WO2023140875A1 - Utilisations d'agents de liaison ilt3 - Google Patents

Utilisations d'agents de liaison ilt3 Download PDF

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WO2023140875A1
WO2023140875A1 PCT/US2022/025327 US2022025327W WO2023140875A1 WO 2023140875 A1 WO2023140875 A1 WO 2023140875A1 US 2022025327 W US2022025327 W US 2022025327W WO 2023140875 A1 WO2023140875 A1 WO 2023140875A1
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variable region
chain variable
seq
amino acid
acid sequence
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PCT/US2022/025327
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Hung-I Harry CHEN
Varun KAPOOR
Jeong Kim
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Ngm Biopharmaceuticals, Inc.
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Publication of WO2023140875A1 publication Critical patent/WO2023140875A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the present disclosure generally relates to agents that bind immunoglobulin-like transcript 3 (ILT3), particularly antibodies that bind human ILT3, as well as compositions comprising the ILT3-binding agents and methods of using the agents and compositions.
  • ILT3 binding agents in combination with Pembrolizumab to treat a disease or disorder such as advanced or metastatic cancers and tumors.
  • 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.
  • 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, 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).
  • LILRB leukocyte Ig-like receptor subfamily B
  • ITIMs cytoplasmic immunoreceptor tyrosine-based inhibitory motifs
  • This group of ITIM- containing receptors includes 5 members: LILRB 1 (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).
  • LILRB 1 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.
  • the biological functions and clinical significance of many of these LILRB s (ILTs) are still being investigated.
  • 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 cells have developed mechanisms and/or hijacked normal inhibitory mechanisms to evade the immune system which can allow for uninhibited growth of tumors.
  • Cancer/tumor immunotherapy 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.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a binding agent that specifically binds human immunoglobulin-like transcript 3 (ILT3), wherein the ILT3 binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising an amino acid sequence identical or substantially identical to TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising an amino acid sequence identical or substantially identical to REWRMTLYAMDY (SEQ ID NO:29) or REWRYTLYAMDY (SEQ ID NO: 105), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to RASES VDSYGNSFMH (SEQ ID NO:
  • a method of inhibiting tumor growth in a subject comprising administering to the subject a therapeutically effective amount of a binding agent that specifically binds human immunoglobulin-like transcript 3 (ILT3), wherein the ILT3 binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising an amino acid sequence identical or substantially identical to TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising an amino acid sequence identical or substantially identical to REWRMTLYAMDY (SEQ ID NO:29) or REWRYTLYAMDY (SEQ ID NO: 105), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to RASESVDSYGNSFMH (SEQ ID NO:
  • a method of inhibiting tumor relapse or tumor regrowth in a subject comprising administering to the subject a therapeutically effective amount of a binding agent that specifically binds human immunoglobulin-like transcript 3 (ILT3), wherein the ILT3 binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising an amino acid sequence identical or substantially identical to TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising an amino acid sequence identical or substantially identical to REWRMTLYAMDY (SEQ ID NO:29) or REWRYTLYAMDY (SEQ ID NO: 105), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to RASESVDSYGN
  • RASES VESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising an amino acid sequence identical or substantially identical to LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising an amino acid sequence identical or substantially identical to QQNNEDPFT (SEQ ID NO:32); and wherein the tumor comprises a microsatellite instability - high (MSI-H) tumor, a deficient mismatch repair (dMMR) positive tumor or tumor mutation burden - high (TMB-H).
  • MSI-H microsatellite instability - high
  • dMMR deficient mismatch repair
  • TMB-H tumor mutation burden - high
  • a binding agent that specifically binds human immunoglobulin-like transcript 3 (ILT3) for treatment of biliary duct cancer, cholangiocarcinoma, or mesothelioma
  • the ILT3 binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising an amino acid sequence identical or substantially identical to TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising an amino acid sequence identical or substantially identical to REWRMTLYAMDY (SEQ ID NO:29) or REWRYTLYAMDY (SEQ ID NO: 105), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to RASESVDSYGNSFMH (SEQ ID NO:
  • RASES VESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising an amino acid sequence identical or substantially identical to LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising an amino acid sequence identical or substantially identical to QQNNEDPFT (SEQ ID NO:32).
  • a binding agent that specifically binds human immunoglobulin-like transcript 3 (ILT3) in the manufacture of a medicament for treatment of biliary duct cancer, cholangiocarcinoma, or mesothelioma
  • the ILT3 binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising an amino acid sequence identical or substantially identical to TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising an amino acid sequence identical or substantially identical to REWRMTLYAMDY (SEQ ID NO:29) or REWRYTLYAMDY (SEQ ID NO: 105), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to RASE
  • a binding agent that specifically binds human immunoglobulin-like transcript 3 (ILT3), for treatment of a microsatellite instability - high (MSI-H) tumor, a deficient mismatch repair (dMMR) positive tumor or tumor mutation burden - high (TMB-H), wherein the ILT3 binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising an amino acid sequence identical or substantially identical to TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising an amino acid sequence identical or substantially identical to REWRMTLYAMDY (SEQ ID NO:29) or REWRYTLYAMDY (SEQ ID NO: 105), and (b) a light chain variable region comprising a light chain variable region CDR1
  • a binding agent that specifically binds human immunoglobulin-like transcript 3 (ILT3), in the manufacture of a microsatellite instability - high (MSLH) tumor, a deficient mismatch repair (dMMR) positive tumor or tumor mutation burden - high (TMB-H), wherein the ILT3 binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising an amino acid sequence identical or substantially identical to GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising an amino acid sequence identical or substantially identical to TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising an amino acid sequence identical or substantially identical to REWRMTLYAMDY (SEQ ID NO:29) or REWRYTLYAMDY (SEQ ID NO: 105), and (b) a light chain variable region comprising a light chain variable region CDR1
  • the method or use comprises (a) the heavy chain variable region comprises the heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), the heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), and the heavy chain variable region CDR3 comprising the amino acid sequence REWRMTLYAMDY (SEQ ID NO:29), and (b) the light chain variable region comprises the light chain variable region CDR1 comprising the amino acid sequence RASESVDSYGNSFMH (SEQ ID NO:30), the light chain variable region CDR2 comprising the amino acid sequence LTSNLES (SEQ ID NO:31), and the light chain variable region CDR3 comprising the amino acid sequence QQNNEDPFT (SEQ ID NO:32).
  • the heavy chain variable region has at least 80% sequence identity to SEQ ID NO: 111. In some embodiments of the method or use, the light chain variable region has at least 80% sequence identity to SEQ ID NO: 112. In some embodiments of the method or use, the heavy chain variable region has at least 80% sequence identity to SEQ ID NO: 111 and the light chain variable region has at least 80% sequence identity to SEQ ID NO: 112.
  • the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 111. In some embodiments of the method or use, the light chain variable region has at least 90% sequence identity to SEQ ID NO: 112. In some embodiments of the method or use, the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 111 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 112.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 111. In some embodiments of the method or use, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments of the method or use, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 111 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 112.
  • the heavy chain variable region comprises the heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), the heavy chain variable region CDR2 comprises the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), and the heavy chain variable region CDR3 comprises the amino acid sequence REWRYTLYAMDY (SEQ ID NO: 105), and (b) the light chain variable region comprises the light chain variable region CDR1 comprises the amino acid sequence RASESVESYGSSFMH (SEQ ID NO: 106), the light chain variable region CDR2 comprises the amino acid sequence LTSNLES (SEQ ID NO:31), and the light chain variable region CDR3 comprises the amino acid sequence QQNNEDPFT (SEQ ID NO:32).
  • the heavy chain variable region has at least 80% sequence identity to SEQ ID NO: 123. In some embodiments of the method or use, the light chain variable region has at least 80% sequence identity to SEQ ID NO: 124. In some embodiments of the method or use, the heavy chain variable region has at least 80% sequence identity to SEQ ID NO: 123 and the light chain variable region has at least 80% sequence identity to SEQ ID NO: 124.
  • heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 123.
  • the light chain variable region has at least 90% sequence identity to SEQ ID NO: 124.
  • the heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 123 and the light chain variable region has at least 90% sequence identity to SEQ ID NO: 124.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 123. In some embodiments of the method or use, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 124. In some embodiments of the method or use, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 123 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 124.
  • the binding agent comprises a heavy chain variable region comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO: 111 and a light chain variable region comprising a VL-CDR1, a VL- CDR2, and a VL-CDR3 from the amino acid sequence of SEQ ID NO: 112.
  • the binding agent comprises a heavy chain variable region comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 from the amino acid sequence of SEQ ID NO: 123 and a light chain variable region comprising a VL-CDR1, a VL- CDR2, and a VL-CDR3 from a light chain variable region having the amino acid sequence of SEQ ID NO: 124.
  • the binding agent is administered once or sequentially. [0024] In some embodiments of the method or use, the binding agent is administered at a dose of 20 mg, 60 mg, 200 mg, 600 mg, 1500 mg, or 2100 mg.
  • the binding agent is administered once every 3 weeks.
  • the binding agent is administered over 30 minutes, 60 minutes or 90 minutes.
  • the binding agent is administered over 30 minutes at a dose of ⁇ 600 mg.
  • the binding agent is administered over 60 minutes at a dose of > 600 mg.
  • the binding agent is administered over 90 minutes at a dose of > 1800 mg.
  • the dose comprises a starting dose.
  • the dose is modified relative to the starting dose.
  • the dose is modified to be about two thirds 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.
  • the binding agent is administered as part of a combination therapy.
  • the binding agent is administered in combination with a PD-1 inhibitor.
  • the PD-1 inhibitor comprises a PD-1 antibody.
  • the PD-1 antibody comprises pembrolizumab.
  • pembrolizumab is administered once or sequentially.
  • pembrolizumab is administered at a dose of 200 mg.
  • treatment with pembrolizumab lasts for up to 2 years.
  • pembrolizumab is administered before the binding agent is administered. [0043] In some embodiments of the method or use, pembrolizumab is administered 30 minutes, 60 minutes or 90 minutes before the binding agent is administered.
  • pembrolizumab is administered after the binding agent is administered.
  • pembrolizumab is administered 30 minutes, 60 minutes or 90 minutes after the binding agent is administered.
  • the combination therapy comprises administration of the binding agent, followed by pembrolizumab, and followed again by the binding agent.
  • pembrolizumab is administered intravenously, subcutaneously, or intra-peritoneally.
  • the subject is human.
  • Figure 1 Schematic of the Phase 1/lb, open-label, multicenter, dose-escalation, doseexpansion, study to determine the safety, PK, and pharmacodynamics of HZ5A7.V5 when given alone or in combination with pembrolizumab.
  • the present disclosure provides novel agents, including but not limited to polypeptides such as antibodies, that bind immunoglobulin-like transcript 3 (ILT3).
  • the ILT3-binding agents include, but are not limited to, polypeptides, antibodies (including antigen-binding fragments thereof), scaffold proteins, and heterodimeric molecules.
  • ILT3-binding agents include, but are not limited to, antagonists of ILT3 activity, inhibitors of ILT3 activity, and/or agents that inhibit ILT3 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. Methods of using the novel ILT3-binding agents are also provided.
  • antibody is used in the broadest sense and includes, for example, an intact immunoglobulin, and an antibody fragment containing an antigen binding portion.
  • Traditional antibody structural units typically comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains.
  • the present invention is directed to antibodies or antibody fragments (antibody monomers) that generally are based on the IgG class, which has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4.
  • IgGl, IgG2 and IgG4 are used more frequently than IgG3.
  • IgGs have different allotypes. All IgGs allotypes can be used for this invention.
  • IgGl has polymorphisms at 356 (D or E) and 358 (L or M). The sequences depicted herein use the 356E/358M allotype, however, the other allotypes are included herein.
  • VH variable heavy region
  • VL variable light region
  • Percent (%) amino acid sequence identity “Percent (%) sequence identity” or “Percent (%) identity” with respect to a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific (parental) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • One particular program is the ALIGN-2 program outlined at paragraphs [0279] to [0280] of US Pub. No. 20160244525, hereby incorporated by reference.
  • Another approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics, 2:482-489 (1981).
  • This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M.O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986).
  • Fc or “Fc region” or “Fc domain” as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CHI) and in some cases, part of the hinge.
  • the Fc domain comprises immunoglobulin domains CH2 and CH3 (Cy2 and Cy3) and the lower hinge region between CHI (Cyl) and CH2 (Cy2).
  • the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat.
  • CH domains in the context of IgG are as follows: “CHI” refers to positions 118- 215 according to the EU index as in Kabat. “Hinge” refers to positions 216-230 according to the EU index as in Kabat. “CH2” refers to positions 231-340 according to the EU index as in Kabat, and “CH3” refers to positions 341-447 according to the EU index as in Kabat.
  • the “Fc domain” includes the -CH2-CH3 domain, and optionally a hinge domain (hinge-CH2-CH3).
  • amino acid modifications are made to the Fc region, for example to alter binding to one or more FcyR receptors or to the FcRn receptor.
  • myeloid cells as used herein refer to any cells having myeloid origin, including but not limited to monocytes, macrophages, and dendritic cells.
  • substantially identical refers to at least 1 amino acid modification (e.g., 1, 2, 3, 4, 5, or 6 amino acid modifications) in the CDR sequence, wherein the modification of the CDR sequence in combination with the rest five CDRs (with or without further modifications in any one or more of the five CDRs) do not change the affinity (KD measured by e.g., SPR technology in a Biacore system) of the resulting antigen binding domain more than 50 folds in comparison to the antigen binding domain comprising the six original CDRs.
  • affinity measured by e.g., SPR technology in a Biacore system
  • Amino acid (aa) sequences for human ILT3 (UniProtKB No. Q8NHJ6) and cynomolgus monkey (“cyno”) ILT3 (NCBI Ref No. XP_015297198) are provided herein as SEQ ID NO:1 and SEQ ID NO:6, respectively.
  • reference to amino acid positions of ILT3 refer to the numbering of amino acid sequences including the signal sequence.
  • ILT3 is a single pass type I transmembrane protein with a predicted molecular weight of approximately 47 kDa. ILT3 has been observed to be predominantly expressed on myeloid antigen presenting cells, such as normal monocytes, macrophages, and dendritic cells. ILT3 is characterized by an extracellular domain comprising two Ig-like C2 type domains, a transmembrane domain, and a long cytoplasmic domain containing 3 ITIM domains (see, e.g., Celia et al., 1997, J. Exp. Med., 185:1743-1751). The two Ig-like C2-type domains may be referred to herein as Domain 1 (DI) and Domain 2 (D2).
  • DI Domain 1
  • D2 Domain 2
  • DI is situated at the N-terminal portion of the protein and D2 is situated closest to the transmembrane region.
  • human ILT3 is a protein of 448 amino acids (aa) - the signal sequence is aa 1-21, the extracellular domain is aa 22-259, the transmembrane region is aa 260-280, and the cytoplasmic domain is aa 281-448.
  • DI is aa 27-188
  • D2 is aa 124-218, and the “stem region” is aa 219-259.
  • ITIMs are aa 358-363, 410-415, and 440-445.
  • an ILT3-binding agent binds ILT3 or a fragment of ILT3.
  • a fragment of ILT3 comprises the extracellular domain.
  • a fragment of ILT3 comprises one of the Ig-like C2 type domains (e.g., DI or D2).
  • a fragment of ILT3 comprises both of the Ig-like C2 type domains (e.g., D1-D2).
  • a fragment of ILT3 comprises both of the Ig-like C2 type domains and the stem region (e.g., Dl-D2-stem).
  • a fragment of ILT3 comprises one of the Ig-like C2 type domains and the stem region (e.g., DI -stem or D2-stem).
  • the extracellular domain of human ILT3 comprises amino acids 22-259 of SEQ ID NO: 1.
  • DI of human ILT3 comprises amino acids 27-118 of SEQ ID NO: 1.
  • D2 of human ILT3 comprises amino acids 124-218 of SEQ ID NO: 1.
  • D1-D2 of human ILT3 comprises amino acids 27-218 of SEQ ID NO: 1.
  • Dl-D2-stem of human ILT3 comprises amino acids 27-259 of SEQ ID NO: 1.
  • D2-stem of human ILT3 comprises amino acids 124-259 of SEQ ID NO: 1.
  • a fragment of human ILT3 comprises the amino acid sequence of SEQ ID NO:3.
  • a fragment of human ILT3 comprises the amino acid sequence of SEQ ID NO:4.
  • a fragment of human ILT3 comprises the amino acid sequence of SEQ ID NO:5.
  • a fragment of human ILT3 comprises the amino acid sequence of SEQ ID NO:4 and SEQ ID NO:5.
  • the extracellular domain of cyno ILT3 comprises amino acids 22-259 of SEQ ID NO:6.
  • DI of cyno ILT3 comprises amino acids 27-118 of SEQ ID NO:6.
  • D2 of cyno ILT3 comprises amino acids 124-218 of SEQ ID NO:6.
