at 4 mg/kg. In some embodiments, the dose is a body weight-based dose.
  • the subject is treated with a single dose of between about 60 mg and about 1800 mg, between about 60 mg and about 1500 mg, between about 60 mg and about 1200 mg, between about 60 mg and about 900 mg, between about 60 mg and about 800 mg, between about 60 mg and about 600 mg, between about 60 mg and about 400 mg, between about 60 mg and about 200 mg, between about 200 mg and about 1800 mg, between about 200 mg and about 1500 mg, between about 200 mg and about 1200 mg, between about 200 mg and about 900 mg, between about 200 mg and about 800 mg, between about 200 mg and about 600 mg, between about 200 mg and about 400 mg, between about 400 mg and about 1800 mg, between about 400 mg and about 1500 mg, between about 400 mg and about 1200 mg, between about 400 mg and about 900 mg, between about 400 mg and about 800 mg, between about 400 mg and about 600 mg, between about 600 mg and about 1800 mg, between about 400 mg and about 1500 mg, between about 400 mg and about 1200 mg, between about 400 mg and about 900 mg, between about 400 mg and about 800 mg, between about
  • the subject is treated with a single dose of about 1 mg, about 3 mg, about 5 mg, about 6 mg, about 20 mg, about 60 mg, about 200 mg, about 600 mg, about 1200 mg, about 1500 mg, about 1800 mg, about 2100 mg, or about 2400 mg.
  • the subject is treated with a single dose of between about 400 mg and about 700 mg, between about 500 mg and about 800 mg, or between about 500 mg and about 700 mg.
  • the subject is treated with a single dose of about 600 mg.
  • the subject is treated with a single dose of between about 1000 mg and about 1300 mg, between about 1100 mg and about 1400 mg, or between about 1100 mg and about 1300 mg.
  • the subject is treated with a single dose of about 1200 mg. In some embodiments of the method or use, the dose is a flat dose.
  • at least one sample e.g., a blood sample or a tissue biopsy
  • at least one sample is collected immediately following the end of infusion into the subject of the binding agent, or a 4 hours, 24 hours, 72 hours, 168 hours, or 336 hours following the end of infusion.
  • At least one sample e.g., a blood sample or a tissue biopsy
  • second and subsequent infusions of the binding agent are administered about 14 days, 21 days, 28 days, or 35 days after the previous infusion of binding agent.
  • the binding agent or antibody is administered at a dose that achieves or can achieve between 1% and 99% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 1% receptor occupancy in immune cells.
  • the binding agent or antibody is administered at a dose that achieves or can achieve at least 5% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 15% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 20% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 25% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 30% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 35% receptor occupancy in immune cells.
  • the binding agent or antibody is administered at a dose that achieves or can achieve at least 40% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 45% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 50% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 55% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 60% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 65% receptor occupancy in immune cells.
  • the binding agent or antibody is administered at a dose that achieves or can achieve at least 70% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or is administered at a dose that achieves or can achieve at least 75% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 80% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 85% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 90% receptor occupancy in immune cells.
  • the binding agent or antibody is administered at a dose that achieves or can achieve at least 95% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 96% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 97% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 98% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve at least 99% receptor occupancy in immune cells. In some embodiments, the binding agent or antibody is administered at a dose that achieves or can achieve 100% receptor occupancy in immune cells.
  • the binding agent or antibody is administered at a dose that achieves or can achieve at least about 95% (e.g., 95%, 96%, 97%, 98%, 99%, 100%) receptor occupancy in immune cells.
  • the binding agent is administered at a dose that achieves at least about 95% of receptor occupancy in immune cells within 1 hour, within 2 hours, within 3 hours, within 4 hour, within 5 hour, within 6 hour, within 8 hour, within 10 hour, within 12 hour, within 14 hour, within 16 hour, within 18 hour, within 20 hour, within 22 hour, or within 24 hour of administration.
  • the binding agent is administered at a dose that achieves at least about 95% of receptor occupancy in immune cells within 24 hours of administration.
  • the binding agent is administered at a dose that achieves at least about 75% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more) receptor occupancy in immune cells.
  • the binding agent is administered at a dose that achieves at least about 75% receptor occupancy in immune cells 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or 25 days after administration.
  • the binding agent is administered at a dose that achieves at least about 75% receptor occupancy in immune cells 21 days after administration.
  • the binding agent is administered at a dose that achieves at least about 75% receptor occupancy in immune cells 3 weeks after administration.
  • the immune cells are monocytes.
  • the immune cells are myeloid derived suppressor cells (MDSCs).
  • the immune cells are pan-myeloid cells.
  • the immune cells are CD33+ myeloid cells.
  • the immune cells are B cells.
  • the immune cells are T cells.
  • the immune cells are natural killer cells.
  • the immune cells are granulocytes.
  • the immune cells are myeloid antigen presenting cells. 5.6.
  • compositions comprising an ILT- binding agent described herein and a pharmaceutically acceptable vehicle.
  • the present disclosure also provides pharmaceutical compositions comprising an ILT2-binding agent described herein and a pharmaceutically acceptable vehicle.
  • pharmaceutical compositions comprising an ILT4-binding agent described herein and a pharmaceutically acceptable vehicle.
  • pharmaceutical compositions comprising an ILT2/ILT4-binding agent described herein and a pharmaceutically acceptable vehicle.
  • Formulations are prepared for storage and use by combining a purified antibody or agent of the present disclosure with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient).
  • Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin
  • the formulation is in the form of an aqueous solution. In some embodiments, the formulation is stored in a lyophilized or in an alternative dried form. [00426]
  • the ILT-binding agents of the present disclosure can be formulated in any suitable form for delivery to a target cell/tissue.
  • the ILT-binding agent (e.g., the ILT2-binding agent, the ILT4-binding agent, or the ILT2/ILT4-binding agent) can be formulated as a liposome, microparticle, microcapsule, albumin microsphere, microemulsion, nanoparticle, nanocapsule, or macroemulsion.
  • an ILT-binding agent e.g., the ILT2-binding agent, an ILT4- binding agent, or an ILT2/ILT4-binding agent
  • liposomes Methods to produce liposomes are known to those of skill in the art.
  • an ILT-binding agent e.g., the ILT2-binding agent, an ILT4- binding agent, or an ILT2/ILT4-binding agent
  • a sustained-release preparation e.g., the sustained-release preparation.
  • sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing an agent, where the matrices are in the form of shaped articles (e.g., films or microcapsules).
  • Sustained-release matrices include but are not limited to polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
  • polyesters such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic
  • compositions or formulations of the present disclosure can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular). 5.7.
  • the present disclosure further provides methods for predicting a subject’s responsiveness to a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4 (e.g., a binding agent disclosed in Section 5.2 herein), where the subject (e.g., a human subject) has a cancer or a tumor.
  • the present disclosure further provides methods for determining the efficacy of a therapy in a subject (e.g., a human subject) having a cancer or a tumor, where the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4 (e.g., a binding agent disclosed in Section 5.2 herein).
  • CD163 Cluster of Differentiation 163 also known as M130, MM130, and SCARI1, is a protein that is encoded by the CD163 gene in human.
  • CD163 functions as an innate immune sensor for gram-positive and gram-negative bacteria
  • the present disclosure provides a method for identifying a subject having a cancer or a tumor as more likely to respond to a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, wherein the method comprises: comparing the expression level of CD163 in a sample obtained from the subject before receiving the therapy to a CD163 reference level, wherein the subject is identified as more likely to respond to the therapy if the expression level of CD163 is higher than the CD163 reference level.
  • the method further comprises administering the therapy to the subject identified as more likely to respond to the therapy.
  • the present disclosure provides a method for identifying a subject having a cancer or a tumor as more likely to respond to a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, where the method comprises: (a) measuring the expression level of CD163 in a sample obtained from the subject before receiving the therapy; (b) comparing the expression level of CD163 in (a) to a CD163 reference level; and (c) identifying the subject as more likely to respond to the therapy if the expression level of CD163 is higher than the CD163 reference level. In certain embodiments, the method further comprises administering the therapy to the subject identified as more likely to respond to the therapy.
  • the present disclosure provides a method for treating a subject having a cancer or a tumor, comprising: (a) comparing the expression level of CD163 in a sample obtained from the subject before receiving a therapy to a CD163 reference level, wherein the therapy comprises a binding agent specifically binding to human ILT2 and/or human ILT4; and (b) administering the therapy to the subject if the expression level of CD163 is higher than the CD163 reference level.
  • the present disclosure provides a method for treating a subject having a cancer or a tumor, comprising: (a) measuring the expression level of CD163 in a sample obtained from the subject before receiving a therapy, wherein the therapy comprises a binding agent specifically binding to human ILT2 and/or human ILT4; (b) comparing the expression level of CD163 to a CD163 reference level; and (c) administering the therapy to the subject if the expression level of CD163 is higher than the CD163 reference level.
  • the present disclosure provides a method for selectively treating a cancer or a tumor in a subject identified to have a higher expression level of CD163 than a CD163 reference level, the method comprising administering a therapeutically effective amount of a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4 to said subject.
  • the CD163 reference level is the expression level of CD163 in a sample from a healthy and/or non-diseased part of the body (e.g., tissue or cells) of the same subject.
  • the CD163 reference level is the expression level of CD163 in a sample from the same subject collected prior to having the cancer or tumor.
  • the CD163 reference level is the expression level of CD163 in a sample from a healthy or non-diseased subject. In certain embodiments, the CD163 reference level is the expression level of CD163 (e.g., a median or a mean value) in samples from healthy or non- diseased subjects. In certain embodiments, the CD163 reference level is a predetermined value based on the expression levels of CD163 in health or non-diseased populations. In certain embodiments, the sample for generating the CD163 reference level is from the same tissue (e.g., both are blood samples, liver samples, pancreas samples) as the sample of the subject having the cancer or tumor.
  • tissue e.g., both are blood samples, liver samples, pancreas samples
  • the present disclosure provides a method for determining the efficacy of a therapy in a subject having a cancer or a tumor, wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, and the method comprises: comparing (a) the expression level of CD163 in a sample obtained from the subject before receiving the therapy and (b) the expression level of CD163 in a sample obtained from the subject after receiving the therapy, wherein the therapy is determined to be effective if the expression level of CD163 in (b) is reduced as compared to the expression level of CD163 in (a). In certain embodiments, the method further comprises continuing administering to the subject the therapy determined to be effective.
  • the present disclosure provides a method for determining the efficacy of a therapy in a subject having a cancer or a tumor, wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, and the method comprises: (a) measuring (i) the expression level of CD163 in a sample obtained from the subject before receiving the therapy, and (ii) the expression level of CD163 in a sample obtained from the subject after receiving the therapy; (b) comparing the expression level of CD163 in (a- i) to the expression level of CD163 in (a-ii); and (c) determining the therapy is effective if the expression level of CD163 in (a-ii) is reduced as compared to the expression level of CD163 in (a-i).