  • D1-D2 of cyno ILT3 comprises amino acids 27-218 of SEQ ID NO:6.
  • Dl-D2-stem of cyno ILT3 comprises amino acids 27-259 of SEQ ID NO:6.
  • D2-stem of cyno ILT3 comprises amino acids 124-259 of SEQ ID NO:6.
  • a fragment of cyno ILT3 comprises the amino acid sequence of SEQ ID NO: 8.
  • a fragment of cyno ILT3 comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, a fragment of cyno ILT3 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, a fragment of cyno ILT3 comprises the amino acid sequence of SEQ ID NO:9 and SEQ ID NO: 10. It is understood that the domains of ILT3 (e.g., human ILT3 or cyno ILT3) may be defined differently by those of skill in the art, therefore the N-terminal amino acids and the C-terminal amino acids of any ILT3 domain or region may vary by 1, 2, 3, 4, 5, or more amino acid residues.
  • an ILT3-binding agent binds a fragment of ILT3. In some embodiments, an ILT3- binding agent binds within a specific region of ILT3. In some embodiments, an ILT3-binding agent binds within the extracellular domain of ILT3. In some embodiments, an ILT3-binding agent binds within the DI domain of ILT3. In some embodiments, an ILT3 -binding agent binds within the D2 domain of ILT3. In some embodiments, an ILT3-binding agent binds within the D2-stem region of ILT3.
  • an ILT3-binding agent binds within the junction between DI andD2 domains of ILT3. In some embodiments, an ILT3 -binding agent binds an epitope on ILT3. In some embodiments, an ILT3-binding agent binds a conformational epitope on ILT3. In some embodiments, an ILT3-binding agent does not bind other human LILRB proteins (e.g., ILT2, ILT4, ILT5, or LILRB5). In some embodiments, an ILT3-binding agent does not bind human LILRA proteins (e.g., LILRA1, LILRA2, LILRA4, LILRA5, or LILRA6).
  • human LILRB proteins e.g., LILRA1, LILRA2, LILRA4, LILRA5, or LILRA6
  • an ILT3-binding agent binds human ILT3. In some embodiments, an ILT3-binding agent binds cyno ILT3. In some embodiments, an ILT3-binding agent binds human ILT3 and cyno ILT3. In some embodiments, an ILT3-binding agent binds SEQ ID NO: 1. In some embodiments, an ILT3-binding agent binds SEQ ID NO:2. In some embodiments, an ILT3-binding agent binds within amino acids 22-259 of SEQ ID NO: 1. In some embodiments, an ILT3-binding agent binds within amino acids 27-118 of SEQ ID NO: 1.
  • an ILT3 -binding agent binds within amino acids 124-218 of SEQ ID NO: 1. In some embodiments, an ILT3-binding agent binds within amino acids 124-259 of SEQ ID NO: 1. In some embodiments, an ILT3-binding agent binds within amino acids 27-218 of SEQ ID NO: 1. In some embodiments, an ILT3 -binding agent binds SEQ ID NO:3. In some embodiments, an ILT3-binding agent binds SEQ ID NON. In some embodiments, an ILT3- binding agent binds SEQ ID NO: 5.
  • an ILT3 -binding agent binds a fragment of ILT3 that comprises SEQ ID NON and SEQ ID NON. In some embodiments, an ILT3-binding agent binds SEQ ID NON. In some embodiments, an ILT3-binding agent binds SEQ ID NO:7. In some embodiments, an ILT3-binding agent binds within amino acids 22-259 of SEQ ID NON. In some embodiments, an ILT3-binding agent binds within amino acids 27- 118 of SEQ ID NON. In some embodiments, an ILT3-binding agent binds within amino acids 124-218 of SEQ ID NON.
  • an ILT3-binding agent binds within amino acids 27-218 of SEQ ID NON. In some embodiments, an ILT3-binding agent binds SEQ ID NON. In some embodiments, an ILT3-binding agent binds SEQ ID NON. In some embodiments, an ILT3-binding agent binds SEQ ID NO: 10. In some embodiments, an ILT3- binding agent binds a fragment of ILT3 that comprises SEQ ID NON and SEQ ID NO: 10. [0064] In some embodiments, an ILT3-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO:2.
  • an ILT3-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NON. In some embodiments, an ILT3-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NON. In some embodiments, an ILT3-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NON. In some embodiments, an ILT3 -binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NON and the amino acid sequence of SEQ ID NON. In some embodiments, an ILT3-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NON.
  • an ILT3-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NON. In some embodiments, an ILT3-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NON. In some embodiments, an ILT3-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, an ILT3-binding agent binds a polypeptide comprising the amino acid sequence of SEQ ID NON and the amino acid sequence of SEQ ID NONO.
  • an ILT3-binding agent binds an epitope comprising amino acids within SEQ ID NON. In some embodiments, an ILT3-binding agent binds an epitope comprising amino acids within SEQ ID NON. In some embodiments, an ILT3-binding agent binds an epitope comprising amino acids within SEQ ID NON. In some embodiments, an ILT3 -binding agent binds an epitope comprising amino acids within SEQ ID NON. In some embodiments, an ILT3 -binding agent binds an epitope comprising amino acids within SEQ ID NON and SEQ ID NON.
  • an ILT3-binding agent binds an epitope comprising amino acids within SEQ ID NO:7. In some embodiments, an ILT3-binding agent binds an epitope comprising amino acids within SEQ ID NO:8. In some embodiments, an ILT3- binding agent binds an epitope comprising amino acids within SEQ ID NO:9. In some embodiments, an ILT3 -binding agent binds an epitope comprising amino acids within SEQ ID NO: 10. In some embodiments, an ILT3-binding agent binds an epitope comprising amino acids within SEQ ID NO:9 and SEQ ID NO: 10.
  • an ILT3 -binding agent binds human ILT3 and has at least one or more of the following properties: (i) binds cyno ILT3; (ii) binds human and cyno ILT3; (iii) does not bind ILT2, ILT4, ILT5, and LILRB5; (iv) does not bind LILRA1, LILRA2, LILRA4, LILRA5, and LILRA6; (v) is an ILT3 antagonist; (vi) inhibits ILT3 activity; (vii) inhibits ILT3 signaling in cells that express ILT3; (viii) inhibits binding of ILT3 to APOE; (ix) inhibits binding of ILT3 to fibronectin; (x) inhibits binding of ILT3 to CNTFR; (xi) inhibits ILT3 -induced suppression of myeloid cells; (xii) inhibits ILT3-induced suppression of myeloid cell activity; (xiii) restore
  • an ILT3-binding agent is an antibody.
  • the antibody is a recombinant antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a chimeric antibody.
  • the antibody is a humanized antibody.
  • the antibody is a human antibody.
  • the antibody is an IgG antibody.
  • the antibody is an IgGl antibody.
  • the antibody is an IgG2 antibody.
  • the antibody is an IgG3 antibody.
  • the antibody is an IgG4 antibody.
  • the antibody comprises an IgG heavy chain.
  • the antibody comprises an IgGl 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 kappa light chain. In some embodiments, the antibody comprises a 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 is an antibody fragment comprising an antigenbinding site. In some embodiments, the antibody is an scFv. In some embodiments, the antibody is a disulfide-linked scFv.
  • 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, Atorney Docket No. 13370-156-228 the antibody is a multivalent antibody. In some embodiments, the antibody is a bivalent antibody. In some embodiments, the antibody is a tetravalent antibody.
  • the antibody is isolated. In some embodiments, the antibody is substantially pure.
  • an ILT3-binding agent is a polyclonal antibody.
  • 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.
  • the antigen is conjugated to a carrier such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.
  • KLH keyhole limpet hemocyanin
  • 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.
  • an ILT3-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.
  • the immunizing antigen is a cyno protein or a fragment 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 directed specifically 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).
  • 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.
  • the isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E. coh. simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.
  • 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.
  • an ILT3-binding agent is a humanized antibody.
  • 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.
  • variable region framework sequence is selected from the consensus sequences of all human antibodies of a particular subgroup of light or heavy chains.
  • 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.
  • HSC Human String Content
  • an ILT3-binding agent is a human antibody.
  • 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. In any case, 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.
  • 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.
  • an ILT3-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 an 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 [0080]
  • an ILT3-binding agent is an scFv antibody.
  • the scFv is a disulfide-linked scFv (dsscFv), which is an scFv comprising an engineered disulfide bond between the light chain variable region and heavy chain variable region of the scFv.
  • dsscFv disulfide-linked scFv
  • the disulfide bond increases stability of the scFv molecule.
  • the disulfide bond increases thermostability of the scFv molecule.
  • an ILT3-binding agent is an Fv. In some embodiments, an ILT3-binding agent is an Fab. In some embodiments, an ILT3-binding agent is a F(ab')2. In some embodiments, an ILT3-binding agent is a F(ab').
  • an ILT3-binding agent is a bispecific antibody. Bispecific antibodies are capable of recognizing and binding at least two different antigens or epitopes. In some embodiments, an ILT3-binding agent is a multispecific antibody. Multispecific antibodies are capable of recognizing and binding at least three different antigens or epitopes.
  • the different epitopes can either be within the same molecule (e.g., two or more epitopes on ILT3) or on different molecules (e.g., one epitope on ILT3 and the rest of the epitope(s) on one or more target(s) other than ILT3).
  • a bispecific or multispecific antibody has enhanced potency as compared to an individual antibody or to a combination of more than one antibody.
  • a bispecific or multispecific 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 or multispecific antibody has the ability to synchronize the PK of two or more active binding agents wherein the two or more individual binding agents have different PK profiles.
  • a bispecific or multispecific antibody has the ability to concentrate the actions of two or more agents in a common area (e.g., tissue) in a subject (e.g., a human).
  • a bispecific or multispecific antibody has the ability to concentrate the actions of two or more agents to a common target (e.g., a specific cell type).
  • a bispecific or multispecific antibody has the ability to target the actions of two or more agents to more than one biological pathway or function.
  • a bispecific or multispecific antibody has the ability to target two or more different cells and bring them closer together.
  • a bispecific or multispecific antibody has decreased toxicity and/or side effects. In some embodiments, a bispecific or multispecific antibody has decreased toxicity and/or side effects as compared to a mixture of the two or more individual antibodies or the antibodies as single agents. In some embodiments, a bispecific or multispecific antibody has an increased therapeutic index. In some embodiments, a bispecific or multispecific antibody has an increased therapeutic index as compared to a mixture of the two or more individual antibodies or the antibodies as single agents. [0085] Many techniques for making bispecific or multispecific antibodies are known to those skilled in the art. In some embodiments, a bispecific or multispecific antibody comprises heavy chain constant regions with modifications in the amino acids that are part of the interface between the two heavy chains.
  • the bispecific or multispecific antibody is generated using a knobs-into-holes (KIH) strategy.
  • the bispecific or multispecific antibody comprises variant hinge regions incapable of forming disulfide linkages between identical heavy chains (e.g., reduce homodimer formation).
  • the bispecific or multispecific antibody comprises heavy chains with changes in amino acids that result in altered electrostatic interactions.
  • the bispecific or multispecific antibodies comprise heavy chains with changes in amino acids that result in altered hydrophobic/hydrophilic interactions.
  • the Bispecific or multispecific antibodies can be intact antibodies or antibody fragments comprising antigen-binding sites.
  • an ILT3-binding agent is an antibody that binds ILT3.
  • an anti-ILT3 antibody binds human ILT3.
  • an anti-ILT3 antibody binds cyno ILT3.
  • an anti-ILT3 antibody binds human ILT3 and cyno ILT3.
  • an anti-ILT3 antibody binds an ILT3 epitope.
  • an anti-ILT3 antibody binds an ILT3 epitope within the extracellular domain of human ILT3.
  • an anti-ILT3 antibody binds an ILT3 epitope within the extracellular domain of cyno ILT3.
  • an anti-ILT3 antibody binds an epitope comprising at least one amino acid within amino acids 22-259 of SEQ ID NO: 1. In some embodiments, an anti-ILT3 antibody binds an epitope comprising at least one amino acid within amino acids 22-120 of SEQ ID NO:1. In some embodiments, an anti-ILT3 antibody binds an epitope comprising at least one amino acid within amino acids 27-118 of SEQ ID NO:1. In some embodiments, an anti-ILT3 antibody binds an epitope comprising at least one amino acid within amino acids 121-259 of SEQ ID NO:1.
  • an anti-ILT3 antibody binds an epitope comprising at least one amino acid within amino acids 124-218 of SEQ ID NO:1. In some embodiments, an anti-ILT3 antibody binds an epitope comprising at least one amino acid within amino acids 124-259 of SEQ ID NO:1. In some embodiments, an anti-ILT3 antibody binds an epitope comprising amino acids within SEQ ID NO:3. In some embodiments, an anti- ILT3 antibody binds an epitope comprising amino acids within SEQ ID NON. In some embodiments, an anti-ILT3 antibody binds an epitope comprising amino acids within SEQ ID NO:5.
  • an anti-ILT3 antibody binds an epitope comprising amino acids within SEQ ID NON and SEQ ID NON. In some embodiments, the epitope is a conformational epitope. In some embodiments, the epitope is a linear epitope. [0088] In some embodiments, an anti-ILT3 antibody competes with a second agent for binding within the extracellular domain of human ILT3. In some embodiments, an anti-ILT3 antibody competes with a second agent for binding within the extracellular domain of cyno ILT3. In some embodiments, an anti-ILT3 antibody competes with a second agent for binding within amino acids 22-259 of SEQ ID NO: 1.
  • an anti-ILT3 antibody competes with a second agent for binding within amino acids 22-120 of SEQ ID NO: 1. In some embodiments, an anti-ILT3 antibody competes with a second agent for binding within amino acids 27-118 of SEQ ID NO: 1. In some embodiments, an anti-ILT3 antibody competes with a second agent for binding within amino acids 121-259 of SEQ ID NO: 1. In some embodiments, an anti-ILT3 antibody competes with a second agent for binding within amino acids 124-218 of SEQ ID NO: 1. In some embodiments, an anti-ILT3 antibody competes with a second agent for binding within amino acids 124-259 of SEQ ID NO: 1.
  • an anti-ILT3 antibody competes with a second agent for binding within amino acid sequence SEQ ID NO:3. In some embodiments, an anti-ILT3 antibody competes with a second agent for binding within amino acid sequence SEQ ID NON. In some embodiments, an anti-ILT3 antibody competes with a second agent for binding within amino acid sequence SEQ ID NO:5. In some embodiments, an anti-ILT3 antibody competes with a second agent for binding within amino acid sequences SEQ ID NON and SEQ ID NON.
  • an ILT3-binding agent is an anti-ILT3 antibody described herein.
  • the ILT3-binding agent is a variant of an anti-ILT3 antibody described herein.
  • the variant of an ILT3 antibody retain one or more binding characteristics of the ILT3 -binding agent described herein.
  • a variant of an anti-ILT3 antibody comprises one to thirty amino acid substitutions.
  • a variant of the anti-ILT3 antibody comprises one to twenty-five amino acid substitutions.
  • a variant of the anti-ILT3 antibody comprises one to twenty amino acid substitutions.
  • a variant of the anti-ILT3 antibody comprises one to fifteen amino acid substitutions. In some embodiments, a variant of the anti- ILT3 antibody comprises one to ten amino acid substitutions. In some embodiments, a variant of the anti-ILT3 antibody comprises one to five amino acid substitutions. In some embodiments, the variant of the anti-ILT3 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.
  • an ILT3-binding agent comprises one or more (e.g., 1, 2, 3, 4, 5, or 6 etc.) amino acid substitutions in a CDR of an antibody described herein while retaining one or more binding characteristics of the ILT3-binding agent described herein.
  • the amino acid substitutions are conservative substitutions.
  • a CDR comprises one amino acid substitution.
  • a CDR comprises two amino acid substitutions.
  • a CDR comprises three amino acid substitutions.
  • a CDR comprises four amino acid substitutions.
  • the CDR is a heavy chain variable region CDR1.
  • the CDR is a heavy chain variable region CDR2.
  • the CDR is a heavy chain variable region CDR3. In some embodiments, the CDR is a light chain variable region CDR1. In some embodiments, the CDR is a light chain variable region CDR2. In some embodiments, the CDR is a light chain variable region CDR3. In some embodiments, one or more of such six CDRs have no more than six amino acids substitutions. In some embodiments, the substitutions are made as part of a humanization process. In some embodiments, 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.
  • 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.
  • Software programs e.g., abYsis
  • an anti-ILT3 antibody described herein comprises the six CDRs of antibody 3A3, 5A7, Hz5A7, 12A12, 16C5, 45G10, 48A6, or 53F10 based on the Kabat definition. In some embodiments, an anti-ILT3 antibody described herein comprises the six CDRs of antibody 3A3, 5A7, Hz5A7, 12A12, 16C5, 45G10, 48A6, or 53F10 based on the Chothia definition. In some embodiments, an anti-ILT3 antibody described herein comprises the six CDRs of antibody 3A3, 5A7, Hz5A7, 12A12, 16C5, 45G10, 48A6, or 53F10 based on the AbM definition.
  • an anti-ILT3 antibody described herein comprises the six CDRs of antibody 3A3, 5A7, Hz5A7, 12A12, 16C5, 45G10, 48A6, or 53F10 based on the IMGT definition. In some embodiments, an anti-ILT3 antibody described herein comprises the six CDRs of antibody 3A3, 5A7, Hz5A7, 12A12, 16C5, 45G10, 48A6, or 53F10 based on the Contact definition. In some embodiments, an anti-ILT3 antibody described herein comprises the six CDRs of antibody 3A3, 5A7, Hz5A7, 12A12, 16C5, 45G10, 48A6, or 53F10 based on the Exemplary definition.
  • an ILT3-binding agent is an anti-ILT3 antibody that comprises one, two, three, four, five, and/or six CDRs of any one of the antibodies described herein.
  • an anti-ILT3 antibody comprises (i) a heavy chain variable region comprising one, two, and/or three heavy chain variable region CDRs from Table 1, and/or (ii) a light chain variable region comprising one, two, and/or three light chain variable region CDRs from Table 1.
  • an anti-ILT3 antibody comprises (i) a heavy chain variable region comprising one, two, and/or three heavy chain variable region CDRs from Table 2, and/or (ii) a light chain variable region comprising one, two, and/or three light chain variable region CDRs from Table 2.
  • an anti-ILT3 antibody comprises (i) a heavy chain variable region comprising one, two, and/or three heavy chain variable region CDRs from Table 3, and/or (ii) a light chain variable region comprising one, two, and/or three light chain variable region CDRs from Table 3.
  • an anti-ILT3 antibody comprises (i) a heavy chain variable region comprising one, two, and/or three heavy chain variable region CDRs from Table 4, and/or (ii) a light chain variable region comprising one, two, and/or three light chain variable region CDRs from Table 4. In some embodiments, an anti-ILT3 antibody comprises (i) a heavy chain variable region comprising one, two, and/or three heavy chain variable region CDRs from Table 5, and/or (ii) a light chain variable region comprising one, two, and/or three light chain variable region CDRs from Table 5.
  • an anti-ILT3 antibody comprises (i) a heavy chain variable region comprising one, two, and/or three heavy chain variable region CDRs from Table 6, and/or (ii) a light chain variable region comprising one, two, and/or three light chain variable region CDRs from Table 6. In some embodiments, an anti-ILT3 antibody comprises (i) a heavy chain variable region comprising one, two, and/or three heavy chain variable region CDRs from Table 7, and/or (ii) a light chain variable region comprising one, two, and/or three light chain variable region CDRs from Table 7.
  • an anti-ILT3 antibody comprises (i) a heavy chain variable region comprising three heavy chain variable region CDRs from Table 1, and (ii) a light chain variable region comprising three light chain variable region CDRs from Table 1.
  • an anti-ILT3 antibody comprises (i) a heavy chain variable region comprising three heavy chain variable region CDRs from Table 2, and (ii) a light chain variable region comprising three light chain variable region CDRs from Table 2.
  • an anti- ILT3 antibody comprises (i) a heavy chain variable region comprising three heavy chain variable region CDRs from Table 3, and (ii) a light chain variable region comprising three light chain variable region CDRs from Table 3.
  • an anti- ILT3 antibody comprises (i) a heavy chain variable region comprising three heavy chain variable region CDRs from Table 7, and (ii) a light chain variable region comprising three light chain variable region CDRs from Table 7.
  • an anti-ILT3 antibody comprises (i) a heavy chain variable region comprising three heavy chain variable region CDRs from Table 8, and (ii) a light chain variable region comprising three light chain variable region CDRs from Table 8.
  • an ILT3-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from an antibody described herein. In some embodiments, an ILT3-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 from an antibody described herein.
  • an ILT3 -binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1, CDR2, and CDR3; and (b) a light chain variable region comprising a light chain variable region CDR1, CDR2, and CDR3 from an antibody described herein.
  • an ILT3-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from antibody 5A7, a humanized version thereof, or variants thereof.
  • an ILT3- binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3 from antibody 5A7.
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 5A7.
  • an ILT3-binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3; and (b) a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 5A7.
  • an ILT3-binding agent is a humanized version of antibody 5A7 (e.g., Hz5A7).
  • an ILT3-binding agent is a variant of antibody 5A7 or humanized 5A7 e.g., Hz5A7.v5).
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3 from antibody Hz5A7.v5.
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody Hz5A7.v5.
  • an ILT3-binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3; and (b) a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody Hz5A7.v5.
  • an ILT3-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from antibody 3 A3, a humanized version thereof, or variants thereof.
  • an ILT3- binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3 from antibody 3 A3.
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 3 A3.
  • an ILT3-binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3; and (b) a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 3 A3.
  • an ILT3-binding agent is a humanized version of antibody 3 A3.
  • an ILT3-binding agent is a variant of antibody 3 A3.
  • an ILT3-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from antibody 12A12, a humanized version thereof, or variants thereof.
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3 from antibody 12A12.
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 12A12.
  • an ILT3-binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3; and (b) a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 12A12.
  • an ILT3-binding agent is a humanized version of antibody 12A12.
  • an ILT3-binding agent is a variant of antibody 12A12.
  • an ILT3-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from antibody 16C5, a humanized version thereof, or variants thereof.
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3 from antibody 16C5.
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 16C5.
  • an ILT3-binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3; and (b) a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 16C5.
  • an ILT3-binding agent is a humanized version of antibody 16C5.
  • an ILT3-binding agent is a variant of antibody 16C5.
  • an ILT3-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from antibody 45G10, a humanized version thereof, or variants thereof.
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3 from antibody 45G10.
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 45G10.
  • an ILT3-binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3; and (b) a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 45G10.
  • an ILT3-binding agent is a humanized version of antibody 45G10.
  • an ILT3-binding agent is a variant of antibody 45G10.
  • an ILT3-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from antibody 48A6, a humanized version thereof, or variants thereof.
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3 from antibody 48A6.
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 48A6.
  • an ILT3-binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3; and (b) a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 48A6.
  • an ILT3-binding agent is a humanized version of antibody 48A6.
  • an ILT3-binding agent is a variant of antibody 48A6.
  • an ILT3-binding agent comprises a heavy chain variable region CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from antibody 53F10, a humanized version thereof, or variants thereof.
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3 from antibody 53F10.
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 53F10.
  • an ILT3-binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3; and (b) a light chain variable region comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody 53F10.
  • an ILT3-binding agent is a humanized version of antibody 53F10.
  • an ILT3-binding agent is a variant of antibody 53F10.
  • an ILT3-binding agent (e.g., an antibody) comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising the amino acid sequence REWRMTLYAMDY (SEQ ID NO:29) or REWRYTLYAMDY (SEQ ID NO: 105), and a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASESVDSYGNSFMH (SEQ ID NO:30) or RASESVESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising the amino acid sequence LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising the amino acid sequence QQNNEDP
  • an ILT3-binding agent (e.g., an antibody) comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising the amino acid sequence REWRMTLYAMDY (SEQ ID NO:29) or REWRYTLYAMDY (SEQ ID NO: 105), and/or (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASESVDSYGNSFMH (SEQ ID NO: 30) or RASESVESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising the amino acid sequence LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising the amino acid sequence
  • the ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising the amino acid sequence REWRMTLYAMDY (SEQ ID NO:29).
  • the ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising the amino acid sequence REWRYTLYAMDY (SEQ ID NO: 105).
  • the ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASESVDSYGNSFMH (SEQ ID NO:30), a light chain variable region CDR2 comprising the amino acid sequence LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising the amino acid sequence QQNNEDPFT (SEQ ID NO:32).
  • the ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASESVESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising the amino acid sequence LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising the amino acid sequence QQNNEDPFT (SEQ ID NO:32).
  • the ILT3-binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising the amino acid sequence REWRMTLYAMDY (SEQ ID NO:29), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASES VDSYGNSFMH (SEQ ID NO:30), a light chain variable region CDR2 comprising the amino acid sequence LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising the amino acid sequence QQNNEDPFT (SEQ ID NO:32).
  • the ILT3-binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising the amino acid sequence REWRYTLYAMDY (SEQ ID NO: 105), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASESVESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising the amino acid sequence LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising the amino acid sequence QQNNEDPFT (SEQ ID NO:32).
  • an ILT3-binding agent (e.g., an antibody) comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), or a variant thereof comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions; a heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), or a variant thereof comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions; and a heavy chain variable region CDR3 comprising the amino acid sequence REWRMTLYAMDY (SEQ ID NO:29), REWRYTLYAMDY (SEQ ID NO: 105), or a variant thereof comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions; and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASES VDSYGNSFMH (SEQ ID NO:30), RASESVESYGS
  • an ILT3-binding agent e.g., an antibody
  • 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 (Gin 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 light chain variable region CDR1, CDR2, and/or CDR3 of an antibody described herein is modified to reduce deamidation. In some embodiments, the light chain variable region CDR1, CDR2, and/or CDR3 of an antibody described herein is modified to reduce deamidation. Any one of the heavy chain variable region CDRs and light chain variable region CDRs of antibody 5A7, 3A3, Hz5A7, 12A12, 16C5, 45G10, 48A6, or 53F10 can be modified to reduce deamidation.
  • an ILT3-binding agent e.g., an antibody
  • Isomerization is a chemical process by which a compound is transformed into any of its isomeric forms, ie., forms with the same chemical composition but with different structure or configuration and, potentially with different physical and chemical properties. Studies have shown that aspartate (Asp or D) isomerization within a CDR can impact antibody binding and/or stability.
  • the heavy chain variable region CDR1, CDR2, and/or CDR3 of an antibody described herein is modified to reduce isomerization.
  • the light chain variable region CDR1, CDR2, and/or CDR3 is modified to reduce isomerization. Any one of the heavy chain variable region CDRs and light chain variable region CDRs of antibody 5A7, 3A3, Hz5A7, 12A12, 16C5, 45G10, 48A6, or 53F10 can be modified to reduce isomerization.
  • an ILT3-binding agent e.g., an antibody
  • 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.
  • the heavy chain variable region CDR1, CDR2, and/or CDR3 of an antibody described herein is modified to reduce oxidation (e.g., methionine oxidation).
  • the light chain variable region CDR1, CDR2, and/or CDR3 of an antibody described herein is modified to reduce oxidation (e.g., methionine oxidation). Any one of the heavy chain variable region CDRs and light chain variable region CDRs of antibody 5A7, 3A3, Hz5A7, 12A12, 16C5, 45G10, 48A6, or 53F10 can be modified to reduce methionine oxidation.
  • an anti-ILT3 binding agent comprises a heavy chain variable region comprising the amino acid sequence of heavy chain variable region CDR1, CDR2, and CDR3 of antibody 5A7 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% identity to the sequence of SEQ ID NO: 111 and a light chain variable region comprising the amino acid sequence of light chain variable region CDR1, CDR2, and CDR3 of antibody 5A7 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% identity to the sequence of SEQ ID NO: 112.
  • an anti-ILT3 binding agent comprises a heavy chain variable region comprising the amino acid sequence of heavy chain variable region CDR1, CDR2, and CDR3 of antibody Hz5A7.v5 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% identity to the sequence of SEQ ID NO: 123 and a light chain variable region comprising the amino acid sequence of light chain variable region CDR1, CDR2, and CDR3s of antibody Hz5A7.v5 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% identity to the sequence of SEQ ID NO: 124.
  • an ILT3-binding agent (e.g., an antibody) comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 111.
  • an ILT3- binding agent comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 112.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 123. In some embodiments, an ILT3-binding agent comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 124.
  • an ILT3-binding agent (e.g., an antibody) comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 111. In some embodiments, an ILT3-binding agent comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 112. In some embodiments, an ILT3-binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 123. In some embodiments, an ILT3-binding agent comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 124.
  • an ILT3-binding agent (e.g., an antibody) comprises a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 111 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 112.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 111 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 112.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 111 and a light chain variable region having at least 95% sequence identity to SEQ ID NO: 112.
  • an ILT3 -binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:111 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 112.
  • an ILT3-binding agent (e.g., an antibody) comprises a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 123 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 124.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 123 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 124.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 123 and a light chain variable region having at least 95% sequence identity to SEQ ID NO: 124.
  • an ILT3 -binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 123 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 124.
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising TISGGGSYTNYPDSVKG (SEQ ID NO:28), a heavy chain variable region CDR3 comprising REWRYTLYAMDY (SEQ ID NO: 105), and a light chain variable region comprising a light chain variable region CDR1 comprising RASES VESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising QQNNEDPFT (SEQ ID NO:32), wherein the heavy chain variable region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97% identity to the sequence of SEQ ID NO: 123, and wherein the light chain variable region
  • an ILT3-binding agent comprises a heavy chain comprising a heavy chain variable region CDR1 comprising GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising TISGGGSYTNYPDSVKG (SEQ ID NO:28), a heavy chain variable region CDR3 comprising REWRYTLYAMDY (SEQ ID NO: 105), and a light chain comprising a light chain variable region CDR1 comprising RASES VESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising QQNNEDPFT (SEQ ID NO:32), wherein the heavy chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the sequence of SEQ ID NO: 126, and wherein the light chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at
  • an ILT3-binding agent comprises a heavy chain comprising a heavy chain variable region CDR1 comprising GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising TISGGGSYTNYPDSVKG (SEQ ID NO:28), a heavy chain variable region CDR3 comprising REWRYTLYAMDY (SEQ ID NO: 105), and a light chain comprising a light chain variable region CDR1 comprising RASES VESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising QQNNEDPFT (SEQ ID NO:32), wherein the heavy chain comprises at least 95% identity to the sequence of SEQ ID NO: 126, and wherein the light chain comprises at least 95% identity to the sequence of SEQ ID NO: 128.
  • an ILT3-binding agent comprises (a) a heavy chain comprising the amino acids of SEQ ID NO: 126 and (b) a light chain comprising a light chain variable region CDR1 comprising RASES VESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising QQNNEDPFT (SEQ ID NO:32), wherein the light chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the sequence of SEQ ID NO: 128.
  • an ILT3-binding agent comprises (a) a heavy chain comprising a heavy chain variable region CDR1 comprising GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising TISGGGSYTNYPDSVKG (SEQ ID NO:28), a heavy chain variable region CDR3 comprising REWRYTLYAMDY (SEQ ID NO: 105), wherein the heavy chain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the sequence of SEQ ID NO: 126, and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 128.
  • an ILT3-binding agent is an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 126 and a light chain comprising the amino acid sequence of SEQ ID NO: 128.
  • an ILT3-binding agent comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFSLTSYGVH (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence VIWPGGTINYNSALMS (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence DKYDGGWFAY (SEQ ID NO: 13), and a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence KASQNVRTAVA (SEQ ID NO: 14), a light chain variable region CDR2 comprising the amino acid sequence LASNRHT (SEQ ID NO: 15), and a light chain variable region CDR3 comprising the amino acid sequence GFSLTSYGVH (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence VIWPGGTINYNSALMS (SEQ ID NO: 12), and a heavy chain variable region CDR3 compris
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFSLTSYGVH (SEQ ID NO:11), a heavy chain variable region CDR2 comprising the amino acid sequence VIWPGGTINYNSALMS (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence DKYDGGWFAY (SEQ ID NO: 13).
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence KASQNVRTAVA (SEQ ID NO: 14), a light chain variable region CDR2 comprising the amino acid sequence LASNRHT (SEQ ID NO: 15), and a light chain variable region CDR3 comprising the amino acid sequence LQHLNYPLT (SEQ ID NO: 16).
  • an ILT3-binding agent comprises (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFSLTSYGVH (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence VIWPGGTINYNSALMS (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence DKYDGGWFAY (SEQ ID NO: 13), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence KASQNVRTAVA (SEQ ID NO: 14), a light chain variable region CDR2 comprising the amino acid sequence LASNRHT (SEQ ID NO: 15), and a light chain variable region CDR3 comprising the amino acid sequence LQHLNYPLT (SEQ ID NO: 16).
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 109. In some embodiments, an ILT3-binding agent comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 110. In some embodiments, an ILT3 -binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 109.
  • an ILT3-binding agent comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO:110.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 109 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 110.
  • an ILT3- binding agent comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 109 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 110.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 109 and a light chain variable region having at least 95% sequence identity to SEQ ID NO: 110. In some embodiments, an ILT3- binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 109 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 110.
  • the ILT3-binding agent is antibody 3 A3. In some embodiments, the ILT3-binding agent is a humanized version of antibody 3 A3. In some embodiments, the ILT3-binding agent is a variant of antibody 3 A3 or humanized antibody 3 A3.