  • the method further comprises continuing administering to the subject the therapy determined to be effective.
  • the present disclosure provides a method for determining the efficacy of a therapy in a subject having a cancer or a tumor, wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, and the method comprises: comprises comparing (a) the expression level of CD163 in a sample obtained from the subject before receiving the therapy and (b) the expression level of CD163 in a sample obtained from the subject after receiving the therapy, wherein the therapy is determined to be not effective if the expression level of CD163 in (b) is not reduced (e.g., increased) as compared to the expression level of CD163 in (a).
  • the method further comprises increasing the dose or dosing frequency of the binding agent where the therapy is determined to be not effective.
  • the present disclosure provides a method for determining the efficacy of a therapy in a subject having a cancer or a tumor, wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, and the method comprises: (a) measuring (i) the expression level of CD163 in a sample obtained from the subject before receiving the therapy, and (ii) the expression level of CD163 in a sample obtained from the subject after receiving the therapy; (b) comparing the expression level of CD163 in (a- i) to the expression level of CD163 in (a-ii); and (c) determining the therapy is not effective if the expression level of CD163 in (a-ii) is not reduced (e.g., increased) as compared to the expression level of CD163 in (a-i).
  • the method further comprises increasing the dose or dosing frequency of the binding agent where the therapy is determined to be not effective.
  • the present disclosure further provides a method of treating a subject having a cancer or a tumor, wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, and the method comprises: comparing (a) the expression level of CD163 in a sample obtained from the subject before receiving the therapy and (b) the expression level of CD163 in a sample obtained from the subject after receiving the therapy; and continuing administering the therapy to the subject if the expression level of CD163 in (b) is reduced as compared to the expression level of CD163 in (a).
  • the present disclosure further provides a method of treating a subject having a cancer or a tumor, comprising: (a) measuring (i) the expression level of CD163 in a sample obtained from the subject before receiving a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, and (ii) the expression level of CD163 in a sample obtained from the subject after receiving the therapy; (b) comparing the expression level of CD163 in (a- i) to the expression level of CD163 in (a-ii); and (c) continuing administering the therapy to the subject if the expression level of CD163 in (a-ii) is reduced as compared to the expression level of CD163 in (a-i).
  • the present disclosure further provides a method of treating a subject having a cancer or a tumor, wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, and the method comprises: comparing (a) the expression level of CD163 in a sample obtained from the subject before receiving the therapy and (b) the expression level of CD163 in a sample obtained from the subject after receiving the therapy; and increasing the dose or dosing frequency of the binding agent if the expression level of CD163 in (b) is not reduced (e.g., increased) as compared to the expression level of CD163 in (a).
  • the present disclosure further provides a method of treating a subject having a cancer or a tumor, comprising; (a) measuring (i) the expression level of CD163 in a sample obtained from the subject before receiving a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, and (ii) the expression level of CD163 in a sample obtained from the subject after receiving the therapy; (b) comparing the expression level of CD163 in (a- i) to the expression level of CD163 in (a-ii); and (c) increasing the dose or dosing frequency of the binding agent if the expression level of CD163 in (a-ii) is not reduced (e.g., increased) as compared to the expression level of CD163 in (a-i).
  • the sample is a peripheral blood sample.
  • the sample is a tissue biopsy.
  • the sample is a tumor biopsy.
  • the sample is a whole blood lysate.
  • the sample comprises cells isolated from peripheral blood or a tissue biopsy (e.g., tumor biopsy) of the subject.
  • the isolated cells are selected from the group consisting of monocytes, MDSCs, pan-myeloid cells, pan-dendritic cells, and combinations thereof.
  • the monocytes are selected from the group consisting of classical monocytes (CD14 + CD16-), non-classical monocytes (CD14- CD16 + ), intermediate monocytes (CD14 + CD16 + ), and combinations thereof.
  • the pan-myeloid cells are selected from the group consisting of CD11b + CD15- cells, CD19-HLA.DR + cells, CD3-CD19-CD56-CD15-cells, and combinations thereof.
  • the pan dendritic cells are selected from the group consisting of D1 cells, D2 cells, and combinations thereof.
  • the expression level of CD163 is a protein expression level of CD163.
  • the expression level of CD163 is an mRNA expression level of CD163.
  • Any suitable methods known in the art can be used with the presently disclosed subject matter for measuring the expression level of CD163.
  • the protein expression level of CD163 can be measured by methods including, but not limited to, flow cytometry, immunohistochemistry, Western Blot, and enzyme-linked immunosorbent assay (ELISA).
  • the mRNA expression level can be measured by methods including, but not limited to, PCR, qPCR, quantitative reverse- transcriptase PCR (RT-qPCR), microarray, Northern blot, and RNA sequencing.
  • the therapy comprises a dosing regimen comprising administering to the subject multiple doses of the binding agent (e.g., a dosing regimen disclosed in Section V of the present disclosure).
  • the sample obtained from the subject after receiving the therapy is a sample obtained after the subject received one dose (e.g., a first dose) of the binding agent.
  • the sample obtained from the subject after receiving the therapy is a sample obtained after the subject received multiple doses (e.g., one, two, three, four, five, six, or more doses) of the binding agent.
  • the sample obtained from the subject after receiving the therapy is a sample obtained after the subject received a first dose of the binding agent. In certain embodiments, the sample obtained from the subject after receiving the therapy is a sample obtained after the subject received a second dose of the binding agent. [00452] In certain embodiments, the sample obtained from the subject after receiving the therapy is a sample obtained between 1 day and 40 days after the subject received a dose (e.g., a first dose) of the binding agent. In certain embodiments, the sample obtained from the subject after receiving the therapy is a sample obtained from the subject between 15 days and 36 days after the subject receiving a dose (e.g., a first dose) of the binding agent.
  • the sample obtained from the subject after receiving the therapy is a sample obtained from the subject between 15 days after the subject receiving the first dose and 15 days after the subject receiving the second dose (e.g., between 15 days and 36 days after the subject receiving a first dose of the binding agent when the agent is administered once every 21 days).
  • the sample obtained from the subject after receiving the therapy is a sample obtained at least 7 days (e.g., 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, or more) after the subject received a dose (e.g., a first dose) of the binding agent.
  • a dose e.g., a first dose
  • the sample obtained from the subject before receiving the therapy is a sample obtained from the subject before receiving any dose of the binding agent.
  • the sample obtained from the subject before receiving the therapy is a sample obtained from the subject before receiving a first dose of the binding agent.
  • the sample obtained from the subject before receiving the therapy is a sample obtained from the subject before receiving a dose of the binding agent but after the subject has received a prior dose of the binding agent.
  • the subject is a human subject.
  • the subject having a cancer selected from the group consisting of hepatocellular carcinoma, small cell lung cancer, thyroid cancer, mesothelioma, glioblastoma, renal cell carcinoma, non-small cell lung cancer, melanoma (skin cutaneous and uveal), pancreatic ductal adenocarcinoma, gastric cancer, squamous cell carcinoma of the head and neck, biliary duct cancer, breast cancer, triple-negative breast cancer, ovarian cancer, cervical cancer, endocervical cancer, colorectal cancer, esophageal cancer, gastroesophageal junction adenocarcinoma, squamous cell carcinoma of head and neck, bladder cancer, uterine cancer, cholangiocarcinoma, and prostate cancer.
  • a cancer selected from the group consisting of hepatocellular carcinoma, small cell lung cancer, thyroid cancer, mesothelioma, glioblastoma, renal cell carcinoma, non-small cell lung cancer,
  • the subject having a tumor selected from the group consisting of a hepatocellular carcinoma, a small cell lung tumor, a thyroid tumor, a mesothelioma, a glioblastoma, a renal cell carcinoma, a non-small cell lung tumor, a melanoma (skin cutaneous and uveal), a pancreatic ductal adenocarcinoma, a gastric tumor, a squamous cell carcinoma of the head and neck, a biliary duct tumor, a breast tumor, a triple-negative breast tumor, an ovarian tumor, a cervical tumor, an endocervical tumor, a colorectal tumor, an esophageal tumor, a gastroesophageal junction adenocarcinoma, a squamous cell carcinoma of head and neck, a bladder tumor, an uterine tumor, a cholangiocarcinoma, or a prostate tumor.
  • a tumor selected from the group
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of (a) a binding agent comprising (i) a heavy chain variable region comprising a heavy chain variable region CDR1 (VH-CDR1), a heavy chain variable region CDR2 (VH-CDR2), and a heavy chain variable region CDR3 (VH-CDR3) from the amino acid sequence of SEQ ID NO:144; and a light chain variable region comprising a light chain variable region CDR1 (VL-CDR1), a light chain variable region CDR2 (VL-CDR2), and a light chain variable region CDR3 (VL-CDR3) from the amino acid sequence of SEQ ID NO:145; or (ii) a heavy chain variable region comprising a heavy chain variable region CDR1 (VH-CDR1), a heavy chain variable region CDR2 (VH-CDR2),
  • the cancer is hepatocellular carcinoma, small cell lung cancer, thyroid cancer, mesothelioma, glioblastoma, renal cell carcinoma, non-small cell lung cancer, melanoma (skin cutaneous and uveal), pancreatic ductal adenocarcinoma, gastric cancer, squamous cell carcinoma of the head and neck, biliary duct cancer, breast cancer, triple- negative breast cancer, ovarian cancer, cervical cancer, endocervical cancer, colorectal cancer, esophageal cancer, gastroesophageal junction adenocarcinoma, squamous cell carcinoma of head and neck, bladder cancer, uterine cancer, cholangiocarcinoma, or prostate cancer.
  • the cancer is hepatocellular carcinoma, small cell lung cancer, thyroid cancer, mesothelioma, glioblastoma, renal cell carcinoma, non-small cell lung cancer, melanoma (skin cutaneous and uveal), pan
  • the immune cells are selected from the group consisting of granulocytes, myeloid antigen presenting cells, natural killer cells, monocytes, CD33 + myeloid cells, T cells, and B cells.
  • the binding agent is administered once every week, 2 weeks, 3 weeks, or 4 weeks.
  • the method of embodiment 10 is administered once every 3 weeks.
  • any one of embodiments 1-3 wherein the binding agent is administered over about 30 minutes at a dose of ⁇ 600 mg. 14. The method of any one of embodiments 1-3, wherein the binding agent is administered over about 60 minutes at a dose of ⁇ 600 mg. 15. The method of any one of embodiments 1-14, wherein the binding agent is administered intravenously. 16. The method of any one of embodiments 1-3, wherein the binding agent is administered intravenously at a dose of about 600 mg once every 3 weeks. 17. The method of any one of embodiments 1-3, wherein the binding agent is administered intravenously at a dose of about 1200 mg once every 3 weeks. 18.
  • the binding agent is administered intravenously at a dose of about 1800 mg once every 3 weeks. 19.