  • an ILT3-binding agent (e.g., an antibody) comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GYTFTDYNMD (SEQ ID NO:43), a heavy chain variable region CDR2 comprising the amino acid sequence YIYPNNGGTGYNQKFNS (SEQ ID NO:44), and a heavy chain variable region CDR3 comprising the amino acid sequence SPYYDYVGSYAMDY (SEQ ID NO:45), and a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence TASSSVSSSYLH (SEQ ID NO:46), a light chain variable region CDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:47), and a light chain variable region CDR3 comprising the amino acid sequence HQYHRSPRT (SEQ ID NO:48); (b) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GY
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GYTFTDYNMD (SEQ ID NO:43), a heavy chain variable region CDR2 comprising the amino acid sequence YIYPNNGGTGYNQKFNS (SEQ ID NO:44), and a heavy chain variable region CDR3 comprising the amino acid sequence SPYYDYVGSYAMDY (SEQ ID NO:45).
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence TASSSVSSSYLH (SEQ ID NO:46), a light chain variable region CDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:47), and a light chain variable region CDR3 comprising the amino acid sequence HQYHRSPRT (SEQ ID NO:48).
  • an ILT3-binding agent comprises (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GYTFTDYNMD (SEQ ID NO:43), a heavy chain variable region CDR2 comprising the amino acid sequence YIYPNNGGTGYNQKFNS (SEQ ID NO:44), and a heavy chain variable region CDR3 comprising the amino acid sequence SPYYDYVGSYAMDY (SEQ ID NO:45), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence TASSSVSSSYLH (SEQ ID NO:46), a light chain variable region CDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:47), and a light chain variable region CDR3 comprising the amino acid sequence HQYHRSPRT (SEQ ID NO:48).
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 113.
  • an ILT3 -binding agent comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 114.
  • an ILT3 -binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 113.
  • an ILT3-binding agent comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO:114.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 113 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 114.
  • an ILT3- binding agent comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 113 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 114.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 113 and a light chain variable region having at least 95% sequence identity to SEQ ID NO: 114. In some embodiments, an ILT3- binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 113 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 114.
  • the ILT3-binding agent is antibody 12A12. In some embodiments, the ILT3-binding agent is a humanized version of antibody 12A12. In some embodiments, the ILT3-binding agent is a variant of antibody 12A12 or humanized antibody 12A12.
  • an ILT3-binding agent (e.g., an antibody) comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GYTFTDYNMD (SEQ ID NO:43), a heavy chain variable region CDR2 comprising the amino acid sequence YIYPSNGGTGYNQKFKS (SEQ ID NO:59), and a heavy chain variable region CDR3 comprising the amino acid sequence VPYYDYLYYYAMDY (SEQ ID NO:60), and a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASSSVSFMH (SEQ ID NO:61), a light chain variable region CDR2 comprising the amino acid sequence ATSNLAS (SEQ ID NO:62), and a light chain variable region CDR3 comprising the amino acid sequence QQWSTNPYMYT (SEQ ID NO:63); (b) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GYTFTDYNMD (SEQ ID NO:43), a heavy chain variable region CDR2 comprising the amino acid sequence YIYPSNGGTGYNQKFKS (SEQ ID NO:59), and a heavy chain variable region CDR3 comprising the amino acid sequence VPYYDYLYYYAMDY (SEQ ID NO:60).
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASSSVSFMH (SEQ ID NO:61), a light chain variable region CDR2 comprising the amino acid sequence ATSNLAS (SEQ ID NO:62), and a light chain variable region CDR3 comprising the amino acid sequence QQWSTNPYMYT (SEQ ID NO:63).
  • an ILT3-binding agent comprises (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GYTFTDYNMD (SEQ ID NO:43), a heavy chain variable region CDR2 comprising the amino acid sequence YIYPSNGGTGYNQKFKS (SEQ ID NO:59), and a heavy chain variable region CDR3 comprising the amino acid sequence VPYYDYLYYYAMDY (SEQ ID NO:60), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASSSVSFMH (SEQ ID NO:61), a light chain variable region CDR2 comprising the amino acid sequence ATSNLAS (SEQ ID NO:62), and a light chain variable region CDR3 comprising the amino acid sequence QQWSTNPYMYT (SEQ ID NO:63).
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 115. In some embodiments, an ILT3 -binding agent comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 116. In some embodiments, an ILT3 -binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 115.
  • an ILT3-binding agent comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO:116.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 115 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 116.
  • an ILT3- binding agent comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 115 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 116.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 115 and a light chain variable region having at least 95% sequence identity to SEQ ID NO: 116. In some embodiments, an ILT3- binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 115 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 116.
  • the ILT3-binding agent is antibody 16C5. In some embodiments, the ILT3-binding agent is a humanized version of antibody 16C5. In some embodiments, the ILT3-binding agent is a variant of antibody 16C5 or humanized antibody 16C5.
  • an ILT3-binding agent (e.g., an antibody) comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSDYGMH (SEQ ID NO:71), a heavy chain variable region CDR2 comprising the amino acid sequence YIFSGSSTIYYADTVKG (SEQ ID NO:72), and a heavy chain variable region CDR3 comprising the amino acid sequence ADGRGAMDY (SEQ ID NO:73), and a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASQDISKFLN (SEQ ID NO:74), a light chain variable region CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO:75), and a light chain variable region CDR3 comprising the amino acid sequence QQGNTLPWT (SEQ ID NO:76); (b) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSDYGMH (S
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSDYGMH (SEQ ID NO:71), a heavy chain variable region CDR2 comprising the amino acid sequence YIFSGSSTIYYADTVKG (SEQ ID NO:72), and a heavy chain variable region CDR3 comprising the amino acid sequence ADGRGAMDY (SEQ ID NO:73).
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASQDISKFLN (SEQ ID NO:74), a light chain variable region CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO:75), and a light chain variable region CDR3 comprising the amino acid sequence QQGNTLPWT (SEQ ID NO:76).
  • an ILT3-binding agent comprises (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSDYGMH (SEQ ID NO:71), a heavy chain variable region CDR2 comprising the amino acid sequence YIFSGSSTIYYADTVKG (SEQ ID NO:72), and a heavy chain variable region CDR3 comprising the amino acid sequence ADGRGAMDY (SEQ ID NO:73), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASQDISKFLN (SEQ ID NO:74), a light chain variable region CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO:75), and a light chain variable region CDR3 comprising the amino acid sequence QQGNTLPWT (SEQ ID NO:76).
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 117. In some embodiments, an ILT3-binding agent comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO:118. In some embodiments, an ILT3 -binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 117.
  • an ILT3-binding agent comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO:118.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 117 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 118.
  • an ILT3- binding agent comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 117 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 118.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 117 and a light chain variable region having at least 95% sequence identity to SEQ ID NO: 118. In some embodiments, an ILT3- binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 117 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 118.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence selected from SEQ ID NOs: 162-163. In some embodiments, an ILT3- binding agent comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 164. In some embodiments, an ILT3-binding agent comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 162-163. In some embodiments, an ILT3- binding agent comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 164.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to an amino acid sequence selected from SEQ ID Nos: 162-163, and a light chain variable region having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to SEQ ID NO: 164.
  • an ILT3- binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 162 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 164.
  • an ILT3-binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 163 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 164.
  • the ILT3-binding agent is antibody 45G10. In some embodiments, the ILT3-binding agent is a humanized version of antibody 45G10. In some embodiments, the ILT3-binding agent is a variant of antibody 45G10 or humanized antibody 45G10.
  • an ILT3-binding agent e.g., an antibody
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISSGGTYTFYPDSVKG (SEQ ID NO:87), and a heavy chain variable region CDR3 comprising the amino acid sequence RGWLLHYYAMDY (SEQ ID NO:88).
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RPSESVDSFGNSFMH (SEQ ID NO: 89), a light chain variable region CDR2 comprising the amino acid sequence LSSKLES (SEQ ID NOVO), and a light chain variable region CDR3 comprising the amino acid sequence QQHNEDPFT (SEQ ID NO:91).
  • an ILT3-binding agent comprises (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISSGGTYTFYPDSVKG (SEQ ID NO:87), and a heavy chain variable region CDR3 comprising the amino acid sequence RGWLLHYYAMDY (SEQ ID NO:88), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RPSESVDSFGNSFMH (SEQ ID NO:89), a light chain variable region CDR2 comprising the amino acid sequence LSSKLES (SEQ ID NO:90), and a light chain variable region CDR3 comprising the amino acid sequence QQHNEDPFT (SEQ ID NO:91).
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 119. In some embodiments, an ILT3-binding agent comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 120. In some embodiments, an ILT3 -binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 119.
  • an ILT3-binding agent comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 120.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 119 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 120.
  • an ILT3- binding agent comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 119 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 120.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 119 and a light chain variable region having at least 95% sequence identity to SEQ ID NO: 120. In some embodiments, an ILT3- binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 119 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 120.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence selected from SEQ ID NOs: 156-160. In some embodiments, an ILT3- binding agent comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 161. In some embodiments, an ILT3-binding agent comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 156-160. In some embodiments, an ILT3- binding agent comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 161.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to an amino acid sequence selected from SEQ ID Nos: 156-160, and a light chain variable region having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to SEQ ID NO: 161.
  • an ILT3- binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 156 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 161.
  • an ILT3-binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 157 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 161. In some embodiments, an ILT3-binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 158 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 161. In some embodiments, an ILT3-binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 159 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 161. In some embodiments, an ILT3-binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 160 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 161.
  • the ILT3-binding agent is antibody 48A6. In some embodiments, the ILT3-binding agent is a humanized version of antibody 48A6. In some embodiments, the ILT3-binding agent is a variant of antibody 48A6 or humanized antibody 48A6.
  • an ILT3-binding agent (e.g., an antibody) comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSDYGMH (SEQ ID NO:71), a heavy chain variable region CDR2 comprising the amino acid sequence YISTGIITVYYADTVKG (SEQ ID NO:99), and a heavy chain variable region CDR3 comprising the amino acid sequence ADGRGAMDY (SEQ ID NO:73), and a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASQDISNFLN (SEQ ID NO: 100), a light chain variable region CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO:75), and a light chain variable region CDR3 comprising the amino acid sequence QQGNTLPWT (SEQ ID NO:76); (b) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence
  • an ILT3-binding agent comprises a heavy chain variable region comprising a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSDYGMH (SEQ ID NO:71), a heavy chain variable region CDR2 comprising the amino acid sequence YISTGIITVYYADTVKG (SEQ ID NO:99), and a heavy chain variable region CDR3 comprising the amino acid sequence ADGRGAMDY (SEQ ID NO:73).
  • an ILT3-binding agent comprises a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASQDISNFLN (SEQ ID NO: 100), a light chain variable region CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO:75), and a light chain variable region CDR3 comprising the amino acid sequence QQGNTLPWT (SEQ ID NO:76).
  • an ILT3- binding agent comprises (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSDYGMH (SEQ ID NO:71), a heavy chain variable region CDR2 comprising the amino acid sequence YISTGIITVYYADTVKG (SEQ ID NO:99), and a heavy chain variable region CDR3 comprising the amino acid sequence ADGRGAMDY (SEQ ID NO:73), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASQDISNFLN (SEQ ID NO: 100), a light chain variable region CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO:75), and a light chain variable region CDR3 comprising the amino acid sequence QQGNTLPWT (SEQ ID NO:76).
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 121.
  • an ILT3 -binding agent comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the sequence of SEQ ID NO: 122.
  • an ILT3 -binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 121.
  • an ILT3-binding agent comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 122.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 121 and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 122.
  • an ILT3- binding agent comprises a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 121 and a light chain variable region having at least 90% sequence identity to SEQ ID NO: 122.
  • an ILT3-binding agent comprises a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 121 and a light chain variable region having at least 95% sequence identity to SEQ ID NO: 122. In some embodiments, an ILT3- binding agent comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 121 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 122.
  • the ILT3-binding agent is antibody 53F10. In some embodiments, the ILT3-binding agent is a humanized version of antibody 53F10. In some embodiments, the ILT3-binding agent is a variant of antibody 53F10 or humanized antibody 53F10.
  • agents that compete with one or more of the binding agents e.g., antibodies
  • an agent competes with one of more of the antibodies described herein for binding to ILT3.
  • an agent that competes with one of 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. Antibodies and antigen-binding fragments that compete with or bind the same epitope as the antibodies described herein are expected to show similar functional properties.
  • an agent e.g., an antibody
  • the reference antibody comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising the amino acid sequence REWRMTLYAMDY (SEQ ID NO:29) or REWRYTLYAMDY (SEQ ID NO: 105), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASESVDSYGNSFMH (SEQ ID NO:30) or RASESVESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising the amino acid sequence LTSNLES (SEQ ID NO:31), and a light chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO
  • an agent e.g., an antibody
  • the reference antibody comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), and a heavy chain variable region CDR3 comprising the amino acid sequence REWRMTLYAMDY (SEQ ID NO:29) or REWRYTLYAMDY (SEQ ID NO: 105), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASESVDSYGNSFMH (SEQ ID NO:30) or RASESVESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising the amino acid sequence LTSNLES (SEQ ID NO:31), and a light chain variable region C
  • an agent e.g., an antibody
  • the reference antibody comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFSLTSYGVH (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence VIWPGGTINYNSALMS (SEQ ID NO: 12), and a heavy chain variable region CDR3 comprising the amino acid sequence DKYDGGWFAY (SEQ ID NO: 13), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence KASQNVRTAVA (SEQ ID NO: 14), a light chain variable region CDR2 comprising the amino acid sequence LASNRHT (SEQ ID NO: 15), and a light chain variable region CDR3 comprising the amino acid sequence LQHLNYPLT (SEQ ID NO: 16).
  • an agent e.g., an antibody
  • competes for binding to human ILT3 with a reference antibody wherein the reference antibody comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GYTFTDYNMD (SEQ ID NO:43), a heavy chain variable region CDR2 comprising the amino acid sequence YIYPNNGGTGYNQKFNS (SEQ ID NO:44), and a heavy chain variable region CDR3 comprising the amino acid sequence SPYYDYVGSYAMDY (SEQ ID NO:45), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence TASSSVSSSYLH (SEQ ID NO:46), a light chain variable region CDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:47), and a light chain variable region CDR3 comprising the amino acid sequence HQYHRSPRT (SEQ ID NO:48).
  • the reference antibody comprises: (a)
  • an agent e.g., an antibody
  • the reference antibody comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GYTFTDYNMD (SEQ ID NO:43), a heavy chain variable region CDR2 comprising the amino acid sequence YIYPSNGGTGYNQKFKS (SEQ ID NO:59), and a heavy chain variable region CDR3 comprising the amino acid sequence VPYYDYLYYYAMDY (SEQ ID NO:60), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASSSVSFMH (SEQ ID NO:61), a light chain variable region CDR2 comprising the amino acid sequence ATSNLAS (SEQ ID NO: 62), and a light chain variable region CDR3 comprising the amino acid sequence QQWSTNPYMYT (SEQ ID NO:63).
  • an agent e.g., an antibody
  • the reference antibody comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSDYGMH (SEQ ID NO:71), a heavy chain variable region CDR2 comprising the amino acid sequence YIFSGSSTIYYADTVKG (SEQ ID NO:72), and a heavy chain variable region CDR3 comprising the amino acid sequence ADGRGAMDY (SEQ ID NO:73), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASQDISKFLN (SEQ ID NO:74), a light chain variable region CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO:75), and a light chain variable region CDR3 comprising the amino acid sequence QQGNTLPWT (SEQ ID NO:76).
  • an agent e.g., an antibody
  • the reference antibody comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISSGGTYTFYPDSVKG (SEQ ID NO:87), and a heavy chain variable region CDR3 comprising the amino acid sequence RGWLLHYYAMDY (SEQ ID NO:88), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RPSESVDSFGNSFMH (SEQ ID NO:89), a light chain variable region CDR2 comprising the amino acid sequence LSSKLES (SEQ ID NO:90), and a light chain variable region CDR3 comprising the amino acid sequence QQHNEDPFT (SEQ ID NO:91).
  • an agent e.g., an antibody
  • competes for binding to human ILT3 with a reference antibody wherein the reference antibody comprises: (a) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSDYGMH (SEQ ID NO:71), a heavy chain variable region CDR2 comprising the amino acid sequence YISTGIITVYYADTVKG (SEQ ID NO:99), and a heavy chain variable region CDR3 comprising the amino acid sequence ADGRGAMDY (SEQ ID NO:73), and (b) a light chain variable region comprising a light chain variable region CDR1 comprising the amino acid sequence RASQDISNFLN (SEQ ID NO: 100), a light chain variable region CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO: 75), and a light chain variable region CDR3 comprising the amino acid sequence QQGNTLPWT (SEQ ID NO:76).
  • the reference antibody comprises: (a) a heavy chain variable region compris
  • an ILT3-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 (CHI, 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.
  • 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 (ACH2 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.
  • 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.
  • binding of the Cl 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 cell expressing a Fc receptor (FcR).
  • Fc receptors that are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and 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.
  • IgG gamma receptors
  • IgE epsilon receptors
  • IgA alpha receptors
  • IgM mi receptors
  • an ILT3-binding agent (e.g., an antibody) comprises a variant Fc region.
  • the amino acid sequences of the Fc region of human IgGl, IgG2, IgG3, and IgG4 are known to those of ordinary skill in the art (e.g., a representative human IgGl region is SEQ ID NO: 129).
  • 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: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, and SEQ ID NO: 134.
  • a modified antibody (e.g., comprising a modified Fc region) 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.
  • a modified antibody does not have one or more effector functions. In some embodiments, a modified antibody does not have any detectable effector functions (e.g., “effectorless” antibodies). In some embodiments, a modified antibody has no ADCC activity and/or no CDC activity. In some embodiments, a modified antibody does not bind an Fc receptor and/or complement factors. In some embodiments, a modified antibody has no effector function(s). In some embodiments, 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. In some embodiments, the constant region is modified to add/substitute one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites.
  • an ILT3-binding agent (e.g., an antibody) 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: 126.
  • an ILT3-binding agent (e.g., an antibody) 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: 128.
  • an ILT3 -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: 126 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: 128.
  • an ILT3-binding agent comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 126.
  • an ILT3-binding agent comprises a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 128.
  • an ILT3-binding agent comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 126 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 128. In some embodiments, an ILT3- binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 126. In some embodiments, an ILT3-binding agent comprises a light chain comprising the amino acid sequence of SEQ ID NO: 128. In some embodiments, an ILT3-binding agent comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 126 and a light chain comprising the amino acid sequence of SEQ ID NO: 128.
  • an ILT3-binding agent is an antibody that comprises a heavy chain of SEQ ID NO: 126 and/or a light chain of SEQ ID NO: 128. In some embodiments, an ILT3-binding agent is an antibody that comprises a heavy chain of SEQ ID NO: 126. In some embodiments, an ILT3-binding agent is an antibody that comprises a light chain of SEQ ID NO: 128. In some embodiments, an ILT3-binding agent is an antibody that comprises a heavy chain of SEQ ID NO: 126 and a light chain of SEQ ID NO: 128. [00197] 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. Using these engineering techniques to modify an antibody it may be possible to disrupt the activity or effector function provided by a specific sequence or region while substantially maintaining the structure, binding activity, and other desired characteristics of the modified antibody.
  • 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.
  • it is desirable to improve the binding affinity of the antibody.
  • 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 a substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide that results in a change in 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.
  • the length of additional amino acids residues may range from one residue to a hundred or more residues.
  • 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.
  • one or more cysteine residues are added to create disulfide bond(s) to improve stability.
  • 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.
  • 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.
  • an ILT3-binding agent described herein is chemically modified.
  • an ILT3-binding agent is an anti-ILT3 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.
  • an ILT3-binding agent is an 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, a Fc region or its variants, a human serum albumin, a CH3 domain of an immunoglobulin, polyethylene glycol (PEG), a PEG mimetic, XTEN®, serum albumin, polysialic acid, N-(2-hydroxypropyl)methacrylamide, or dextran.
  • an antibody fragment e.g., scFv, Fv, Fab, F(ab')2, or F(ab')
  • the antibody fragment is attached (either directly or indirectly) to a half-life extending moiety including, but not limited to, a Fc region or its variants, a human serum albumin, a CH3 domain of an immunoglobulin, poly
  • the present disclosure encompasses ILT3-binding agents built upon nonimmunoglobulin backbones, wherein the agents bind the same epitope or essentially the same epitope as an anti-ILT3 antibody disclosed herein.
  • a non- immunoglobulin-based binding agent is an agent that competes with an anti-ILT3 antibody described herein in a competitive binding assay.
  • alternative ILT3-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 a-helices; (2) small scaffolds with few secondary structures or an irregular architecture of a-helices and P-sheets; and (3) scaffolds consisting of predominantly P-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 y-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 P-strand protein fold; and Kunitz domain inhibitor scaffold
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 shown in Table 1.
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFSLTSYGVH (SEQ ID NO: 11), a heavy chain variable region CDR2 comprising the amino acid sequence VIWPGGTINYNSALMS (SEQ ID NO: 12), a heavy chain variable region CDR3 comprising the amino acid sequence DKYDGGWFAY (SEQ ID NO: 13), a light chain variable region CDR1 comprising the amino acid sequence KASQNVRTAVA (SEQ ID NO: 14), a light chain variable region CDR2 comprising the amino acid sequence LASNRHT (SEQ ID NO: 15), and a light chain variable region CDR3 comprising the amino acid sequence LQHLNYPLT (SEQ ID NO: 11), a heavy chain
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 shown in Table 2.
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), a heavy chain variable region CDR3 comprising the amino acid sequence REWRMTLYAMDY (SEQ ID NO:29), a light chain variable region CDR1 comprising the amino acid sequence RASESVDSYGNSFMH (SEQ ID NO:30), a light chain variable region CDR2 comprising the amino acid sequence LTSNLES (SEQ ID NO: 31), and a light chain variable region CDR3 comprising the amino acid sequence QQNNEDPFT
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 from antibody 5A7. In some embodiments, an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 shown in Table 8.
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISGGGSYTNYPDSVKG (SEQ ID NO:28), a heavy chain variable region CDR3 comprising the amino acid sequence REWRYTLYAMDY (SEQ ID NO: 105), a light chain variable region CDR1 comprising the amino acid sequence RASES VESYGSSFMH (SEQ ID NO: 106), a light chain variable region CDR2 comprising the amino acid sequence LTSNLES (SEQ ID NO:31), and a light chain variable region CDR3 comprising the amino acid sequence QQNNEDPFT (SEQ ID NO:32).
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 from antibody Hz5A7.v5.
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 shown in Table 3.
  • an ILT3 -binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1 comprising the amino acid sequence GYTFTDYNMD (SEQ ID NO:43), a heavy chain variable region CDR2 comprising the amino acid sequence YIYPNNGGTGYNQKFNS (SEQ ID NO:44), a heavy chain variable region CDR3 comprising the amino acid sequence SPYYDYVGSYAMDY (SEQ ID NO:45), a light chain variable region CDR1 comprising the amino acid sequence TASSSVSSSYLH (SEQ ID NO:46), a light chain variable region CDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:47), and a light chain variable region CDR3 comprising the amino acid
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 shown in Table 4.
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1 comprising the amino acid sequence GYTFTDYNMD (SEQ ID NO:43), a heavy chain variable region CDR2 comprising the amino acid sequence YIYPSNGGTGYNQKFKS (SEQ ID NO:59), a heavy chain variable region CDR3 comprising the amino acid sequence VPYYDYLYYYAMDY (SEQ ID NO:60), a light chain variable region CDR1 comprising the amino acid sequence RASSSVSFMH (SEQ ID NO:61), a light chain variable region CDR2 comprising the amino acid sequence ATSNLAS (SEQ ID NO:62), and a light chain variable region CDR3 comprising the amino acid sequence
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 shown in Table 5.
  • an ILT3 -binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSDYGMH (SEQ ID NO:71), a heavy chain variable region CDR2 comprising the amino acid sequence YIFSGSSTIYYADTVKG (SEQ ID NO:72), a heavy chain variable region CDR3 comprising the amino acid sequence ADGRGAMDY (SEQ ID NO:73), a light chain variable region CDR1 comprising the amino acid sequence RASQDISKFLN (SEQ ID NO:74), a light chain variable region CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO:75), and a light chain variable region CDR3 comprising the amino acid sequence QQGNTLPW
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 shown in Table 6.
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSSYGMS (SEQ ID NO:27), a heavy chain variable region CDR2 comprising the amino acid sequence TISSGGTYTFYPDSVKG (SEQ ID NO:87), a heavy chain variable region CDR3 comprising the amino acid sequence RGWLLHYYAMDY (SEQ ID NO:88), a light chain variable region CDR1 comprising the amino acid sequence RPSESVDSFGNSFMH (SEQ ID NO:89), a light chain variable region CDR2 comprising the amino acid sequence LSSKLES (SEQ ID NO:90), and a light chain variable region CDR3 comprising the amino acid sequence QQHNEDP
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 shown in Table 7.
  • an ILT3-binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1 comprising the amino acid sequence GFTFSDYGMH (SEQ ID NO:71), a heavy chain variable region CDR2 comprising the amino acid sequence YISTGIITVYYADTVKG (SEQ ID NO:99), a heavy chain variable region CDR3 comprising the amino acid sequence ADGRGAMDY (SEQ ID NO:73), a light chain variable region CDR1 comprising the amino acid sequence RASQDISNFLN (SEQ ID NO: 100), a light chain variable region CDR2 comprising the amino acid sequence YTSRLHS (SEQ ID NO:75), and a light chain variable region CDR3 comprising the amino acid sequence QQGNTLPWT
  • an ILT3- binding agent comprises an engineered scaffold protein comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region CDR1, CDR2, and CDR3 from antibody 53F10.
  • a composition comprises an ILT3-binding agent described herein.
  • a composition comprises an anti-ILT3 antibody described herein.
  • a composition comprises a monoclonal anti-ILT3 antibody described herein.
  • a pharmaceutical composition comprises an ILT3-binding agent described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprises an anti-ILT3 antibody described herein and a pharmaceutically acceptable carrier.
  • a composition comprises a monoclonal anti-ILT3 antibody described herein and a pharmaceutically acceptable carrier.
  • an ILT3-binding agent is isolated. In some embodiments, an ILT3-binding agent is substantially pure.
  • antigen-antibody interactions are non-covalent and reversible, formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic and van der Waals forces.
  • affinity and/or avidity are commonly used.
  • the binding of an antibody to its antigen is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (KD).
  • KD is the ratio of an antibody dissociation rate (k O ff) (how quickly it dissociates from its antigen) to the antibody association rate (kon) (how quickly it binds to its antigen).
  • KD values are determined by measuring the k on and k O ff rates of a specific antibody/antigen interaction and then using a ratio of these values to calculate the KD value.
  • KD values may be used to evaluate and rank order the strength of individual antibody/antigen interactions. The lower the KD of an antibody, the higher the affinity of the antibody for its target.
  • affinity is measured using SPR technology in 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.
  • an ILT3-binding agent binds ILT3 with a dissociation constant (KD) of 1 pM 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.
  • KD dissociation constant
  • an ILT3-binding agent binds ILT3 with a KD of about 20 nM or less.
  • an ILT3 -binding agent binds ILT3 with a KD of 10 nM or less.
  • an ILT3 -binding agent binds ILT3 with a KD of 5 nM or less, In some embodiments, an ILT3 -binding agent binds ILT3 with a KD of 3 nM or less, In some embodiments, an ILT3 -binding agent binds ILT3 with a KD of 2 nM or less, In some embodiments, an ILT3 -binding agent binds ILT3 with a KD of 1 nM or less. In some embodiments, an ILT3-binding agent binds ILT3 with a KD of 0.5 nM or less.
  • an ILT3-binding agent binds ILT3 with a KD of 0.1 nM or less. In some embodiments, an ILT3-binding agent binds ILT3 with a KD of 50 pM or less. In some embodiments, an ILT3 -binding agent binds ILT3 with a KD of 25 pM or less. In some embodiments, an ILT3 -binding agent binds ILT3 with a KD of 10 pM or less. In some embodiments, an ILT3 -binding agent binds ILT3 with a KD of 1 pM or less.
  • an ILT3-binding agent binds ILT3 with a KD of 0.01 nM to 2.5 nM. In some embodiments, an ILT3-binding agent binds ILT3 with a KD of 0.1 nM to 5 nM. In some embodiments, an ILT3 -binding agent binds ILT3 with a KD of 1 nM to 5 nM.
  • the dissociation constant of the binding agent for ILT3 is the dissociation constant determined using an ILT3 protein immobilized on a Biacore chip and the binding agent flowed over the chip. In some embodiments, the dissociation constant of the binding agent for ILT3 is the dissociation constant determined using the binding agent captured by an anti-human IgG antibody on a Biacore chip and soluble ILT3 flowed over the chip.
  • an ILT3 -binding agent binds ILT3 with a half maximal effective concentration (EC50) of 1 pM 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.
  • an ILT3-binding agent binds human ILT3 with an EC50 of 1 pM 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.
  • an ILT3-binding agent binds cyno ILT3 and/or human ILT3 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. In some embodiments, an ILT3-binding agent binds ILT3 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.
  • an ILT3 -binding agent binds human ILT3 and has at least one or more of the following properties: (i) binds cyno ILT3; (ii) binds human and cyno ILT3; (iii) does not bind ILT2, ILT4, ILT5, and LILRB5; (iv) does not bind LILRA1, LILRA2, LILRA4, LILRA5, and LILRA6; (v) is an ILT3 antagonist; (vi) inhibits ILT3 activity; (vii) inhibits ILT3 signaling in cells that express ILT3; (viii) inhibits binding of ILT3 to APOE; (ix) inhibits binding of ILT3 to fibronectin; (x) inhibits binding of ILT3 to CNTFR; (xi inhibits ILT3 -induced suppression of myeloid cells; (xii) inhibits ILT3-induced suppression of myeloid cell activity; (xiii) restore
  • the ILT3-binding agents (e.g., antibodies) 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.
  • a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest.
  • 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. For example, several small 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.
  • 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 ILT3 -binding agents described herein.
  • recombinant expression vectors can be replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding one or more polypeptide chain(s) of an ILT3 -binding agent, such as an anti- ILT3 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 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. coh.
  • an ILT3 -binding agent e.g., an antibody
  • an ILT3 -binding agent of the present disclosure is expressed from one or more vectors.
  • a heavy chain variable region is expressed by one vector and a light chain variable region is expressed by a second vector.
  • a heavy chain variable region and a light chain variable region are expressed by one vector.
  • a vector encodes a heavy chain variable region of an ILT3 -binding agent described herein.
  • a vector encodes a light chain variable region of an ILT3 -binding agent described herein. In some embodiments, a vector encodes a heavy chain variable region and a light chain variable region of an ILT3 -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 ILT3 -binding agent described herein. In some embodiments, a vector encodes a light chain polypeptide of an ILT3 -binding agent described herein. In some embodiments, a vector encodes a heavy chain polypeptide and a light chain polypeptide of an ILT3 -binding agent described herein.
  • Suitable host cells for expression of an ILT3-binding agent (e.g., an antibody) or a ILT3 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.
  • Suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney- derived), L-929 (murine fibroblast-derived), Cl 27 (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 nontranscribed 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' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • nontranscribed 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' nontranslated 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 also offers a robust method for producing correctly folded and biologically functional proteins.
  • 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 ILT3-binding agents described herein.
  • the cells produce the ILT3-binding agents described herein.
  • the cells produce an antibody.
  • the cells produce an antibody that binds human ILT3.
  • the cells produce an antibody that binds cyno ILT3.
  • the cells produce an antibody that binds human ILT3 and cyno ILT3.
  • the cells produce an antibody designated 5A7.
  • the cells produce a humanized version of antibody 5A7, referred to as Hz5A7.
  • the cells produce a variant of Hz5A7, for example, Hz5A7.v5. In some embodiments, the cells produce an antibody designated 3 A3. In some embodiments, the cells produce an antibody designated 12A12. In some embodiments, the cells produce an antibody designated 16C5. In some embodiments, the cells produce an antibody designated 45G10. In some embodiments, the cells produce a humanized version of antibody 45G10, referred to as Hz45G10. In some embodiments, the cells produce an antibody designated 48 A6. In some embodiments, the cells produce a humanized version of antibody 48A6, referred to as Hz48A6. In some embodiments, the cells produce an antibody designated 53F10.
  • the cell is a prokaryotic cell (e.g., E. colt). In some embodiments, the cell is an eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, 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: 154), 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.
  • SEC size exclusion chromatography
  • MS mass spectrometry
  • NMR nuclear magnetic resonance
  • IEF isoelectric focusing
  • HPLC high performance liquid chromatography
  • x-ray crystallography x-ray crystallography
  • 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.
  • a suitable purification matrix for example, 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 HIC 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.
  • ILT3-binding agents e.g., antibodies
  • ILT3-binding agents e.g., antibodies
  • an anti-ILT3 antibody is tested for its ability to bind ILT3 (e.g., human ILT3 and/or cyno ILT3). Binding assays include, but are not limited to, SPR (e.g., Biacore), ELISA, and FACS.
  • an anti-ILT3 antibody is tested for its ability to inhibit, reduce, or block binding to fibronectin, APOE, and/or CNTFR.
  • antibodies may be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency.
  • monoclonal antibodies generated against ILT3 are grouped based upon the epitope each individual antibody recognizes, a process known as “epitope binning”.
  • epitope binning antibodies are tested in a pairwise combinatorial manner and antibodies that compete with each other are grouped together into bins.
  • 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.
  • the antigen is immobilized on a surface and the two antibodies are flowed over the immobilized antigen and compete to bind.
  • antibodies that block one another can be identified.
  • a competitive blocking profile is created for each antibody relative to the other antibodies.