  • the heavy chain variable region of the binding agent comprises the VH-CDR1 having the amino acid sequence of SEQ ID NO:115, the VH-CDR2 having the amino acid sequence of SEQ ID NO:112, and the VH-CDR3 having the amino acid sequence of SEQ ID NO:107; and the light chain variable region of the binding agent comprises the VL-CDR1 having the amino acid sequence of SEQ ID NO:89, the VL-CDR2 having the amino acid sequence of SEQ ID NO:90, and the VL-CDR3 having the amino acid sequence of SEQ ID NO:91; (2) the heavy chain variable region of the binding agent comprises the VH-CDR1 having the amino acid sequence of SEQ ID NO:111, the VH-CDR2 having the amino acid sequence of SEQ ID NO:112, and the VH-CDR3 having the amino
  • the heavy chain variable region of the binding agent comprises the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2, and the VL-CDR3 comprising amino acid sequences of any one of the five CDR definitions set forth in Table 8A; or (2) the heavy chain variable region of the binding agent comprises the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2, and the VL-CDR3 comprising amino acid sequences of any one of the five CDR definitions set forth in Table 8B. 21.
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:144;
  • the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:145; or
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:144 and the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:145. 22.
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:143;
  • the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:142; and/or (c) the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:143 and the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:142.
  • the heavy chain variable region of the binding agent comprises the VH-CDR1 having the amino acid sequence of SEQ ID NO:115, the VH-CDR2 having the amino acid sequence of SEQ ID NO:112, and the VH-CDR3 having the amino acid sequence of SEQ ID NO:107; and the light chain variable region of the binding agent comprises the VL-CDR1 having the amino acid sequence of SEQ ID NO:89, the VL-CDR2 having the amino acid sequence of SEQ ID NO:90, and the VL-CDR3 having the amino acid sequence of SEQ ID NO:91; (2) the heavy chain variable region of the binding agent comprises the VH-CDR1 having the amino acid sequence of SEQ ID NO:111, the VH-CDR2 having the amino acid sequence of SEQ ID NO:112, and the VH-CDR3 having the amino acid sequence of SEQ ID NO:107; and the light chain variable region of the binding agent comprises the VL-CDR1 having the amino acid sequence of SEQ ID NO:
  • the heavy chain variable region of the binding agent comprises the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2, and the VL-CDR3 comprising amino acid sequences of any one of the five CDR definitions set forth in Table 8A; or (2) the heavy chain variable region of the binding agent comprises the VH-CDR1, the VH-CDR2, the VH-CDR3, the VL-CDR1, the VL-CDR2, and the VL-CDR3 comprising amino acid sequences of any one of the five CDR definitions set forth in Table 8B. 28.
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:144;
  • the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:145; or
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:144 and the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:145.
  • the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:143;
  • the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:142; and/or (c) the heavy chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:143 and the light chain variable region of the binding agent comprises an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:142.
  • the heavy chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:143; (b) the light chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:142; and/or (c) the heavy chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:143 and the light chain variable region of the binding agent comprises the amino acid sequence of SEQ ID NO:142.
  • the tumor comprises a microsatellite instability – high (MSI-H) tumor or a deficient mismatch repair (dMMR) positive tumor.
  • the tumor is a hepatocellular carcinoma, a small cell lung tumor, a thyroid tumor, a mesothelioma, a glioblastoma, a renal cell carcinoma, a non-small cell lung tumor, a melanoma (skin cutaneous and uveal), a pancreatic ductal adenocarcinoma, a gastric tumor, a squamous cell carcinoma of the head and neck, a biliary duct tumor, a breast tumor, a triple-negative breast tumor, an ovarian tumor, a cervical tumor, an endocervical tumor, a colorectal tumor, an esophageal tumor, a gastroesophageal junction adenocarcinoma, a squamous cell carcinoma of head and neck, a bladder tumor, an uterine tumor, a cholangiocarcinoma, or a prostate tumor.
  • the tumor is a hepatocellular carcinoma, a small cell lung
  • the binding agent is (a) an antibody; (b) a recombinant antibody; (c) an antibody fragment comprising at least one antigen-binding site; (d) a chimeric antibody; (e) a humanized antibody; (f) a bispecific or multispecific antibody; or (g) attached to a half-life extending moiety.
  • the binding agent is an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody; optionally wherein the binding agent is a human IgG1 antibody, a human IgG2 antibody, or a human IgG4 antibody; further optionally the human IgG1 antibody has reduced or no effector function.
  • the binding agent comprises a kappa light chain or a lambda light chain
  • the antibody comprises a human kappa light chain or a human lambda light chain.
  • the antibody fragment is a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, a (scFv)2, a single chain antibody, a dual variable region antibody, a diabody, or a nanobody. 38.
  • the binding agent comprises a heavy chain comprising an amino acid sequence with 80% identity to the sequence of SEQ ID NO:156 and a light chain comprising an amino acid sequence with 80% identity to the sequence of SEQ ID NO:157.
  • the binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:156 and a light chain comprising the amino acid sequence of SEQ ID NO:157. 40.
  • the binding agent has one or more of the following properties: (1) binds rhesus ILT2; (2) binds cyno ILT2; (3) does not bind ILT3, ILT5, and LILRB5; (4) does not bind LILRA2, LILRA4, LILRA5, and LILRA6; (5) is an ILT2 antagonist; (6) is an ILT4 antagonist, (7) inhibits ILT2 activity; (8) inhibits ILT4 activity; (9) inhibits ILT2 signaling in cells that express ILT2; (10) inhibits ILT4 signaling in cells that express ILT4; (11) inhibits binding of ILT2 to MHC I molecules; (12) inhibits binding of ILT4 to MHC I molecules; (13) inhibits ILT2-induced suppression of myeloid cells; (14) inhibits ILT4-induced suppression of myeloid cells; (15) inhibits ILT2-induced suppression of myeloid cell activity; (16) inhibits ILT4-induced suppression of myeloid cell activity
  • a method for identifying a subject having a cancer or a tumor as more likely to respond to a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, the method comprising comparing the expression level of CD163 in a sample of the subject before receiving the therapy to a CD163 reference level, wherein the subject is identified as more likely to respond to the therapy if the expression level of CD163 is higher than the CD163 reference level. 43.
  • a method for identifying a subject having a cancer or a tumor as more likely to respond to a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, the method comprising: (a) measuring the expression level of CD163 in a sample of the subject before receiving the therapy; (b) comparing the expression level of CD163 in (a) to a CD163 reference level; and (c) identifying the subject as more likely to respond to the therapy if the expression level of CD163 is higher than the CD163 reference level.
  • a method for selectively treating a cancer or a tumor in a subject identified to have a higher expression level of CD163 than a CD163 reference level comprising administering a therapeutically effective amount of a binding agent specifically binding to human ILT2 and/or human ILT4 to the subject.
  • a method for treating a subject having a cancer or a tumor comprising: (a) comparing the expression level of CD163 in a sample of the subject before receiving a therapy to a CD163 reference level, wherein the therapy comprises a binding agent specifically binding to human ILT2 and/or human ILT4; and (b) administering a therapeutically effective amount of the binding agent to the subject if the expression level of CD163 is higher than the CD163 reference level. 46.
  • a method for treating a subject having a cancer or a tumor comprising: (a) measuring the expression level of CD163 in a sample obtained from the subject before receiving a therapy, wherein the therapy comprises a binding agent specifically binding to human ILT2 and/or human ILT4; (b) comparing the expression level of CD163 to a CD163 reference level; and (c) administering the therapy to the subject if the expression level of CD163 is higher than the CD163 reference level.
  • the CD163 reference level is the expression level of CD163 in a sample from a healthy or non-diseased subject, or the expression level of CD163 in samples from a population of healthy or non-diseased subjects, wherein the sample from the healthy or non-diseased subject or the samples from a population of healthy or non-diseased subjects are from the same source as the sample from the subject having the cancer or tumor.
  • a method for determining the efficacy of a therapy in a subject having a cancer or a tumor wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, the method comprising comparing (a) the expression level of CD163 in a sample of the subject before receiving the therapy and (b) the expression level of CD163 in a sample of the subject after receiving the therapy, wherein the therapy is determined to be effective if the expression level of CD163 in (b) is reduced as compared to the expression level of CD163 in (a).
  • a method of treating a subject having a cancer or a tumor wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, and the method comprises: (a) comparing (i) the expression level of CD163 in a sample of the subject before receiving the therapy and (ii) the expression level of CD163 in a sample of the subject after receiving the therapy; and (b) continuing administering the therapy to the subject if the expression level of CD163 in (a-ii) is reduced as compared to the expression level of CD163 in (a-i). 51.
  • a method of treating a subject having a cancer or a tumor comprising: (a) measuring (i) the expression level of CD163 in a sample of the subject before receiving a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, and (ii) the expression level of CD163 in a sample of the subject after receiving the therapy; (b) comparing the expression level of CD163 in (a-i) to the expression level of CD163 in (a-ii); and (c) continuing administering the therapy to the subject if the expression level of CD163 in (a-ii) is reduced as compared to the expression level of CD163 in (a-i). 52.
  • a method of treating a subject having a cancer or a tumor wherein the therapy comprises a binding agent that specifically binds to human ILT2 and/or human ILT4, and the method comprises: (a) comparing (i) the expression level of CD163 in a sample of the subject before receiving the therapy and (ii) the expression level of CD163 in a sample of the subject after receiving the therapy; and (b) increasing the dose or dosing frequency of the binding agent if the expression level of CD163 in (a-ii) is not reduced as compared to the expression level of CD163 in (a-i). 53.
  • a method of treating a subject having a cancer or a tumor comprising: (a) measuring (i) the expression level of CD163 in a sample of the subject before receiving a therapy comprising a binding agent specifically binding to human ILT2 and/or human ILT4, and (ii) the expression level of CD163 in a sample of the subject after receiving the therapy; (b) comparing the expression level of CD163 in (a-i) to the expression level of CD163 in (a-ii); and (c) increasing the dose or dosing frequency of the binding agent if the expression level of CD163 in (a-ii) is not reduced as compared to the expression level of CD163 in (a-i). 54.
  • any one of embodiments 42-53 wherein the sample is a peripheral blood sample or a tumor biopsy.
  • the sample comprises cells isolated from peripheral blood or a tumor biopsy of the subject.
  • the isolated cells are selected from the group consisting of monocytes, MDSCs, pan-myeloid cells, pan-dendritic cells, and combinations thereof.
  • the expression level of CD163 is a protein expression level of CD163.
  • the protein expression level is measured by flow cytometry, immunohistochemistry, Western Blot, or enzyme-linked immunosorbent assay (ELISA). 59.
  • the mRNA expression level is measured by quantitative reverse-transcriptase PCR (RT-qPCR), microarray, Northern blot, or RNA sequencing.
  • RT-qPCR quantitative reverse-transcriptase PCR
  • microarray microarray
  • Northern blot or RNA sequencing.