  • the blocking results determine which bin each antibody is placed in.
  • High-throughput methods of epitope binning are known in the art and allow for screening and characterization of large numbers of antibodies within a short period of time.
  • Antibodies that bind similar epitopes often share similar functions and/or capabilities. Conversely, antibodies that bind different epitopes may have different functional activities.
  • an epitope bin comprises at least one antibody from the group consisting of 3A3, 5A7, 12A12, 16C5, 45G10, 48A6, and 53F10. In some embodiments, an epitope bin comprises at least antibodies 5A7 and 48A6. In some embodiments, an epitope bin comprises at least antibodies 12A12 and 16C5. In some embodiments, an epitope bin comprises at least antibodies 45G10 and 53F10. In some embodiments, an epitope bin comprises at least antibodies 12A12 and 16C5.
  • 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. These methods include (i) mutagenesis, including but not limited to, shotgun mutagenesis, site-directed mutagenesis, and alanine scanning; (ii) domain or fragment scanning; (iii) peptide scanning (e.g., Pepscan technology); (iv) display methods, including but not limited to, phage display, microbial display, and ribosome/mRNA display; (v) methods involving proteolysis and mass spectroscopy; (vi) methods involving amide hydrogen/deuterium exchange; and (vii) structural determination, including but not limited to, x-ray crystallography and NMR.
  • purified anti-ILT3 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 cells function include, but are not limited to, cell activation assays (e.g., cell proliferation assays), cytotoxic T-cell (CTL) assays, mixed lymphocyte reaction (MLR) assays, cytokine/chemokine production assays, FcR binding assays, and cell migration assays.
  • cell activation assays e.g., cell proliferation assays
  • CTL cytotoxic T-cell
  • MLR mixed lymphocyte reaction
  • cytokine/chemokine production assays cytokine/chemokine production assays
  • FcR binding assays FcR binding assays
  • cell migration assays include, but are not limited to, cell activation assays (e.g., cell proliferation assays), cytotoxic T-cell (CTL) assays, mixed lymphocyte reaction (MLR) assays, cytokine/chemokine production assays, FcR binding assays, and cell migration
  • ILT3 generally acts a negative regulator/inhibitory molecule, in some embodiments, inhibiting, reducing, blocking, antagonizing, suppressing, and/or interfering with ILT3 activity results in a release of ILT3 -induced suppression of a biological function (e.g., an activation signal).
  • ILT3 is expressed on myeloid cells, such as monocytes, macrophages, dendritic cells (DCs), and myeloid APCs.
  • ILT3 is highly expressed on suppressive myeloid cells such as tolerogenic dendritic cells (tolDCs) and myeloid-derived suppressor cells (MDSCs).
  • ILT3 activity or ILT3 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 ILT3 activity results in a release of ILT3 -induced suppression of an activation signal.
  • an anti-ILT3 antibody inhibits ILT3 signaling.
  • an anti-ILT3 antibody inhibits ILT3 signaling thereby reversing an ILT3 -induced suppressive effect.
  • an anti-ILT3 antibody inhibits an ILT3 -induced extinction signal.
  • an anti-ILT3 antibody disrupts the ILT3 signaling pathway. In some embodiments, an anti-ILT3 antibody disrupts the ILT3 signaling pathway and activates myeloid cells. In some embodiments, an anti-ILT3 antibody disrupts the ILT3 signaling pathway and activates myeloid APCs. In some embodiments, an anti-ILT3 antibody disrupts the ILT3 signaling pathway and activates dendritic cells. In some embodiments, an anti-ILT3 antibody disrupts the ILT3 signaling pathway and activates or reactivates tolDCs.
  • an anti-ILT3 antibody disrupts the ILT3 signaling pathway and restores the ability of tolDCs to respond to stimuli (e.g., LPS). In some embodiments, an anti-ILT3 antibody disrupts the ILT3 signaling pathway and activates primary dendritic cells.
  • the terms “inhibiting”, “reducing”, “blocking”, “antagonizing”, “suppressing”, and “interfering” are relative to levels and/or activity in the absence of treatment with the ILT3 -binding agent. In some embodiments, the terms “inhibiting”, “reducing”, “blocking”, “antagonizing”, “suppressing”, and “interfering” are relative to levels and/or activity prior to treatment with the ILT3 -binding agent.
  • an anti-ILT3 antibody that inhibits ILT3 activity is antibody 3 A3. In some embodiments, an anti-ILT3 antibody that inhibits ILT3 activity is antibody 5A7. In some embodiments, an anti-ILT3 antibody that inhibits ILT3 activity is antibody Hz5A7.v5. In some embodiments, an anti-ILT3 antibody that inhibits ILT3 activity is antibody 12A12. In some embodiments, an anti-ILT3 antibody that inhibits ILT3 activity is antibody 16C5. In some embodiments, an anti-ILT3 antibody that inhibits ILT3 activity is antibody 45G10. In some embodiments, an anti-ILT3 antibody that inhibits ILT3 activity is antibody 48A6.
  • an anti-ILT3 antibody that inhibits ILT3 activity is antibody 53F10. In some embodiments, an anti-ILT3 antibody that inhibits ILT3 activity is antibody Hz45G10. In some embodiments, an anti-ILT3 antibody that inhibits ILT3 activity is antibody Hz48A6.
  • the present disclosure also provides conjugates comprising an anti-ILT3 antibody described herein.
  • the antibody is attached to a second molecule.
  • the antibody is conjugated to a cytotoxic agent or moiety.
  • 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.
  • 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-ILT3 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 HC1), 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 l,5-difluoro-2,4- dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyridyid
  • an anti-ILT3 antibody described herein is conjugated to a detectable substance or molecule that allows the antibody to be used for diagnosis and/or detection.
  • a labeled anti-ILT3 antibody is used to monitor immune cells in a tumor or in the microenvironment of a tumor.
  • a labeled anti-ILT3 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-ILT3 antibody described herein is used in an immunoassay.
  • Immunoassays are 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-ILT3 antibody described herein is used on a tissue sample or a tumor sample.
  • An anti-ILT3 antibody described herein can also be conjugated to a second antibody to form an antibody heteroconjugate.
  • An anti-ILT3 antibody as described herein may be attached to a solid support.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • immobilized anti- ILT3 antibodies are used in immunoassays.
  • immobilized anti-ILT3 antibodies are used in purification of the target antigen.
  • the ILT3-binding agents (e.g., antibodies) of the disclosure 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 ILT3-binding agent is useful for activating, promoting, increasing, and/or enhancing an immune response to cancer or cancer cells.
  • an ILT3-binding agent 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.
  • a method comprises administering an ILT3-binding agent (e.g., an antibody) described herein in combination with at least one additional therapeutic agent or therapeutic therapy.
  • an ILT3-binding agent e.g., an antibody
  • Treatment with two or more therapeutic agents often uses agents that work by different mechanisms of action, although this is not required.
  • Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects.
  • Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the agent(s).
  • Combination therapy may decrease the likelihood that resistance to an agent will develop.
  • the combination of an ILT3 -binding agent (e.g., an antibody) described herein and at least one additional therapeutic agent results in additive or synergistic results.
  • the combination therapy results in an increase in the therapeutic index of the ILT3 -binding agent.
  • the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s).
  • the combination therapy results in a decrease in the toxicity and/or side effects of the ILT3-binding agent.
  • the combination therapy results in a decrease in the toxicity and/or side effects of the additional therapeutic agent(s).
  • combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells.
  • a combination treatment comprises one additional therapeutic agent or two or more additional therapeutic agents.
  • Useful classes of therapeutic agents include, but are not limited to, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, anti-metabolites, chemotherapy sensitizers, duocarmycins, etoposides, fhiorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.
  • alkylating agents e.g., platinum complexes such as c
  • Therapeutic agents that may be administered in combination with the ILT3-binding agents described herein include chemotherapeutic agents.
  • the method or treatment involves the administration of an ILT3-binding agent of the present disclosure in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents.
  • Chemotherapeutic agents useful in the present disclosure include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil
  • paclitaxel TAXOL
  • docetaxel TAXOTERE
  • chlorambucil gemcitabine
  • 6- thioguanine mercaptopurine
  • platinum analogs such as cisplatin and carboplatin
  • vinblastine platinum
  • etoposide VP- 16
  • ifosfamide mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT 11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA); and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluoromethylomithine
  • XELODA retinoic acid
  • esperamicins capecitabine
  • Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); and anti-androgen
  • the chemotherapeutic agent is a topoisomerase inhibitor.
  • Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II).
  • Topoisomerase inhibitors include, but are not limited to, doxorubicin HC1, daunorubicin citrate, mitoxantrone HC1, actinomycin D, etoposide, topotecan HC1, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.
  • the additional therapeutic agent is irinotecan.
  • the chemotherapeutic agent is an anti-metabolite.
  • An antimetabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of cells, such as cell division.
  • Anti-metabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.
  • the additional therapeutic agent is gemcitabine.
  • the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin.
  • the agent is a taxane.
  • the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel.
  • the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel; ABRAXANE), DHA-paclitaxel, or PG-paclitaxel.
  • the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof.
  • the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plkl.
  • the additional therapeutic agent is paclitaxel.
  • the additional therapeutic agent is nab-paclitaxel.
  • an additional therapeutic agent comprises an agent such as a small molecule.
  • treatment can involve the combined administration of an ILT3-binding agent of the present disclosure with a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF.
  • tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF.
  • an ILT3-binding agent of the present disclosure is administered in combination with a protein kinase inhibitor selected from the group consisting of: gefitinib (IRES SA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B).
  • a protein kinase inhibitor selected from the group consisting of: gefitinib (IRES SA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B).
  • a protein kinase inhibitor selected from the group consisting of:
  • an additional therapeutic agent comprises a biological molecule, such as an antibody.
  • treatment can involve the combined administration of an ILT3-binding agent of the present disclosure with antibodies against tumor-associated antigens including, but not limited to, antibodies that bind EGFR, HER2/ErbB2, and/or VEGF.
  • the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody).
  • the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).
  • the additional therapeutic agent is an antibody that modulates the immune response.
  • the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, or an anti-TIGIT antibody.
  • treatment with an ILT3 -binding agent described herein can include combination treatment with other biologic molecules, such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or can be accompanied by surgical removal of tumors, removal of cancer cells, or any other therapy deemed necessary by a treating physician.
  • the additional therapeutic agent is an immunotherapeutic agent.
  • an ILT3-binding agent is combined with a growth factor selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-a, TGF-P, TNF-a, VEGF, PIGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.
  • a growth factor selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropo
  • the additional therapeutic agent is an immunotherapeutic agent.
  • the immunotherapeutic agent is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL- 3), interleukin 12 (IL-12), interleukin 1 (IL-1), interleukin 2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4- IBB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-Ll antibody, anti -LAG-3 antibody, and anti-TIM-3 antibody.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • IL- 3 interleukin 12
  • IL-1 interleukin 1
  • IL-2
  • an immunotherapeutic agent is selected from the group consisting of: a modulator of PD-1 activity, a modulator of PD-L1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4- IBB activity, an modulator of 0X40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDO 1 activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor
  • an immunotherapeutic agent is selected from the group consisting of: a PD-1 antagonist, a PD-L1 antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD 80 antagonist, a CD 86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, and/or an IDO1 antagonist.
  • the PD-1 antagonist is an antibody that specifically binds PD-1.
  • the antibody that binds PD-1 is pembrolizumab (KEYTRUDA, MK-3475), pidilizumab (CT-011), nivolumab (OPDIVO, BMS- 936558, MDX-1106), MEDI0680 (AMP-514), REGN2810, BGB-A317, PDR-001, or STI- Al l 10.
  • the antibody that binds PD-1 is described in PCT Publication WO 2014/179664, for example, an antibody identified as APE2058, APE1922, APE1923, APE1924, APE 1950, or APE1963, or an antibody containing the CDR regions of any of these antibodies.
  • the PD-1 antagonist is a fusion protein that includes PD-L2, for example, AMP -224.
  • the PD-1 antagonist is a peptide inhibitor, for example, AU P-12.
  • the PD-L1 antagonist is an antibody that specifically binds PD- Ll.
  • the antibody that binds PD-L1 is atezolizumab (TECENTRIQ, RG7446, MPDL3280A), MEDI4736, BMS-936559 (MDX-1105), avelumab (BAVENCIO, MSB0010718C), durvalumab (IMFINZI), KD033, the antibody portion of KD033, or STI- A1014.
  • the antibody that binds PD-L1 is described in PCT Publication WO 2014/055897, for example, Ab-14, Ab-16, Ab-30, Ab-31, Ab-42, Ab-50, Ab-52, or Ab-55, or an antibody that contains the CDR regions of any of these antibodies.
  • the CTLA-4 antagonist is an antibody that specifically binds CTLA-4.
  • the antibody that binds CTLA-4 is ipilimumab (YERVOY) or tremelimumab (CP-675,206).
  • the CTLA-4 antagonist a CTLA-4 fusion protein, for example, KAHR-102.
  • the LAG3 antagonist is an antibody that specifically binds LAG3.
  • the antibody that binds LAG3 is IMP701, IMP731, BMS-986016, LAG525, and GSK2831781.
  • the LAG3 antagonist includes a soluble LAG3 receptor, for example, IMP321.
  • the KIR antagonist is an antibody that specifically binds KIR.
  • the antibody that binds KIR is lirilumab.
  • an immunotherapeutic agent is selected from the group consisting of: a CD28 agonist, a 4-1BB agonist, an 0X40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, and a GITR agonist.
  • the 0X40 agonist includes 0X40 ligand, or an OX40-binding portion thereof.
  • the 0X40 agonist may be MEDI6383.
  • the 0X40 agonist is an antibody that specifically binds 0X40.
  • the antibody that binds 0X40 is MEDI6469, MEDI0562, or MOXR0916 (RG7888).
  • the 0X40 agonist is a vector (e.g., an expression vector or virus, such as an adenovirus) capable of expressing 0X40 ligand.
  • the OX40-expressing vector is Delta -24 - RGDOX or DNX2401.
  • the 4-1BB (CD137) agonist is a binding molecule, such as an anticalin.
  • the anticalin is PRS-343.
  • the 4-1BB agonist is an antibody that specifically binds 4- IBB.
  • antibody that binds 4- IBB is PF-2566 (PF-05082566) or urelumab (BMS-663513).
  • the CD27 agonist is an antibody that specifically binds CD27.
  • the antibody that binds CD27 is varlilumab (CDX-1127).
  • the GITR agonist comprises a GITR ligand or a GITR-binding portion thereof.
  • the GITR agonist is an antibody that specifically binds GITR.
  • the antibody that binds GITR is TRX518, MK-4166, or INBRX- 110.
  • immunotherapeutic agents include, but are not limited to, cytokines such as chemokines, interferons, interleukins, lymphokines, and members of the tumor necrosis factor (TNF) family.
  • immunotherapeutic agents include immunostimulatory oligonucleotides, such as CpG dinucleotides.
  • an immunotherapeutic agent includes, but is not limited to, anti- PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti- CD28 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti -4- IBB antibodies, anti- 0X40 antibodies, anti-KIR antibodies, anti-Tim-3 antibodies, anti-LAG3 antibodies, anti-CD27 antibodies, anti-CD40 antibodies, anti-GITR antibodies, anti-TIGIT antibodies, anti-CD20 antibodies, anti-CD96 antibodies, or anti -IDO 1 antibodies.
  • treatment with an ILT3 -binding agent can occur prior to, concurrently with, or subsequent to administration of the additional therapeutic agents.
  • combined administration includes co-admini strati on, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities.
  • preparation of agents and/or dosing schedules for additional therapeutic agents are according to manufacturers' instructions or as determined empirically by the skilled practitioner.
  • an ILT3 -binding agent e.g., an antibody
  • a subject e.g., a human
  • an ILT3 -binding agent e.g., an antibody
  • at least one additional therapeutic agent may be administered in any order or concurrently.
  • an ILT3-binding agent is administered to subjects that have previously undergone treatment with a therapeutic agent.
  • an ILT3- binding agent and a second therapeutic agent are administered substantially simultaneously or concurrently.
  • a subject may be given an ILT3-binding agent while undergoing a course of treatment with a second therapeutic agent (e.g., a chemotherapeutic agent).
  • a second therapeutic agent e.g., a chemotherapeutic agent
  • an ILT3-binding agent is administered within 1 year of the treatment with a second therapeutic agent.
  • an ILT3-binding agent is administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In some embodiments, an ILT3 -binding agent is administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, an ILT3-binding agent is administered within 5, 4, 3, 2, or 1 days of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments can be administered to the subject within a matter of hours or minutes (i.e., substantially simultaneously).
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a binding agent provided herein, wherein the cancer comprises a biliary duct cancer, cholangiocarcinoma, or mesothelioma.
  • a method of inhibiting tumor growth in a subject comprising administering to the subject a therapeutically effective amount of a binding agent provided herein, wherein the tumor comprises a microsatellite instability - high (MSI-H) tumor, a deficient mismatch repair (dMMR) positive tumor or tumor mutation burden - high (TMB-H).