  • RNA sequencing RNA sequencing.
  • the sample obtained from the subject after receiving the therapy is a sample obtained after the subject received one dose or multiple doses of the binding agent.
  • any one of embodiments 48-61 wherein the sample obtained from the subject after receiving the therapy is a sample obtained from the subject between 15 days after the subject received a first dose of the binding agent and 15 days after the subject received a second dose of the binding agent. 63.
  • the antigen-binding domain binds specifically to both human ILT2 and human ILT4 and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity-determining region 1 (VH-CDR1), a heavy chain complementarity-determining region 2 (VH-CDR2), and a heavy chain complementarity-determining region 3 (VH-CDR3) from the amino acid sequence of SEQ ID NO:144; and a light chain variable region (VL) comprising a light chain complementarity- determining region 1 (VL-CDR1), a light chain complementarity-determining region 2 (VL- CDR2), and a light chain complementarity-determining region 3 (VL-CDR3) from the amino acid sequence of SEQ ID NO:145.
  • VH heavy chain variable region
  • VH-CDR1 heavy chain complementarity-determining region 1
  • VH-CDR2 heavy chain complementarity-determining region 2
  • VH-CDR3 heavy chain complementarity-determining region 3
  • the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:115
  • the VH- CDR2 comprises the amino acid sequence of SEQ ID NO:112
  • the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:107
  • the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:89
  • the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:90
  • the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:91
  • the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:111
  • the VH- CDR2 comprises the amino acid sequence of SEQ ID NO:112
  • the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:107
  • the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:89
  • the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:90
  • the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:91
  • the antigen-binding domain comprises (a) a heavy chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:156, and/or a light chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:157; or (b) a heavy chain comprising the amino acid sequence of SEQ ID NO:156, and/or a light chain comprising the amino acid sequence of SEQ ID NO:157. 67.
  • the antigen-binding domain binds specifically to both human ILT2 and human ILT4 and comprises: (i) a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO:139; and a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from the amino acid sequence of SEQ ID NO:140; (ii) a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO:133; and a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from the amino acid sequence of SEQ ID NO:134; (iii) a VH comprising a VH-CDR1, a VH-CDR2, and a VVH-CDR3 from the amino acid sequence of S
  • the VH comprises the VH-CDR1, the VH-CDR2, and the VH-CDR3 from the amino acid sequence of SEQ ID NO:139; and the VL comprises the VL-CDR1, the VL-CDR2, and the VL-CDR3 from the amino acid sequence of SEQ ID NO:140, and wherein (a) the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:95, the VH- CDR2 comprises the amino acid sequence of SEQ ID NO:87, the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:102, the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:103, the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:90, and the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:91; (b) the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:86, the VH-CDR2 comprises the amino acid
  • the antigen-binding domain comprises: (a) a heavy chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:148, and/or a light chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:149; (b) a heavy chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:152, and/or a light chain comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:153; (c) a heavy chain comprising the amino acid sequence of SEQ ID NO:148, and/or a light chain comprising the amino acid sequence of SEQ ID NO:149; or (d) a heavy chain comprising the amino acid sequence of SEQ ID NO:152, and/or a light chain comprising the amino acid sequence of SEQ ID NO:153.
  • the antigen-binding domain binds specifically to human ILT2 and comprises a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO:125; and a VL comprising a VL- CDR1, a VL-CDR2, and a VL-CDR3 from the amino acid sequence of SEQ ID NO:126. 72.
  • the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:31
  • the VH-CDR2 comprises the amino acid sequence of SEQ ID NO:23
  • the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:24
  • the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:25
  • the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:26
  • the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:27
  • the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:22
  • the VH-CDR2 comprises the amino acid sequence of SEQ ID NO:23
  • the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:24
  • the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:25
  • the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:26
  • the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:27
  • the antigen-binding domain binds specifically to human ILT4 and comprises: (a) a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO:129; and a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from the amino acid sequence of SEQ ID NO:130; or (b) a VH comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO:127; and a VL comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 from the amino acid sequence of SEQ ID NO:128.
  • the VH comprises the VH-CDR1, the VH-CDR2, and the VH-CDR3 from the amino acid sequence of SEQ ID NO:129; and the VL comprises the VL-CDR1, the VL-CDR2, and the VL-CDR3 from the amino acid sequence of SEQ ID NO:130, and wherein (i) the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:63, the VH- CDR2 comprises the amino acid sequence of SEQ ID NO:55, and the VH-CDR3 comprises the amino acid sequence of SEQ ID NO:56,the VL-CDR1 comprises the amino acid sequence of SEQ ID NO:57, the VL-CDR2 comprises the amino acid sequence of SEQ ID NO:58, and the VL-CDR3 comprises the amino acid sequence of SEQ ID NO:59; (ii) the VH-CDR1 comprises the amino acid sequence of SEQ ID NO:54, the VH-CDR2 comprises the
  • Anti-ILT antibodies were generated using the extracellular domain of human ILT2, the extracellular domain of human ILT4, and/or the extracellular domain of rhesus ILT2 as the immunogen.
  • anti-ILT2 antibodies were generated using the extracellular domain of human ILT2.
  • anti-ILT4 antibodies were generated using the extracellular domain of human ILT4.
  • Anti-ILT2/ILT4 antibodies were generated using a mixture of the extracellular domain of human ILT2, the extracellular domain of human ILT4, and the extracellular domain of rhesus ILT2.
  • lymphocytes Single cell suspensions of lymphocytes were obtained from the spleens and lymph nodes of immunized mice after the individual animals had been determined to have suitable antibody titers. Lymphocytes were fused with murine myeloma cells by standard methods. Hybridoma fusions were plated onto semi-solid media for HAT selection. After 5-7 days, single colonies were selected using a ClonePixTM system and plated into 96-well plates. [00458] ELISA assays were used to screen antibodies against human ILT2, human ILT4, and rhesus ILT2. Antibodies that bound to human ILT2 only, human ILT4 only, or human ILT2, human ILT4 and rhesus ILT2 were selected.
  • genomic structure was used to identify candidate orthologs of both human ILT2 and ILT4 in cynomolgus monkey.
  • the genomic organization of the locus that contains the LILR family members in human (hg38) and cynomolgus monkey (Macaca fascicularis v5.0.95, “cyno”) were compared using the gene annotations provided by Ensembl. In cases where annotations were not found, manual analysis of the intervening sequence was used to confirm lack of an open reading frame.
  • the predicted protein sequence of genes in cynomolgus monkey without pre-annotated gene symbols was compared to all human genes using blastp tools on UniProt to find the most likely human ortholog based on the identity score.
  • Example 3 Binding Characteristics of Anti-human ILT2, ILT4, and ILT2/ILT4 Antibodies
  • the binding affinities of anti-ILT2 and anti-ILT4 antibodies were measured using a Biacore system (GE Healthcare LifeSciences). Equilibrium dissociation constant (K D ) measurements were carried out with purified antibodies to evaluate their binding to human ILT2, human ILT4, and rhesus ILT2. Briefly, purified anti-ILT2 antibody 27F9, anti-ILT4 antibodies 47C8 and 48A5, and anti-ILT2/ILT4 antibodies 47H6, 51A1, 64A12, 73C4, and 73D1 were captured on a Sensor Chip Protein A (GE HealthCare).
  • K D Equilibrium dissociation constant
  • the surface of the Biacore Sensor Chip Protein A consists of a carboxymethylated dextran matrix with a recombinant Protein A variant covalently attached.
  • Soluble human ILT2-ECD, human ILT4-ECD, or rhesus ILT2-ECD were injected at a flow rate of 30 ⁇ L/min at 25°C.
  • the ILT2 or ILT4 proteins were used at concentrations ranging from 1.6 - 200 nM in PBS-P buffer with 2 fold dilutions.
  • Kinetic data were collected over time and fit using the simultaneous global fit equation to yield affinity constants (K D values) for each antibody. [00463] Binding data is shown in Tables 9A and 9B.
  • Table 9A 1 - Binding assay was performed under low resolution parameters
  • Table 9B 1 - Binding assay was performed under low resolution parameters 7.4
  • Example 4 Sequence Analyses of Anti-ILT2, Anti-ILT4, and Anti-ILT2/ILT4 Antibodies
  • Representative anti-ILT2 antibody 27F9, anti-ILT4 antibodies 47C8 and 48A5, and anti-ILT2/ILT4 antibodies 47H6, 51A1, 64A12, 73C4, and 73D1 were sequenced and the VH and VL amino acid sequences are disclosed herein and summarized in Table 10.
  • Table 10 [00465] The heavy chain and VL-CDRs for the individual antibodies are disclosed in Tables 1- 8 and as SEQ ID NOs:22-124.
  • Example 5 Generation of Humanized Antibodies
  • Several of the anti-ILT2/ILT4 antibodies i.e., 47H6, 64A12, and 73D1 were humanized by methods known to those skilled in the art. These humanized antibodies are referred to herein as Hz47H6.v2, Hz64A12, and Hz73D1.v1, respectively.
  • the VH-CDR2 was modified from DFNPNNGGTTYNQKFEG (SEQ ID NO:71) to DFNPNNAGTTYNQKFEG (SEQ ID NO:118).
  • the VH sequence of Hz47H6.v2 is SEQ ID NO:133 and the VL sequence of Hz47H6.v2 is SEQ ID NO:134; the VH sequence of Hz64A12 is SEQ ID NO:139 and the VL sequence of Hz64A12 is SEQ ID NO:140; and the VH sequence of Hz73D1.v1 is SEQ ID NO:144 and the VL sequence of Hz73D1.v1 is SEQ ID NO:145.
  • the binding affinities of the humanized antibodies to human ILT2 and human ILT4 were measured using a Biacore system as described herein.
  • humanized anti-ILT2/ILT4 antibodies bind to cyno ILT2. Binding affinities of an exemplary clone Hz73D1.v1 measured by a Biacore system are shown in Table 12. In the Biacore system, a humanized anti-ILT2/ILT4 antibody was captured on a Protein A chip. ILT proteins were injected at different concentrations into the flow cells to evaluate kinetic parameters at 25°C. The binding affinity (KD) of Hz73D1.v1 to human ILT2 and ILT4 was determined to be 1.03 and 0.205 nM, respectively. The KD of Hz73D1.v1 to cyno ILT2 was determined to be 19.1 nM.
  • ILT2 and ILT4 Expression in Subsets of Immune Cells [00470] ILT2 and ILT4 expression has been described on various cells of hematopoietic origin, including myeloid cells, granulocytes, and lymphocytes (Colonna M et al. J Exp Med.
  • PBMC or whole blood samples were stained with a fluorescently labeled antibody panel to distinguish various immune subsets (monocytes, B cells, NK cells, CD4+ and CD8+ T cells, neutrophils and eosinophils). Samples were further stained with fluorescently labeled isotype, 27F9 (ILT2 specific), 48A5 (ILT4 specific), 73D1, and Hz73D1.v1. The number of ILT2 and ILT4 molecules per cell of each immune subset was estimated by incubation with Quantum Simply Cellular microspheres followed by flow cytometry analysis.
  • ILT2 As shown in Table 13 and Figure 1, high expression of ILT2 was observed on monocytes, B cells, a subset of NK cells (5-20% total NK cells), and a subset of CD8+ T cells (5-20% total CD8+ T cells). High expression of ILT4 was observed on monocytes, eosinophils and neutrophils. The results showed that cynomolgus monkey peripheral immune cells expressed ILT2 protein with a level of expression and immune cell distribution comparable with the combination of ILT2 and ILT4 in human peripheral blood.
  • the natural ligands of ILT2 and ILT4 include, but are not limited to, HLA class I molecules, including HLA-A, HLA-B, HLA-C, HLA-E, and HLA-G.
  • HLA class I molecules including HLA-A, HLA-B, HLA-C, HLA-E, and HLA-G.
  • Biotinylated HLA-A*1101 was captured in high amounts on a NeutrAvidin chip surface.
  • Antigen-antibody complexes were prepared with (i) ILT2-ECD and anti-ILT2 antibody 27F9, anti-ILT4 antibody 48A5, or anti-ILT2/ILT4 antibody Hz73D1.v1 and (ii) ILT4-ECD and anti-ILT2 antibody 27F9, anti-ILT4 antibody 48A5, or anti-ILT2/ILT4 antibody Hz73D1.v1.
  • the antibody concentration of each antibody was titrated from 0.09 - 100 nM and the concentration of human ILT2-ECD or ILT4-ECD was kept constant at 20 nM.
  • the complexes were mixed in a 96-well microplate and each was injected onto the HLA-A-coated chip surface.
  • the measured signal (response unit, RU) was plotted against the antibody concentration. [00474] The results are shown in Figure 2. A decrease in binding of ILT2 to HLA-A was observed with increasing concentrations of anti-ILT2 antibody 27F9 and anti-ILT2/ILT4 antibody Hz73D1.v1 in a dose-dependent manner. In contrast, anti-ILT4 antibody 48A5 had no effect on the binding of ILT2 to HLA-A. Similarly, a decrease in binding of ILT4 to HLA-A was observed with increasing concentrations of anti-ILT4 antibody 48A5 and anti-ILT2/ILT4 antibody Hz73D1.v1, with no effect by anti-ILT2 antibody 27F9.
  • anti-ILT2 and anti-ILT2/ILT4 antibodies described herein inhibit the interactions between ILT2 and its natural ligands.
  • anti-ILT4 and anti- ILT2/ILT4 antibodies described herein inhibit the interactions between ILT4 and its natural ligands.
  • this experiment also showed that the anti-ILT2/ILT4 antibody not only bound to both targets, i.e., ILT2 and ILT4, but was biologically functional in blocking the interactions of both targets with their ligands.
  • anti-ILT2/ILT4 antibodies may be a potential therapeutic for blocking ILT2-induced and ILT4-induced suppression of immune responses.
  • Example 8 Binding of Anti-ILT2/ILT4 Antibody to Cells [00476] The binding of anti-ILT2/ILT4 antibodies to monocytes was evaluated. The binding of exemplary anti-ILT2/ILT4 antibody 73D1 and the humanized version Hz73D1.v1 to the CD14+ monocyte population of human and cyno PBMC were determined by flow cytometry analysis.
  • PBMCs human or cyno PBMCs (from 2 donors each) were thawed, washed in media (RPMI, 10% FBS, L-glutamine, pen/strep), and resuspended in fresh media.
  • PBMCs were stained with a cocktail of labeled antibodies to differentiate between the different cell types.1 ⁇ 10 6 cells/well were stained with fluorescently-labelled 73D1 or Hz73D1.v1 antibody and incubated on ice. The cells were washed with ice-cold reagents and immediately analyzed by flow cytometry. The geometric mean fluorescence intensity for CD14+ monocytes was calculated for each sample.
  • Binding of anti-ILT2/ILT4 antibodies to ILT family members was evaluated by fluorescent-activated cell sorting (FACS) using 293T cells expressing human and cynomolgus monkey ILT2, ILT4, LILRA1, ILLRB3, ILLRB4, ILLRB5, ILLRA2, ILLRA4, ILLRA5, and ILLRA6 respectively.
  • the binding affinities of an exemplary anti-ILT2/ILT4 antibody Hz73D1.v1 to human ILT2, human ILT4, human LILRA1, and cyno LILRA1 expressed on 293T cells and measured by flow cytometry are shown to be 1.2nM, 1.4nM, 2.64nM and 1.97 nM respectively.
  • Example 9 Inhibition of the Interaction Between ILT2 or ILT4 and MHC I Molecules by Anti-ILT Antibodies
  • the ability of anti-ILT2/ILT4 antibodies to inhibit the interaction between ILT2 and/or ILT4 and MHC I molecules was evaluated using a reporter cell system.
  • the Raji cell line was established over 50 years ago from a Burkitt lymphoma and has been shown to express MHC I and MHC II molecules.
  • the lymphoblastoid cell line (LCL) 721.221 is a mutant of parent LCL 721, wherein LCL 721.221 does not express any MHC I molecules.
  • LCL 721.221 cells were transfected with the non-classical MHC I molecule HLA-G and a stable cell line was established, referred to herein as 721.221-HLA-G.
  • Raji cells or 721.221-HLA-G cells were co- cultured with cells expressing a stable reporter system and a cell surface receptor of interest ("reporter cells").
  • reporter cells a cell surface receptor of interest
  • the extracellular domain of the receptor of interest e.g., ILT2 or ILT4
  • the extracellular domain of the receptor of interest is fused with the transmembrane/intracellular domain of PILR ⁇ that associates with the adaptor protein DAP12.
  • chimeric receptor e.g., ILT2 or ILT-4
  • a ligand e.g., MHC I molecule
  • DAP12 becomes phosphorylated and activates an NFAT-responsive promoter which drives GFP expression (see, e.g., Deng et al., 2014, Blood, 124:924-935).
  • ILT2 and ILT4 reporter cells expressing human ILT2 or human ILT4 were stained with CellTracker Deep Red (ThermoFisher) to distinguish them from Raji or 721.221-HLA-G cells upon analysis.
  • Reporter cells were washed after staining and resuspended at 1 ⁇ 10 6 cells/ml in X-VIVOTM 15 media (Lonza). Raji cells or 721.221-HLA-G cells were washed and resuspended in X-VIVOTM 15 media at 1 ⁇ 10 6 cell/ml.
  • anti-ILT2 antibody 27F9, anti-ILT4 antibody 48A5, anti-ILT2/ILT4 antibody 73D1 and Hz73D1.v1, and a control antibody were serially diluted and 50 ⁇ l was added to each well of a 96-well flat-bottom cell culture plate.
  • anti-ILT4 antibody 48A5, anti- ILT2/ILT4 antibody Hz73D1.v1, and a control antibody were serially diluted and 50 ⁇ l was added to each well of a 96-well flat-bottom cell culture plate.
  • ILT2-expressing or ILT4- expressing reporter cells were co-cultured with Raji cells or 721.221-HLA-G cells (100 ⁇ l/well). The plates were incubated overnight at 37°C. The next day, reporter cells were assayed for GFP expression by FACS.
  • Table 14 shows IC 50 of Hz73D1.v1 for blocking the interaction of human ILT2, human ILT4, and cyno ILT2 with MHC I molecules on Raji cells.
  • Table 15 shows IC50 of Hz73D1.v1 for blocking the interaction of human ILT2, human ILT4 with HLA-G expressed on 721.221 cells.
  • cyno ILT2 reporter cells were stained with CellTracker Deep Red, washed, and resuspended at 1 ⁇ 10 6 cells/ml in X-VIVOTM 15 media (Lonza). Raji cells were washed and resuspended in X-VIVOTM 15 media at 1 ⁇ 10 6 cell/ml.
  • Anti-ILT2/ILT4 antibody 73D1 and Hz73D1.v1 and a control antibody were serially diluted and 50 ⁇ l was added to each well of a 96-well flat-bottom cell culture plate.
  • ILT2-expressing reporter cells 100 ⁇ l/well were co- cultured with Raji cells (100 ⁇ l/well). The plates were incubated overnight at 37°C. The next day, reporter cells were assayed for GFP expression by FACS.
  • expression of GFP was induced when cyno ILT2/4 was expressed on the surface of the reporter cells in the presence of Raji cells.
  • anti-ILT2/ILT4 antibody 73D1 or Hz73D1.v1 the percentage of GFP-expressing cells was decreased in a dose-dependent manner.
  • Table 14 shows IC 50 of Hz73D1.v1 for blocking the interaction of cyno ILT2 with MHC I molecules on Raji cells.
  • Anti-human ILT2 (clone 27F9) or ILT4 (clone 48A5) antibodies were used as tool reagents to dissect ILT2- and ILT4-specific blocking activities.
  • Anti-IL2/ILT4 antibodies produced a dose-dependent reduction in the interaction between ILT2 and HLA-A, as well as between ILT4 and HLA-A. IC50s of the antibodies tested are shown in Table 16. Table 16 7.10 Example 10: Effect of Anti-ILT Antibodies on Activity of NK Cells [00485]
  • NKL is a human natural killer (NK) cell line established from the peripheral blood of a patient with large granular lymphocyte (LGL) leukemia and kindly provided by Dr. Louis Lanier.
  • NK cells express ILT2 but generally do not express ILT4.721.221 cells were transfected with plasmids expressing either HLA-G or HLA-A*0201 and high- expressing pools were enriched by antibiotic selection, generating 721.221-HLA-G and 721.221-A*0201 cell lines.721.221-HLA-G cells (described herein) or 721.221-HLA-A*0201 cells are used as targets in cytolytic cell assays.
  • the target cells were labeled with CellTracker Deep Red (ThermoFisher) to distinguish them from NKL cells (after co-culture) and then resuspended at 5 ⁇ 10 5 cells/ml in assay media (RPMI with 10% FBS, penicillin/streptomycin, L-glutamine, 5% human serum and recombinant human IL-2 (rhIL-2) at 20 ng/ml).
  • NKL cells were suspended at 7.5 ⁇ 10 6 cells/ml in assay media.
  • Sytox Blue will stain cells with compromised cell membranes allowing live cells to be distinguished from dead or damaged cells.
  • Cells were analyzed by FACS and the percent target cell killing was calculated based on the value of a positive control well of target cells permeabilized with detergent (100% lysis).
  • anti-ILT2 antibody 27F9 and anti-ILT2/ILT4 antibodies 73D1 and Hz73D1.v1 enhanced NK cytolytic activity in a dose-dependent manner.