  • MSI-H microsatellite instability - high
  • dMMR deficient mismatch repair
  • TMB-H tumor mutation burden - high
  • a method of inhibiting tumor relapse or tumor regrowth in a subject comprising administering to the subject a therapeutically effective amount of a binding agent provided herein, wherein the tumor comprises a microsatellite instability - high (MSI-H) tumor, a deficient mismatch repair (dMMR) positive tumor or tumor mutation burden - high (TMB-H).
  • MSI-H microsatellite instability - high
  • dMMR deficient mismatch repair
  • TMB-H tumor mutation burden - high
  • a binding agent provided herein for treatment of biliary duct cancer, cholangiocarcinoma, or mesothelioma is provided herein for treatment of biliary duct cancer, cholangiocarcinoma, or mesothelioma.
  • a binding agent provided herein in the manufacture of a medicament for treatment of biliary duct cancer.
  • a binding agent provided herein for treatment of a microsatellite instability - high (MSI-H) tumor, a deficient mismatch repair (dMMR) positive tumor or tumor mutation burden - high (TMB-H).
  • MSI-H microsatellite instability - high
  • dMMR deficient mismatch repair
  • TMB-H tumor mutation burden - high
  • a binding agent provided herein in the manufacture of a microsatellite instability - high (MSI-H) tumor, a deficient mismatch repair (dMMR) positive tumor or tumor mutation burden - high (TMB-H).
  • the binding agent is administered once or sequentially.
  • the binding agent is administered at a dose of 20 mg, 60 mg, 200 mg, 600 mg, 1500 mg, or 2100 mg. [00290] In some embodiments, the binding agent is administered once every 3 weeks.
  • the binding agent is administered over 30 minutes, 60 minutes or 90 minutes.
  • the binding agent is administered over 30 minutes at a dose of ⁇ 600 mg.
  • the binding agent is administered over 60 minutes at a dose of > 600 mg.
  • the binding agent is administered over 90 minutes at a dose of > 1800 mg.
  • the dose comprises a starting dose.
  • the dose is modified relative to the starting dose.
  • the dose is modified to be about two thirds 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.
  • the binding agent is administered as part of a combination therapy.
  • the binding agent is administered in combination with a PD-1 inhibitor.
  • the PD-1 inhibitor comprises a PD-1 antibody.
  • the PD-1 antibody comprises pembrolizumab.
  • pembrolizumab is administered once or sequentially.
  • pembrolizumab is administered at a dose of 200 mg.
  • pembrolizumab lasts for up to 2 years.
  • pembrolizumab is administered before the binding agent is administered.
  • pembrolizumab is administered 30 minutes, 60 minutes or 90 minutes before the binding agent is administered.
  • pembrolizumab is administered after the binding agent is administered.
  • pembrolizumab is administered 30 minutes, 60 minutes or 90 minutes after the binding agent is administered.
  • the combination therapy comprises administration of the binding agent, followed by pembrolizumab, and followed again by the binding agent.
  • pembrolizumab is administered intravenously, subcutaneously, or intra-peritoneally.
  • the binding agent provided herein or a combination treatment comprising same is administered as described in the Examples section below.
  • tumor types include pancreatic, breast cancer, mesothelioma, gastric, NSCLC, cervical, endocervical, biliary duct (cholangiocarcinoma), SCCHN, bladder urothelial, CRC, esophageal, ovarian, RCC, prostate, and melanoma (skin cutaneous).
  • Phase 1 of this study is a first in human (FIH) dose finding study which includes monotherapy dose escalation in Part la and monotherapy expansion cohort in Part 2a.
  • Phase lb of this study is a combination dose finding with pembrolizumab in Part lb.
  • Intra-patient dose escalation may be allowed during Part la and Part lb if specified conditions are met.
  • patients treated with monotherapy who experience disease progression in Part la or Part 2a may be allowed to cross over to a combination treatment regimen with pembrolizumab, if the safety of the combination regimen has been confirmed in Part lb (see Figure 1).
  • [00321] Patients having an ECOG PS of ⁇ 1 are included.
  • Patients having adequate bone marrow function are included.
  • Adequate bone marrow function includes: ANC > 1500/pL; Platelet count > 100 X 10 3 /pL; Hemoglobin > 9.0 g/dL. Limited transfusion to reach this value is allowed. There should not be a chronic need for transfusion in the recent past (approximately 3 months).
  • liver function includes: total bilirubin ⁇ 1.5 X upper limit of normal (ULN) (except patients with Gilbert syndrome who must have total bilirubin ⁇ 3.0 mg/dL); AST ⁇ 2.5 X ULN (if patient has known liver metastases, then ⁇ 5 X ULN); ALT ⁇ 2.5 X ULN (if patient has known liver metastases, then ⁇ 5 X ULN) [00324] Patients having adequate renal function are included. Adequate renal function, includes serum creatinine ⁇ 1.5 X ULN or estimated CrCl > 30 mL/min as calculated using the method standard for the institution. In equivocal cases, a 24-hour urine collection test can be used to estimate the CrCl more accurately.
  • An archival tumor biopsy must be provided and should be taken within 1 year of enrollment. If not available, a fresh tumor biopsy is acceptable.
  • Additional inclusion criteria for Part la includes patients that have evaluable disease (either measurable or non-measurable) per RECIST vl .1 criteria. Also, the following tumor types are allowed: pancreatic, breast, mesothelioma, gastric, NSCLC, cervical and endocervical, biliary duct, SCCHN, bladder urothelial, CRC, esophageal, ovarian, RCC, prostate, and melanoma (skin cutaneous).
  • Additional inclusion criteria for Part lb includes patients that have at least 1 measurable lesion by CT with contrast or MRI (for patients with contrast allergy) per RECIST vl.l criteria. Also, the following tumor types are allowed: PDAC, ovarian, NSCLC, SCCHN, RCC, gastric, melanoma, and any tumor type with MSI-H, dMMR or TMB-H [>10 mutations/megabase (mut/Mb)]. Enrollment of patients with PDAC and ovarian cancer will be capped up to approximately 5 patients in total.
  • tumor types should have received anti-PDl/PDLl directed therapy and progressed: gastric, NSCLC, SCCHN, bladder urothelial, esophageal, and tumor types with MSI-H, dMMR or TMB-H.
  • Additional inclusion criteria for Part 2a includes patients that must have at least 1 measurable lesion by CT with contrast or MRI per RECIST vl.l criteria. Also, the following tumor types are allowed: pancreatic, breast, mesothelioma, gastric, NSCLC, cervical and endocervical, biliary duct (cholangiocarcinoma), SCCHN, bladder urothelial, CRC, esophageal, ovarian, RCC, prostate, and melanoma (skin cutaneous).
  • tumor types are allowed: pancreatic, breast, mesothelioma, gastric, NSCLC, cervical and endocervical, biliary duct (cholangiocarcinoma), SCCHN, bladder urothelial, CRC, esophageal, ovarian, RCC, prostate, and melanoma (skin cutaneous).
  • Patients with active brain or leptomeningeal metastasis are excluded. Patients with known brain metastases are eligible if they have been treated and an MRI shows no evidence of progression for at least 8 weeks after treatment is completed and within 3 weeks prior to first dose of study drug. Patients are not eligible if they require high dose of systemic corticosteroids that could result in immunosuppression (>10 mg/day prednisone equivalents) for at least 2 weeks prior to study drug administration.
  • [00334] Patients receiving treatment with anti-neoplastic therapy including but not limited to cytotoxic chemotherapy, major surgery, radiation, biologic agents, and investigational agents) within 28 days or > 5 X t‘/z, whichever is earlier, before the first dose of study drug, are excluded.
  • Major surgery is defined as any surgical procedure that involves general anesthesia and a significant incision (ie, larger than what is required for the placement of a central venous access, percutaneous feeding tube, or biopsy) within 28 days of first dose of HZ5A7.V5, or anticipated surgery during the study.
  • HIV seropositive patients who are healthy and low-risk for AIDS-related outcomes could be considered eligible. Eligibility criteria for HIV-positive patients should be evaluated and will be based on current and past CD4 and T-cell counts, history (if any) of AIDS-defining conditions (eg, opportunistic infections), and status of HIV treatment.
  • HIV infected patients must be on anti-retroviral therapy (ART) and have well-controlled HIV infection/disease, defined as follows, to be eligible for enrollment: patients on ART must have a CD4+ T-cell count >350 cells/mm3 at the time of screening; patients on ART must have achieved and maintained virologic suppression defined as confirmed HIV RNA level below 50 copies/mL or the lower limit of qualification (below the limit of detection) using the locally available assay at the time of screening and for at least 12 weeks prior to screening; patients on ART must have been on a stable regimen, without changes in drugs or dose modification, for at least 4 weeks prior to study entry (Day 1).
  • ART anti-retroviral therapy
  • Patients who are HBsAg positive are eligible if they have received HBV antiviral therapy for at least 4 weeks and have undetectable HBV viral load prior to randomization. Patients should remain on anti-viral therapy throughout study intervention and follow local guidelines for HBV anti-viral therapy post completion of study intervention.
  • HIV-infected patients with a history of Kaposi sarcoma and/or multicentric Castleman disease are excluded.
  • the baseline uncorrected QT interval is >470 msec, this interval should be rate- corrected using QTcF and the resulting QTcF should be used for decision making and reporting.
  • Patients having any of the following in the previous 6 months are excluded: cerebrovascular accident, transient ischemic attack, symptomatic pulmonary embolism, and/or other clinically significant episodes of thrombo-embolic disease. Patients with venous thromboembolic events that are non- life threatening and stable on low molecular weight heparin or factor Xa inhibitors that can be reversed by andexanet-a are allowed.
  • LVEF left ventricular ejection fraction
  • ECHO screening echocardiogram
  • MUGA multiple gated acquisition
  • Additional exclusion criteria for Part lb includes patients with active, known, or suspected autoimmune disease. Patients with vitiligo, Type I diabetes mellitus, residual hypothyroidism due to autoimmune condition only requiring hormone replacement, psoriasis not requiring systemic treatment, or conditions not expected to recur in the absence of an external trigger are permitted to enroll. Also, patients with diagnosis of prior immunodeficiency or organ transplant requiring immunosuppressive therapy are excluded.
  • Part la will investigate doses of HZ5A7.V5 as monotherapy in sequentially escalating dose levels following intravenous (IV) administration to select the recommended Phase 2 dose (RP2D).
  • the FIH starting dose in Part la will be 20 mg HZ5A7.V5. See Figure 1 and Table 9. At least 6 to 12 patients are to be enrolled at the maximum administered dose/ maximum tolerated dose (MAD/MTD) level. At the time the MTD/MAD is determined, only those patients who consent to a mandatory paired tumor biopsy may be enrolled at dose levels no greater than the MTD/MAD.
  • MAD/MTD maximum administered dose/ maximum tolerated dose
  • V5 will be administered IV every three weeks (Q3W) for the duration of each 21 -day cycle for all cohorts. Part la dose escalation is expected to enroll approximately 35 patients.
  • the monotherapy dose escalation in Part la utilizes a 2-stage design, in which Stage 1 dose escalation is rule-based and guided by an accelerated titration design (ATD) with predefined moderate toxicity (PMT) criteria and Stage 2 is started after either observation of a second patient with a PMT event or the first dose-limiting toxicity (DLT) incidence then guided by the Bayesian logistic regression model (BLRM). PMTs and DLTs will be assessed during the first cycle of treatment (21 days after the first dose). Cohort size will be 1 patient during the accelerated titration stage and 2 to 4 patients during the BLRM stage, with at least 2 DLT- evaluable patients per cohort. Late-onset toxicities will be taken into consideration of what is determined to be the MTD/MAD.
  • ATD accelerated titration design
  • PMT moderate toxicity
  • DLT first dose-limiting toxicity
  • Cohort size will be 1 patient during the accelerated titration stage and 2 to 4 patients during the BLRM stage, with at
  • the length of treatment is estimated to be 6 months, depending on each patient’s disease status and clinical responses to the study treatment.
  • Part lb will enroll patients to further evaluate the safety of HZ5A7.V5 in combination with a fixed dose of pembrolizumab to select the combination RP2D.
  • the combination starting dose in Part lb will be modeled based on the BLRM operating characteristics determined in the ongoing monotherapy dose escalation (Part la) prior to determining the MTD/MAD, the details of which are described immediately below.
  • HZ5A7.V5 Treatment with HZ5A7.V5 in combination with pembrolizumab will continue until completion of 35 administrations of pembrolizumab (approximately 2 years). After 35 administrations of pembrolizumab, HZ5A7.V5 will continue to be administered as a monotherapy.
  • the combination starting dose will be determined based on modeling primarily with DLT information from Part la, with consideration of evolving PK, pharmacodynamic, and other safety profiles including immune activation status assessed in the Part la dose escalation.
  • the modeled starting dose that satisfies the escalation with over-dose control (EWOC) would be used as the starting dose of HZ5A7.V5 to be combined with pembrolizumab (Babb et al., Statistic in Medicine. ( 17)( 10) : 1103-20 (1998)).
  • EWOC over-dose control
  • the starting dose in Part la is HZ5A7.V5 20 mg Q3W.
  • the prior risks of overdosing range from 4.9% to 13.5%, which satisfy the EWOC criterion.
  • the combination starting dose will be determined based on modeling primarily with DLT information from HZ5A7.V5 monotherapy dose escalation, with consideration of evolving PK, pharmacodynamic, and other safety profiles including immune activation status assessed in the Part la dose escalation.
  • prior specification of combination may be modeled using meta-analytic predictive approach and prior distribution of the interaction parameter, based on prior DLT distribution of individual compound, HZ5A7.V5 and pembrolizumab (Neuenschwander et al., CRC Press. 96-132 (2015); Patnaik et al., Clin Cancer Res. 21(19):4286-93 (2015)).
  • the modeled starting dose that satisfies the EWOC would be used as the starting dose (Babb et al., Statistic in Medicine. ( 17)( 10) : 1103-20 (1998)).
  • the next dose level after the stating dose level will be guided by the BLRM.
  • Part lb Detailed BLRM operating characteristics for Part lb would be provided before initiation of Part lb upon request.
  • Part lb starts before the MTD/MAD of the monotherapy is determined, should emerging safety data from ongoing monotherapy at the next dose level suggest the next dose level exceeds MTD, a starting dose lower than the dose modeled above would be considered as an alternative starting dose after evaluating the safety data, including the nature of the DLT, to reduce any potential synergistic safety concerns.
  • DLT information from a minimum of 1 to 2 cohorts of HZ5A7.V5 monotherapy will be required to incorporate into the BLRM modeling before initiation of Part lb.
  • the starting dose of HZ5A7.V5 that will be administered in combination with pembrolizumab will be determined based on the modeling primarily with DLT information from HZ5A7.V5 monotherapy and pembrolizumab, together with evolving PK, pharmacodynamic, and other safety profiles including immune activation status assessed in the Part la dose escalation.
  • Patients will be enrolled in Part 2a if there is no standard therapy available to them for curative purposes, if they have refused standard therapy, or if standard therapy would not be indicated in the opinion of the treating physician.
  • Patients with pancreatic, breast cancer, mesothelioma, gastric, NSCLC, cervical, endocervical, biliary duct (cholangiocarcinoma), SCCHN, bladder urothelial, CRC, esophageal, ovarian, RCC, prostate and melanoma (skin cutaneous) will be treated with HZ5A7.V5 monotherapy at the MTD/MAD that is determined during Part la.
  • Part 2a of the study may commence after determination of the RP2D dose of HZ5A7.V5 monotherapy from Part la. Depending on data from Part la and Part lb, the Part 2a monotherapy dose expansion may or may not commence.
  • the objective of the biomarker assessment is to provide insight into the predictive and pharmacodynamic effects of HZ5A7.V5 as a monotherapy and in combination with pembrolizumab.
  • Archival samples and paired-on treatment biopsies will be collected for RNAseq and IHC analysis, which will be used to analyze gene and protein expression associated with inflammatory myeloid and T-cell phenotypes. These data will be further analyzed with clinical response to identify predictive markers.
  • peripheral blood samples will be collected and analyzed using FACS to monitor changes in dendritic cell activation, macrophage reprogramming, T-cell activation, and modulation in other peripheral blood subsets. Furthermore, peripheral blood will be evaluated for changes in myeloid-associated proteins that have been shown to be modulated by HZ5A7.V5 in preclinical in vitro studies.
  • ALT alanine aminotransferase
  • aPTT activated partial thromboplastin time
  • AST aspartate aminotransferase
  • 0- hCG beta-human chorionic gonadotropin
  • BUN blood urea nitrogen
  • FT3 free triiodothyronine
  • FT4 free thyroxine
  • HBV hepatitis B vims
  • HCV hepatitis C virus
  • HIV human immunodeficiency virus
  • hsCRP high- sensitivity C-reactive protein
  • IFN interferon
  • IL interleukin
  • INR intemational normalized ratio
  • LDH lactate dehydrogenase
  • PT prothrombin time
  • TNF tumor necrosis factor
  • TSH thyroid stimulating hormone
  • WBC white blood cell.