  • EC 50 values for Hz73D1.v1 were 2.4 nM for HLA-G expressing target cells and 0.13 nM for HLA-A*0201 expressing target cells. In comparison, EC50 for 27F9 was 0.064 nM for HLA-G expressing target cells.
  • Anti-ILT4 antibody 48A5 had little to no effect on NK cell activity. These results show that anti-ILT2 antibodies and anti-ILT2/ILT4 antibodies are able to block the functional interaction between ILT2 on NK cells and MHC I molecules on the surface of target cells, leading to the enhancement of cytolytic activity. This supports the theory that anti-ILT2/ILT4 antibodies could enhance killing of tumor cells by inhibiting ILT2-induced suppression of NK cells.
  • NK cells were isolated from PBMC by negative selection, washed in assay media (RPMI with 10% FBS, pen/strep, 5% human serum, rhIL-2 (40 ng/ml), and IL-15 (50 ng/ml)), and resuspended at 7.5 ⁇ 10 6 cells/ml in assay media.721.221-HLA-G target cells were labeled with CellTracker Deep Red (ThermoFisher) and resuspended at 5 ⁇ 10 5 cells/ml in assay media.
  • Target cells and NK cells 50 ⁇ l each) were combined in each well of a V-bottom plate.
  • Anti-ILT2 antibody 27F9, anti-ILT4 antibody 48A5, or anti-ILT2/ILT4 antibodies 47H6, 73D1, or 64A12 were added to wells at a final concentration of 1 ⁇ g/ml. Plates were cultured overnight at 37°C followed by centrifugation at 360 ⁇ g for 8 minutes at room temperature and removal of media. Cells were then resuspended in PBS containing a 1:1,000 dilution of Sytox Blue (ThermoFisher). Cells were analyzed by FACS and the percent target cell killing was calculated based on the value of a positive control well of target cells permeabilized with detergent (100% lysis).
  • NK cell cytolytic activity was evaluated by FACS analysis of expression of activation marker CD137, gating on the CD3 negative, CD56 positive NK population.
  • CD57 staining was performed to enrich for memory (adaptive) NK cells known to preferentially express ILT2 (Lopez-Vergès S et al, Proc.Natl. Acad. Sci. (2011), 108 (36) 14725- 14732).
  • CD137 is upregulated on NK cells during cytolysis, providing a surrogate measure of cell killing.
  • Hz73D1.v1 or anti-ILT2 (27F9), but not anti-ILT4 (48A5) significantly increased the CD137+ population ( Figure 8). This effect was most pronounced in the CD57+ memory NK cell population.
  • anti-IL2/ILT4 antibodies are able to block the interaction of ILT2 on primary NK cells with MHC-I on target cells, leading to enhanced activation and cytolytic activity.
  • ADCC antibody dependent cellular cytotoxicity
  • Anti-EGFR antibody was added to induce ADCC of these EGFR+ CAL-27 cells.
  • CD137 was upregulated on NK cells during cytolysis, providing a surrogate measure of cell killing.
  • Hz73D1.v1 or anti-ILT227F9, but not anti-ILT448A5 significantly increased the percent CD137+ NK cells in the CD57+ population ( Figure 9).
  • an anti-NKG2A antibody significantly increased percent CD137+ in the CD57-, but not the CD57+ population.
  • Both antibodies induced a comparable fold increase in activation (Hz73D1.v1 increased percent CD137+ cells 1.8-fold while anti-NKG2A antibody increased percent CD137+ cells 2.1-fold).
  • An anti-KIR2DL antibody showed no activity in this assay.
  • ovMDSCs human peripheral monocytes were grown in pre-conditioned X-VIVOTM 15 media (Lonza). Pre-conditioned media was generated by culture with the OVISE cell line.
  • the OVISE cell line is an ovarian clear cell adenocarcinoma which secretes a large number of factors into its growth media.
  • Monocytes were cultured in media consisting of 50% OVISE-conditioned media and 50% fresh X-VIVOTM 15 for 5 days; the resulting MDSC-like cells are referred to as ovMDSCs.
  • MDSC-like cells show MDSC functional characteristics, including increased Arg1 and IDO and suppression of T cell proliferation and pro-inflammatory cytokines in MLR (Mixed Lymphocyte Reaction) assays when compared to monocyte derived dendritic cells (data not shown).
  • MLR Mated Lymphocyte Reaction
  • monocyte-derived dendritic cells were generated by culture of human peripheral monocytes with GM-CSF and IL-4 for 5 days.
  • 1 ⁇ 10 5 allogeneic T-cells were co-cultured with 2.5 ⁇ 10 4 ovMDSCs or moDCs in a 96-well round bottom cell culture plate.
  • the cells were co-cultured in the presence of anti-ILT2 antibody 27F9, anti-ILT4 antibody 48A5, anti-ILT2/ILT4 antibodies 47H6 or 73D1, or an isotype control antibody (each at 10 ⁇ g/ml).
  • Controls included T-cells only, moDCs only, moDCs with T-cells (positive MLR), ovMDSCs only, and ovMDSCs with T-cells.
  • Cells were incubated at 37°C and after 5 days 50 ⁇ l of media containing 3 H-Thymidine was added to each well.
  • T-cell proliferation was suppressed by co-culture with ovMDSCs.
  • the presence of anti-ILT4 antibody 48A5 and anti-ILT2/ILT4 antibodies 47H6 and 73D1 increased proliferation of T-cells cultured with ovMDSC to a level equivalent to the MLR response of T-cells with moDCs.
  • Anti-ILT2 antibody 27F9 showed no ability to enhance T-cell proliferation.
  • Another MLR assay was set up wherein 1 ⁇ 10 5 allogeneic T-cells were co-cultured with 2.5 ⁇ 10 4 ovMDSCs in a 96-well round bottom cell culture plate. The cells were co- cultured in the presence of serial dilutions of anti-ILT2 antibody 27F9, anti-ILT4 antibody 48A5, anti-ILT2/ILT4 antibodies 73D1 or Hz73D1.v1, or a control isotype antibody. Cells were incubated at 37°C and after 5 days 50 ⁇ l of supernatant was harvested from each well for cytokine analysis using a Luminex system.
  • anti-ILT4 antibody 48A5 and anti-ILT2/ILT4 antibodies 73D1 and Hz73D1.v1 were shown to increase T-cell proliferation ( Figure 11A).
  • TNF- ⁇ secretion and GM-CSF secretion were increased in a dose-dependent manner in the presence of anti-ILT4 and anti-ILT2/ILT4 antibodies ( Figures 11B-11C). As in the previous experiment, no effect was seen with anti-ILT2 antibodies.
  • anti-ILT4 and anti-ILT2/ILT4 antibodies are able to inhibit and/or block the functional interaction between ILT4 on MDSCs and MHC I molecules on neighboring cells (e.g., APCs), leading to the reversal of MDSC suppression and the enhancement of T-cell activation.
  • the reversal of MDSC-induced suppression also appears to lead to the increased secretion of pro-inflammatory cytokines such as TNF- ⁇ and GM-CSF.
  • LPS Lipopolysaccharide
  • TLR2 and TLR4 toll-like receptors 2 and 4
  • PBMC/LPS assay was performed. Briefly, frozen human PBMCs were thawed, washed in media (RPMI with 10% FBS, L-glutamine and pen/strep) and counted.
  • Anti-ILT2 antibody 27F9, anti-ILT4 antibody 48A5, anti-ILT2/ILT4 antibodies 73D1 and Hz73D1.v1, and a control isotype antibody were serially diluted in media and 50 ⁇ l added to each well of a 96-well round bottom tissue culture plate.
  • PBMCs were added at 1 ⁇ 10 6 cells/ml in a volume of 100 ⁇ l and the plate was incubated at 37°C for 30 minutes.
  • LPS was added in 50 ⁇ l of media (final concentration of 30 ng/ml) and the plate were incubated at 37°C for 2 days. Cell supernatants were removed for analysis of cytokines using a Luminex® system.
  • a LPS assay with tolerogenic dendritic cells was set up. Briefly, isolated human monocytes were plated onto 10 cm tissue culture dishes at 4 ⁇ 10 6 cells/dish in a volume of 20 ml media (X-VIVOTM 15 media (Lonza) supplemented with 50 ng/mL each of recombinant GM-CSF and IL-4 (Peprotech)). The monocytes were incubated for 5-7 days at 37°C and then the cells were harvested.
  • the in vitro generated DCs were suspended in fresh media containing 1:50 dilution of Fc Block (Biolegend) at a concentration of 6 ⁇ 10 5 cells/ml.
  • DCs were seeded in 96-well tissue culture plates at 50 ⁇ l/well and anti-ILT4 antibody 48A5, anti-ILT2/ILT4 antibody Hz73D1.v1, or a control antibody were added in serial dilutions at 50 ⁇ l/well.
  • LPS was added (6 ⁇ g/ml final concentration) together with one of a variety of tolerizing agents in a volume of 50 ⁇ l media.
  • the tolerizing agents included: vitamin D3/dexamethasone (VitD3/Dex) at 100 nM and 10 nM respectively, cyclosporin A at 750 ng/ml, rapamycin at 100 ng/ml, prostaglandin E2 (PGE2) at 1 ⁇ g/ml, IL-6 at 20 ng/ml, IL-10 at 20 ng/ml or TGF ⁇ at 20 ng/ml. Plates were incubated at 37°C for 2 days and supernatants were harvested for analysis using a Luminex® system.
  • anti-ILT4 and anti-ILT2/ILT4 antibodies enhanced LPS- mediated cytokine (e.g.,TNF ⁇ ) secretion in a dose-dependent manner.
  • LPS- mediated cytokine e.g.,TNF ⁇
  • the EC50 values of Hz73D1.v1 induced TNFa secretion in the presence of LPS and an additional tolerizing agent are shown in table 17.
  • TNF- ⁇ there was no increase of TNF- ⁇ in tolDCs treated with a control antibody and anti-ILT2 antibody (data not shown).
  • HMGB1 High mobility group protein B1
  • HMGB1 is a nuclear protein that can be released from cells and binds to TLRs.
  • HMGB1 has been shown to be present at high concentrations in necrotic tumor tissue, providing a potential source of innate stimulation of tumor infiltrating DC, monocytes, and macrophages (see, e.g., Guerriero et al., 2011, J. Immunol., 186:3517-3526).
  • anti-ILT2 antibody 27F9 anti-ILT4 antibody 48A5
  • anti-ILT2/ILT4 antibodies 47H6, 73D1 and 64A12 and a control isotype antibody were serially diluted in media and 50 ⁇ l added to each well of a 96-well round bottom tissue culture plate.
  • PBMCs were added at 1 ⁇ 10 6 cells/ml in a volume of 100 ⁇ l and the plate was incubated at 37°C for 30 minutes.
  • HMGB1 Recombinant HMGB1 (Biolegend) was added at a concentration of 2.5 ⁇ g/ml (a concentration anticipated to be present in the microenvironment of a tumor) and the plate was incubated at 37°C for 2 days. Cell supernatants were removed for analysis of cytokines using a Luminex® system.
  • anti-ILT4 antibody 48A5 and anti-ILT2/ILT4 antibodies 47H6, 73D1, and 64A12 enhanced secretion of TNF- ⁇ .
  • tumor cell necrosis and/or tumor cell death from radiotherapy results in the release of nuclear DNA.
  • Free nuclear DNA can lead to myeloid cell stimulation via STING (stimulator of interferon genes) signaling.
  • STING stimulation of interferon genes
  • anti-ILT antibodies could enhance inflammatory cytokine production from PBMCs treated with a STING agonist. Briefly, PBMCs were plated in 96-well round bottom tissue culture plates and anti-ILT2 antibody 27F9, anti-ILT4 antibody 48A5, and a control isotype antibody were added.
  • STING agonist 2'3'-cGAMP (Invivogen) was added at a concentration of 10 ⁇ g/ml (a concentration anticipated to be present in the microenvironment of a necrotic tumor). Cells were incubated for two days and supernatants were analyzed using a Luminex® system.
  • exemplary anti-ILT4 antibody 48A5 enhanced secretion of IFN ⁇ , TNF- ⁇ , and IL-1 ⁇ .
  • ILT2 or ILT4 may play a role in suppression of T-cell-mediated stimulation of myeloid cells.
  • PBMCs were activated with an anti-CD3 antibody and recombinant IL-2. Briefly, anti-ILT2 antibody 27F9, anti-ILT4 antibody 48A5, anti-ILT2/ILT4 antibodies 47H6 and 64A12, and a control isotype antibody were serially diluted in media and 50 ⁇ l added to each well of a 96-well round bottom tissue culture plate.
  • PBMCs were added at 1 ⁇ 10 6 cells/ml in a volume of 100 ⁇ l and the plate was incubated at 37°C for 30 minutes.
  • Anti-CD3 antibody clone HIT3a (eBioscience) was added at a concentration of 10 ng/ml and recombinant IL-2 was added at a concentration of 100 U/ml (Peprotech). Cells were incubated for two days and supernatants were analyzed using a Luminex® system.
  • anti-ILT4 antibody 48A5 and anti-ILT2/ILT4 antibodies 47H6 and 64A12 enhanced TNF- ⁇ secretion in a dose-dependent manner.
  • Anti-ILT2 antibody 27F9 did not have any effect of cytokine production. These data show that ILT4 blockade enhances the immune response (i.e., pro-inflammatory cytokine secretion) resulting from interaction between activated T-cells and myeloid cells. 7.14
  • Example 14 Effect of Anti-ILT Antibodies on Macrophage Phagocytosis [00508] Phagocytosis assays were performed to further characterize the effect of anti-ILT2, anti-ILT4, and anti-ILT2/ILT4 antibodies on macrophage functions.
  • Macrophages were generated by culture of isolated monocytes in media (RPMI with 10% FBS, L-glutamine and pen/strep) containing 50ng/ml recombinant M-CSF (Peprotech) for 5 days at 37° C in 12-well tissue culture plates. Macrophages were removed from the plate with a cell scraper and plated at 20,000 cells/well in 100 ⁇ l media in a flat bottom 96-well tissue culture plate. Macrophages were incubated overnight at 37° C. The next day, antibody was added to the macrophages at a concentration of 2.5 ⁇ g/ml together with 2.5 ⁇ g/ml of anti-CD47 antibody to induce antibody dependent phagocytosis.
  • media RPMI with 10% FBS, L-glutamine and pen/strep
  • M-CSF Peprotech
  • Raji cells were stained for 1 hour with a 1:4,000 dilution of pH Rodo Red Dye (Essen Bioscience), washed in media and added at 50,000 cells/well to macrophages. Phagocytosis was measured by mean red fluorescence on an Incucyte machine at 45 minute intervals. Test antibody was compared to isotype control antibody at the peak response timepoint (3 hours).
  • anti-ILT2/ILT4 antibodies e.g., Hz73D1.v1
  • anti-ILT2 antibodies e.g., 27F9
  • Anti-ILT4 antibodies (e.g., 48A5) had no effect on phagocytosis by macrophages.
  • antibody 24E7 To control for ILT2 expression by Raji cells, antibody 24E7, an ILT2 binder unable to block MHC-I interaction, was used.
  • anti-ILT2 and anti-ILT2/ILT4 antibodies are able to enhance macrophage phagocytosis by blocking macrophage ILT2 interaction with MHC-I on tumor cells, and inhibiting ILT-2 induced suppression of macrophages, thus increasing macrophage phagocytosis of tumors.
  • Activation of myeloid cells can be achieved by the crosslinking of Fc receptors (FcR) on the cell surface and the crosslinking results in cytokine production; this is used as the basis for a myeloid cell activation assay.
  • FcR Fc receptors
  • Dendritic cells were generated as described herein. Briefly, isolated human monocytes were plated on 10 cm tissue culture dishes with 4 ⁇ 10 6 cells per dish in a volume of 20 ml media (Lonza) supplemented with 50 ng/mL each of recombinant GM- CSF and IL-4 (Peprotech). The monocytes were incubated for 5 - 7 days at 37°C and the resulting dendritic cells were harvested.
  • Example 16 Effect of Anti-ILT Antibodies on Cytokine Secretion from Human and Cyno Blood Cells [00513] To investigate the potential for toxicity related to cytokine release (i.e., a “cytokine storm”), a variety of antibodies were cultured with human or cyno blood cells and cytokine production was analyzed. Briefly, antibodies were added at 10 ⁇ g/ml to 96-well flat-bottom plates and shaken at room temperature for 1 hour.
  • Red blood cells were removed from blood by lysis in RBC lysis buffer (eBioscience), followed by centrifugation, and the remaining cells were washed with culture media. 5 ⁇ 10 5 cells were added per well, with LPS added to replicate wells as a positive control, and plates incubated at 37°C for 24 hours. Supernatants were collected and assayed for cytokine secretion using a Luminex® system.
  • incubation of human blood cells with soluble LPS or plate- coated anti-CD28 antibody induced a significant increase in many cytokines including TNF- ⁇ , GM-CSF, MIP-1 ⁇ , IL-6, IL-1 ⁇ , and IL-10.
  • incubation of cyno blood cells with soluble LPS induced a significant increase in cytokines such as TNF- ⁇ , IL-1 ⁇ , MIP- 1 ⁇ , IL-6 and MIP-1 ⁇ .
  • a further in vitro cytokine release assay was performed to evaluate the capacity of anti- ILT2/ILT4 antibodies to induce pro-inflammatory cytokines IL-2, TNF- ⁇ , IL-6 and IFN- ⁇ .
  • Whole blood samples for 10 healthy donors were used.
  • Anti-ILT2/ILT4 antibodies were compared with a number of positive and negative controls.
  • An anti-KLH isotype control antibody was used as a negative control.
  • a super-agonist anti-CD28 antibody (clone ANC28.1) was used as a positive control (Walker 2011).
  • SEB Staphylococcus enterotoxin B
  • Two assay formats were used.
  • cells were cultured with an antibody (Hz73D1.v1, an isotype control antibody or anti-CD28 antibdy) or SEB at increasing concentrations. Antibodies were tested at 0.1, 1, 10 and 100 ⁇ g/ml. SEB was tested at 0.01, 0.1 and 1 ⁇ g/ml.
  • 96-well tissue culture plates were pre-incubated with an antibody overnight at 4° C, washed and then cells were added and cultured. Cell culture supernatant was then collected and tested for cytokine levels by cytometric bead array assay.
  • Isolated CD8+ T cells were cultured in basic media (Xvivo15 media with 10mM HEPES, 2mM Glutamine, pen/strep and 5% normal human serum) and stimulated for 24 hours at 37° C with CD3/28 beads adding 5 ⁇ l beads per million cells.
  • Activated T cells (2 ⁇ 10 5 ) were then harvested and resuspended in 1 ml of Lentivirus expressing human ILT2 at a viral concentration of 1 ⁇ 10 7 PFU/ml in the presence of polybrene to infect cells.
  • Infected cells were then resuspended in T cell media (basic media supplemented with 5 ng/ml IL-7, 5 ng/ml IL-15 and 25 ng/ml IL-2) and allowed to expand for two weeks. Cells were then FACS sorted for the ILT2+ population and expanded for an additional 6 weeks to generated sufficient cells for the study.
  • T cell media basic media supplemented with 5 ng/ml IL-7, 5 ng/ml IL-15 and 25 ng/ml IL-2
  • Cells were then FACS sorted for the ILT2+ population and expanded for an additional 6 weeks to generated sufficient cells for the study.
  • 2.5 ⁇ 10 4 CellTracker deep red-labelled 721.221-HLAG cells were mixed with 2.5 ⁇ 10 5 ILT2-transduced CD8+ T cells at a 10:1 T cell:Target ratio and 0.1 ⁇ g/ml anti-CD3/CD19 bispecific antibody in RPMI-10% FBS media.
  • Anti-ILT antibodies or control antibodies were added to the cells at a concentration of 10 ⁇ g/ml and incubated for 18 hours. Target cells were analyzed by FACS and percent target cell killing was calculated as the number of dead target cells divided by the total number of target cells.
  • anti-ILT2/ILT4 antibodies e.g., Hz73D1.v1
  • anti-ILT2 antibodies e.g., 27F9
  • anti-ILT4 antibodies e.g., 48A5
  • anti-ILT2 and anti- ILT2/ILT4 antibodies enhance CD8+ T cell cytolytic activity by blocking CD8+ T cell ILT2 interaction with MHC-I on target cells.
  • anti-ILT2 and anti-ILT2/ILT4 antibodies disrupt the immune suppressive activity of ILT2 on cytolytic lymphocytes, in particular effector memory T cells known to be involved in anti-tumor cell killing.
  • Example 18 Anti-ILT2/ILT4 Antibodies Induce M2-like to M1-like Polarization of Monocyte-derived Macrophages
  • Macrophages are traditionally characterized as either pro-inflammatory (M1) or immune suppressive (M2) based on surface expression markers CD80, CD86 (M1), CD163, CD204, and CD206 (M2).
  • M1 pro-inflammatory
  • M2 immune suppressive
  • Anti-IL2/ILT4 antibodies as well as anti-IL2 and anti-ILT4 antibodies were evaluated for their ability to polarize macrophages toward an M1-like or M2-like phenotype.
  • Macrophages were generated by culture of monocytes in media (RPMI with 10% FBS, L-glutamine and pen/strep) containing Hz73D1.v1, 27F9, 48A5 or isotype control antibody (1 ug/ml) and 50 ng/ml recombinant M-CSF (Peprotech) for 5 days at 37° C in 48-well tissue culture plates. On day 5 samples were analyzed by flow cytometry for various surface markers indicative of an M1-like or M2-like phenotype.
  • Hz73D1.v1 induced a decrease in M2-like macrophage phenotypic markers CD163, CD204, and CD206 and additional M2-like markers CD14 and CD209, consistent with an M2-like to M1-like polarization of the monocytes during differentiation.
  • Anti-ILT4 specific antibody 48A5, but not anti-ILT2 specific antibody 27F9 induced an increase in M1-like marker CD86 and a decrease in M2-like markers CD163, CD204, and CD206, and additional M2-like markers CD14, CD209, and CCR5, consistent with an M2-like to M1-like polarization with ILT4 blockade.
  • mice (12-14 weeks of age) were injected subcutaneously with 5 million A375 human melanoma tumor cells on the right flank and tumors allowed to grow to 100-200 mm 3 and randomized into two groups.
  • Group 1 was treated with isotype control antibody while Group 2 was treated with hz73D1.v1 (NGM707). All animals received antibody at 10mg/kg at days 25, 29, 33, and 39.
  • hz73D1.v1 As shown in Figure 24, hz73D1.v1 (NGM707) significantly inhibited tumor growth with a mean tumor growth inhibition of 70% compared to isotype at day 47 post tumor implantation.
  • hz73D1.v1 induces a robust anti-tumor immune response in animals engrafted with a human immune system.
  • Example 20 Blockade of ILT2 and ILT4 Inhibits Tumor Growth Individually and in Combination
  • a human PBMC-engrafted mouse tumor model was established to evaluate the individual and combined effect of anti-ILT2, anti-ILT4, anti-PD1, and hz73D1.v1 (NGM707) on tumor growth.
  • Female NSG mice were purchased from Jackson Laboratories. As illustrated in Figure 25, at 8-10 weeks of age, the animals were injected subcutaneously with A375 melanoma tumor cells.
  • mice were irradiated at 1.8gy using an X-ray irradiator and injected intraperitoneally with 5 million PBMC.
  • the tumors reached 80-100 mm 3 five days later.
  • Anti-ILT2 antibody 27F9, anti-ILT4 antibody 48A5, anti-ILT2/ILT4 antibody hz73D1.v1 (NGM707), and a control isotype antibody were added at 1 ⁇ g/ml final concentration.
  • Monocytes were added at 1 ⁇ 10 6 cells/ml in a volume of 100 ⁇ l and the plate was incubated at 37°C for 30 minutes.
  • LPS was added in 50 ⁇ l of media (final concentration of 30 ng/ml) and the plate were incubated at 37°C for 2 days. Cell supernatants were removed for analysis of cytokines using a Luminex system.
  • Interferon gamma secretion is another characteristic of anti-tumor immunity.
  • Plasma from animals in a separate study following the same protocol was analyzed for secretion of cytokines.
  • a significant increase in interferon gamma was observed between isotype and hz73D1.v1 (NGM707) treated animals.
  • Anti-PD1 and anti-ILT4 treatment did not induce interferon gamma secretion above isotype control levels.
  • Hz73D1.v1 Reverses a Suppressive Myeloid Phenotype Induced by Cancer Associated Fibroblasts
  • CAF Cancer associated fibroblasts
  • a CAF cell line was co-cultured with monocyte derived dendritic cells.
  • Isolated human monocytes were plated onto 10 cm tissue culture dishes at 4 ⁇ 10 6 cells/dish in a volume of 20 ml media (X-VIVOTM 15 media (Lonza) supplemented with 50 ng/mL each of recombinant GM-CSF and IL-4 (Peprotech)). The monocytes were incubated for 5-7 days at 37°C and then the cells were harvested. For the assay, these in vitro generated DCs were co-cultured with a CAF cell line in X-VIVO 15 media for 4 days in 12 well plates. DCs, alone or with CAF, were cultured in the presence of either hz73D1.v1 (NGM707) or isotype control.
  • NVM707 hz73D1.v1
  • NGM707 Monotherapy Using Hz73D1.v1 (NGM707)
  • a multicenter phase 1a human trial was designed to evaluate NGM707 as a monotherapy for various types of ILT2 and ILT4 high expressing cancer, including ovarian cancer, melanoma, gastric cancer, pancreatic cancer, renal cell carcinoma (RCC), rectal cancer, colorectal cancer (CRC), rectosigmoid adenocarcinoma, cholangiocarcinoma, non-small cell lung cancer (NSCLC), cervical cancer, breast cancer (including triple-negative breast cancer), pancreatic ductal adenocarcinoma (PDAC), and head and neck squamous cell carcinomas (HNSCCs).
  • NGM707 doses (6 mg, 20 mg, 60 mg, 200 mg, 600 mg, 1200 mg or 1800 mg) was administered intravenously to 34 human subjects once every 3 weeks. All 34 subjects had received a median of 4 prior therapies (ranging from 1-11) and all had metastatic disease. 1. Safety [00539] Treatment-related adverse events (AE) were characterized by type, frequency, severity (graded by NCI CTCAE v5.0), timing, seriousness, and relationship to the study drug. Laboratory abnormalities in the subjects were characterized by type, frequency, severity (graded by NCI CTCAE v5.0), and timing. Treatment-related adverse events and serious treatment- related adverse events are shown in Table 20. A maximum tolerated dose was not reached in this trial.
  • Anti-Tumor Activity Tumor progression in the subjects was assessed according to the criteria set out from RECIST 1.1. Of 24 response-evaluable subjects, best overall responses were partial response in one subject (1800 mg; metastatic melanoma), stable disease in six subjects and non-complete response/non-progressive disease in one subject. Six subjects had reduced target cancer lesion size, and the maximum reduction of lesion size was observed in one subject reaching 70%. Maximum treatment duration to date was six months, and five subjects remained on study. Objective responses over time and lesion assessment in the subjects are shown in Figure 31. [00541] The profile of chemokines associated with T cell and myeloid cell recruitment was evaluated in the subject showing a partial response.
  • CXCL9, CXCL10, CXCL11, and CCL3 were evaluated at cycle 1 day 4, day 8, and day 15, cycle 2 day 1, and cycle 2 day 3.
  • Each of these cytokines showed a time-dependent and statistically significant increase over the course of therapy. Together, these results are consistent with an NGM707-driven basis for the observed clinical response.
  • the inflammatory immune status and cell proliferation signature was evaluated in biopsy samples obtained from all NGM707 monotherapy patients with evidence of disease control. Inflammatory immune status was evaluated using the 18-gene T Cell Inflammation Signature ("TIS;" Ayers et al., JCI, 2017).
  • the proliferation signature was evaluated using a 10- gene tumor cell panel including ANLN, CCNB1, CDC25C, CDKN1C, CENPF, CEP55, MELK, MKI67, RRM2, and TYMS.
  • the data in Figure 38 demonstrates that patients showing a response of stable disease or better (i.e., stable disease (“SD”), non-complete response/non- progressive disease (“NPD/NCR”) or partial response (“PR”) had a significantly elevated TIS score and significantly reduced tumor cell proliferation score relative to those with progressive disease (“PD”).
  • SD stable disease
  • NPD/NCR non-complete response/non- progressive disease
  • PR partial response
  • NGM707 blood samples from the subjects were collected for measurement of the serum concentrations of NGM707 on day 0 (pre-dosing), day 7 (post- dosing), day 14 (post-dosing) and day 21 (post-dosing) for the first three cycles.
  • concentrations were determined using an electrochemiluminescence immunoassay on the Meso Scale Diagnostics (MSD) platform. Briefly, a biotinylated monoclonal anti-idiotype antibody of the anti-NGM707 antibody was captured onto a streptavidin coated plate. The plate was then incubated with the serum of the subjects.
  • Receptor ILT2 and ILT4 occupancy were analyzed in peripheral immune cells isolated from the subjects administered with different doses of NGM707. Briefly, blood from the patients was drawn into Cyto-Chex BCT direct draw tubes (Streck, Inc.; La Vista, NE, USA) and analyzed within 4 days of collection. A panel of 10 fluorochrome-labeled antibodies was used to identify major blood immune cell populations, including monocytes, CD4+ T cells, CD8+ T cells, total T cells, NK cells, B cells, granulocytes, and total CD33+ myeloid cells.
  • the antibody panel included two fluorochrome-labeled antibodies specific for each target: 1) a competing antibody that only binds free target, and 2) a non- competing antibody that binds all target molecules (independent of NGM707 presence).
  • RO of ILT2 and ILT4 were each calculated based on the relative mean fluorescence intensities of the competing and non-competing antibodies, adjusted for their relative intensities at the baseline (RO measured prior to treatment).
  • dose-dependent receptor occupancy (RO) was observed for both ILT2 and ILT4.
  • RO dose-dependent receptor occupancy
  • Biomarker Identification To identify biomarkers associated with NGM707 treatment in cancer patients, blood was collected from the patients between D15 of cycle 1 and D15 of cycle 2 of dosing with NGM707. CD163 expression on various immune cells was assayed by flow cytometry using an antibody panel.
  • CD163 expression level on peripheral non-classical monocytes was significantly reduced in patients that showed clinical benefits (stable diseases or better) from NGM707 treatment.
  • Paired tumor core needle biopsies baseline and on-treatment were collected from two colorectal cancer patients (patients 11002 and 11004) on day 20-21 after starting the treatment with 600 mg of NGM707.
  • PBMCs Peripheral blood mononuclear cells from healthy blood donors were cultured in vitro for 3 days with 5 ⁇ g/mL of NGM707 or anti-KLH control antibody in the presence of plate-bound IgG (to provide FcR stimulation). After treatment, the cells were stained with a panel of fluorochrome-labeled antibodies including CD14, CD16, and CD163. The Mean Fluorescence Intensity (MFI) of CD163 was assessed on CD14 low CD16+ non-classical monocytes. As illustrated in Figure 36, in vitro treatment with NGM707 reduced CD163 expression on non-classical monocytes.
  • MFI Mean Fluorescence Intensity
  • CD163 is a marker of suppressive M2 macrophages, and these results evidence myeloid reprogramming in response to NGM707 treatment.
  • the results have identified reduction of CD163 expression as a biomarker for assessing efficacy and/or clinical benefits from NGM707 treatment.
  • the results also suggest that CD163 is a predictive marker for efficacy and/or clinical benefits from NGM707 treatment.

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Abstract

La présente divulgation concerne des méthodes de traitement d'un sujet atteint d'un cancer à l'aide d'un agent de liaison (par exemple, des anticorps et des fragments de liaison à l'antigène de ceux-ci) qui se lient spécifiquement à ILT2 et à ILT4. La présente divulgation concerne également des méthodes d'utilisation d'un biomarqueur pour prédire la réactivité d'un sujet à la thérapie présentement divulguée et l'efficacité de la thérapie.
PCT/US2023/067618 2022-05-31 2023-05-30 Méthodes thérapeutiques utilisant des agents de liaison ilt WO2023235703A1 (fr)

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