  • laboratory tests should include albumin, creatine kinase, total bilirubin, direct and indirect bilirubin, gamma-glutamyl transferase, PT/intemational normalized ratio, alkaline phosphatase, total bile acids, and acetaminophen drug and/or protein adduct levels.
  • the FIH starting dose of HZ5A7.V5 20 mg was determined based on consideration of the totality of the nonclinical in vivo and in vitro pharmacology and safety data described below. Particular weight is given to the in vivo pharmacological effect level and safety data, as these data are deemed most relevant for predicting responses in patients.
  • the FIH dose selection strategy seeks to identify a dose that is most likely to be safe for patients, while minimizing the number of patients who would be dosed at subtherapeutic levels. Based on these data, a starting dose of 20 mg IV Q3W is deemed to be reasonable for HZ5A7. V5, an antagonist antibody for the treatment of advanced or metastatic solid tumors.
  • HZ5A7.V5 As part of the 4-week good laboratory practice (GLP)-toxicity study, pharmacodynamic biomarkers of HZ5A7.V5 action were identified through gene expression analysis of the spleen, a major reservoir of ILT3 -expressing immune cells. This analysis found that ILT3 and other immune-related genes were significantly upregulated (as compared with levels in vehicle-treated animals) in response to treatment with 10 or 100 mg/kg of HZ5A7.V5, but not in response to treatment with 1 mg/kg HZ5A7.V5.
  • LPS lipopolysaccharide
  • HZ5A7.V5 Based on the data from these cynomolgus monkey in vivo pharmacology studies, 10 and 100 mg/kg of HZ5A7.V5 were identified as pharmacologically effective doses, while 1 mg/kgof HZ5A7.V5 was below the pharmacological effect level.
  • the human-equivalent dose of the pharmacologically inactive dose of 1 mg/kg in cynomolgus monkey was determined to be 20 mg HZ5A7.V5.
  • Evaluation of the interaction between ILT3 and its ligand fibronectin provides the most direct in vitro functional assay to evaluate the activity of HZ5A7.V5.
  • HZ5A7.V5 was well tolerated in 2 repeat-dose toxicity studies in cynomolgus monkeys at dose levels up to 100 mg/kg, with no treatment-related clinical or histopathological observations.
  • the no-observed-adverse-effect level (NOAEL) was determined to be 100 mg/kg/week for 4 weekly doses.
  • the starting dose of 20 mg is well below the NOAEL observed in cynomolgus monkeys, and the GLP toxicity study predicts a safety margin of ⁇ 300-fold in Cmax and AUC for the FIH dose (Table 11).
  • HZ5A7.V5 The ability of HZ5A7.V5 to induce pro-inflammatory cytokines indicative of CRS was tested in an in vitro cytokine release assay using whole blood from healthy donors. The impact of HZ5A7.V5 on secretion of the cytokines IL-2, IL-6, IFNy, and TNFa was tested in both plate- coated and soluble formats. HZ5A7.V5 did not induce cytokine secretion above the level induced by the isotype control in any of the 10 donors tested.
  • a PK parameters in cynomolgus monkeys (C max and AUC 0-2 id) determined following Day 22 dosing of the ADA-negative recovery group animals in the 4-week GLP toxicity study at NOAEL of 100 mg/kg. Rationale for Dosing Regimen
  • Part la dose escalation The purpose of the Part la dose escalation is to identify the MTD of single agent HZ5A7.V5, which is defined as the highest dose with a true DLT rate in the targeted toxicity interval of[0.16, 0.33],
  • the dose escalation will be initiated by ATD with PMT criteria, which will be converted to and guided by BLRM and the EWOC principle.
  • a 2-parameter BLRM will be used to model the relationship between DLT and the doses of HZ5A7.V5 monotherapy in the dose escalation. Toxicities after completion of the first cycle will be monitored as late-onset toxicities.
  • Part la is initiated by ATD with the PMT.
  • PMT-evaluable patients if there is 1 patient with PMT or DLT at a given dose level, thedose level will enroll another 2 to 4 patients and dose escalation will switch from ATD to BLRM, which enables incorporation of available information about observed DLTs seen in the prior cohorts (retrospectively).
  • the ATD may be converted to the BLRM sooner than the observation of 2 patients with PMT or 1 patient with a DLT.
  • Patients are classified as PMT evaluable if they experience a PMT or if they otherwise, in the absence of a PMT, receive at least 75% intended dose for single doses of the study drug during Cycle 1 in the dose escalation part of the study and have received all scheduled safety assessments during the PMT observation period during Part la. Patients who fail to meet these criteria may be replaced.
  • the PMT observation period will include events occurring in the first cycle of treatment (within 21 days after the start of the study treatment) for each patient.
  • All AEs will be collected from all patients enrolled in the study. The severity of AE will begraded using the NCI CTCAE v5.0.
  • the occurrence of any of the following toxicities during the PMT observation period will beconsidered a PMT: Grade > 3 neutropenia that persists > 3 days; Grade 3 thrombocytopenia that persists >3 days; or Grade 3 anemia.
  • Non-hematologic Toxicities include: Grade > 2 toxicities that are considered clinically significant except those that have not been maximally treated (e.g., nausea, vomiting, diarrhea), can be easilytreated (e.g., electrolyte abnormalities), or those events which are clearly and incontrovertibly due to underlying disease/disease progression or extraneous cause; LFT abnormality (alanine aminotransferase [ALT]/AST >3.0 to 5.0*ULN ifbaseline was normal; >3.0 to 5.0*ULN ifbaseline was abnormal) (For patients with Grade 2 hepatic transaminase at baseline as a result of livermetastasis, a hepatic transaminase level >7*ULN will be considered as a PMT); Grade 3 CRS; or Grade 2 CRS, except if it has improved to baseline within 48 hours.
  • Grade > 2 toxicities that are considered clinically significant except those that have not been maximally treated (e.g., nausea, vomiting, diarrhea), can be
  • AEs will not be adjudicated as PMTs: Grade ⁇ 2 IRRs, allergic reactions, or anaphylaxis will not be considered as PMTs but may be a reason for converting to BLRM; or Isolated Grade 2 laboratory abnormalities that are not associated with clinical sequelae and are corrected with suppl ementation/appropri ate management within72 hours of their onset.
  • a 2-parameter BLRM will be used to model the relationship between DLT and the dose of HZ5A7.V5 monotherapy in Part la.
  • posterior probabilities of having a DLT falling into 3 dosing intervals (underdosing, targeted toxicity, overdosing) will be calculated for all dose levels.
  • a dose may only be used for newly enrolled patients if the risk of overdosing (ie, toxicity higher than 0.33 at that dose) is less than 35% [EWOC], The use of the EWOC principle limits the risk that a potential next dose will exceed the MTD/MAD.
  • Provisional dose levels to be evaluated from the starting dose through the highest dose level are listed in Table 12.
  • the adaptive BLRM permits an intermediate dosing schedule (eg, 900 mg, 1800 mg) based on the observed DLTs. Dose levels may not be skipped during the escalation.
  • a Dose level -1 represents a treatment dose to be explored should predefined moderate toxicity or dose-limiting toxicity occur at the starting dose. Intermediate doses may be explored
  • the proposed dose levels, schedules, and PK time points may be reconsidered during the study based on the emerging safety and PK data and/or in the future (e.g., every 6 weeks).
  • the combination starting dose will be determined based on modeling primarily with DLT information from Part la, with consideration of evolving PK, pharmacodynamic, and other safety profiles including immune activation status assessed in the Part la dose escalation.
  • the modeled starting dose that satisfies the EWOC would be used as the starting dose of HZ5A7.V5 to be combined with pembrolizumab (Babb et al., Statistic in Medicine. (17)(10): 1103-20 (1998)).
  • the next dose level after the starting dose level will be guided by the BLRM.
  • Intra-patient dose escalation to the next higher dose level of HZ5A7.V5 may be considered in Parts la and lb for enrolled patients meeting the following conditions.
  • the patient has completed Cycle 3 administration, PK collection of all time points of Cycle 3 and predose of Cycle 4 Day 1, and first on-treatment tumor assessment.
  • the candidate for intra-patient dose-escalation tolerated the current dose level well, with highest toxicity observed being Grade 2 or below except those eventswhich are clearly and incontrovertibly due to underlying disease/disease progression or extraneous cause.
  • Grade 2 or below except those events which are clearly and incontrovertibly due to underlying disease/disease progression or extraneous cause.
  • isolated Grade 3 laboratory abnormalities that are not associated with clinical sequelae may be allowed.
  • Cross over to a combination treatment regimen with pembrolizumab may be considered for patients enrolled in Part la and Part 2a who experience unequivocal disease progression during monotherapy, if they meet the following conditions.
  • the patient meets all inclusion and exclusion criteria, including Part lb specific criteria except for tumor types, prior to initiating combination therapy with pembrolizumab.
  • the patient may receive combination treatment with pembrolizumab as long as the washout period between the combination treatmentwith pembrolizumab is > 21 days after the prior administration of HZ5A7.V5.
  • Patients may be treated at a lower or higher dose level of HZ5A7.V5, when combined with pembrolizumab, than the originally assigned monotherapy dose.
  • PMT evaluable Patients are classified as PMT evaluable if they experience a PMT or if they otherwise, in the absence of a PMT, receive at least 75% intended dose for single doses of the study drug during Cycle 1 in the dose escalation part of the study and have received all scheduled safety assessments during the PMT observation period during Part la. Patients who fail to meet these criteria may be replaced.
  • the PMT observation period will include events occurring in the first cycle of treatment (within 21 days after the start of the study treatment) for each patient.
  • All AEs will be collected from all patients enrolled in the study. The severity of AE will begraded using the NCI CTCAE v5.0.
  • Hematologic toxicities include Grade > 3 neutropenia that persists > 3 days, Grade 3 thrombocytopenia that persists >3 days or Grade 3 anemia. Other changes in complete blood count within hematological inclusion criteria will not be regarded as a PMT, unless the change of AE grade is > 2 from baseline.
  • Non-hematologic toxicities include: Grade > 2 toxicities that are considered clinically significant except those that have not been maximally treated (e.g., nausea, vomiting, diarrhea), can be easilytreated (e.g., electrolyte abnormalities), or those events which are clearly and incontrovertibly due to underlying disease/disease progression or extraneous cause; LFT abnormality (alanine aminotransferase [ALT]/AST >3.0 to 5.0> ⁇ ULN ifbaseline was normal; >3.0 to 5.0xULN ifbaseline was abnormal); Grade 3 CRS; Grade 2 CRS, except if it has improved to baseline within 48 hours.
  • Grade > 2 toxicities that are considered clinically significant except those that have not been maximally treated (e.g., nausea, vomiting, diarrhea), can be easilytreated (e.g., electrolyte abnormalities), or those events which are clearly and incontrovertibly due to underlying disease/disease progression or extraneous cause
  • LFT abnormality alanine aminotransferase
  • a hepatic transaminase level >7xULN will be considered as a PMT.
  • AEs will not be adjudicated as PMTs: Grade ⁇ 2 IRRs, allergic reactions, or anaphylaxis; or isolated Grade 2 laboratory abnormalities that are not associated with clinical sequelae and are corrected with supplementation/appropriate management within72 hours of their onset. While Grade ⁇ 2 IRRs, allergic reactions, or anaphylaxis will not be considered as PMTs occurrence of these events may be a reason for converting to BLRM
  • Patients are classified as DLT evaluable if they experience a DLT or if they otherwise, in the absence of a DLT, receive at least 75% of the intended dose of the study drug during Cycle 1 of Parts la and lb of the study and have received all scheduled safety assessments during the DLT observation period. Patients who fail to meet these criteria may be replaced.
  • the DLT observation period will include events occurring in the first cycle of treatment (within 21 days after the start of the study treatment) for each patient, which is consistent with the DLT observation period for other immune-oncology antibody therapeutics targeting ILT family members expressed on myeloid cells, including the anti-ILT3 antagonist mAb MK-0482 and the anti-ILT4 antagonists mAbs 10-108 andMK-4830 (ClinicalTrials.gov Identifiers: NCT03978278, NCT05054348, and NCT03564691).
  • DLT can include hematologic toxicities and non-hematologic toxicides. Hematologic DLTs include: Grade 4 neutropenia that persists > 5 days; Grade 4 thrombocytopenia of any duration; Grade > 3 thrombocytopenia with Grade > 2 (clinically significant) bleeding; febrile neutropenia (defined as ANC ⁇ 1000/mm 3 with a single temperature of > 38.3°C [ 101 °F] or a sustained temperature of > 38°C [100.4°] for more thanl hour); or Grade > 3 neutropenia with infection.
  • Non-hematologic DLT include Grade > 3 toxicities that are considered clinically significant except for the following: Grade 3 nausea/vomiting or diarrhea for ⁇ 72 hours with adequate antiemeticand other supportive care; Grade 3 fatigue for ⁇ 1 week; Grade > 3 electrolyte abnormality that lasts up to 72 hours, is not clinically complicated, and resolves spontaneously or responds to conventional medicalinterventions; Grade > 3 amylase or lipase that is not associated with symptoms or clinicalmanifestations of pancreatitis.
  • Another non-hematologic DLT includes confirmed DILI meeting Hy’s Law (concomitant ALT/AST > 3*ULN withbilirubin > 2*ULN without an alternative cause [eg, cholestasis]).
  • ALT/AST > 3*ULN withbilirubin > 2*ULN without an alternative cause [eg, cholestasis]
  • a hepatic transaminase level > 10> ⁇ ULN will be considered as a DLT.
  • Another non-hematologic DLT includes ALT/AST > 8*ULN regardless of duration or ALT/AST 5-8*ULN that persists for > 2 weeks.
  • Treatment-related AEs causing a delay by >14 days in receiving the next scheduled cycle due to persisting toxicities is a DLT.
  • Additional non-hematologic DLTs include Grade 4 CRS or Grade > 3 CRS, except if the Grade 3 CRS has improved to Grade ⁇ 1 within 48 hours or if the Grade ⁇ 3 CRS that has worsened due to not being maximally treated (ie, lack of administration of SOC treatment per guidelines for the management of CRS).
  • DLTs isolated Grade 3 laboratory abnormalities that are not associated with clinical sequelae and are corrected with supplementation/appropriate management within72 hours of their onset; or Grade 3 IRR, allergic reactions, or anaphylaxis. While Grade 3 IRR, allergic reactions, or anaphylaxis will not be considered as DLTs, they may be a reason for discontinuation.
  • the MTD is defined as the highest dose with the probability of DLT from the targeted toxicity interval.
  • the targeted toxicity interval for the DLT rate is defined as [0.16, 0.33], Maximum Administered Dose
  • the MAD will be defined as the highest dose tested.
  • the RP2D for HZ5A7. V5 as monotherapy and in combination with pembrolizumab will be selected based on all available clinical data obtained from Part la and Part lb; all safety, efficacy, PK, and pharmacodynamic data will be aggregated and analyzed. Any late-onset toxicities will be included in this assessment.
  • the preliminary RP2D is selected for further investigation in the Part 2 portion of the study.
  • the preliminary RP2D of HZ5A7.V5 for thePart 2a monotherapy expansion will be equal to or less than the MTD/MAD identified in Part la.
  • the planned dose of pembrolizumab for this study is 200 mg Q3W. This dose is justified as outlined below.
  • PK data in KN001 evaluating target-mediated drug disposition conclusively demonstrated saturation of PD1 in systemic circulation at doses much lower than 200 mg Q3W.
  • a EOT visit Should occur within 1 week after the patient has been discontinued from study treatment and should include complete tumor assessments if not completed within 6 weeks of the EOT visit.
  • b 1-Month and late-onset irAE follow-up At least 28 days and no more than 35 days after discontinuation of treatment or last dose of study drug, whichever is later, patients will return to undergo review of these assessments as well as an evaluation of resolution of any treatment-related toxicity. Patients continuing to experience treatment-related toxicity at this point will continue to be followed at least Q4W until resolution or if no further improvement is expected.
  • To collect additional late irAEs all patients will have a follow up visit 90 and 120 days ( ⁇ 7 days) after the last dose to collect post-treatment related AEs and survival status.
  • Abbreviated physical examination Additional examinations to be performed as clinically indicated.
  • Abbreviated PE includes, but is not limited to, assessment of the skin, lungs, cardiovascular system, and abdomen (liver and spleen).
  • Baseline signs and symptoms On Day 1 prior to the start of study treatment, patients will be asked about any signs and symptoms experienced within the past 14 days. Baseline signs and symptoms will be recorded in the Medical History eCRF.
  • g Vital signs Includes temperature, blood pressure, pulse rate, and respiratory rate to be recorded after the patient has rested for at least 5 minutes in a supine or seated position. In addition to time points noted, also as clinically indicated. Weight measured on Day 1 of each cycle only. Height is measured at the Screening visit only.
  • ECHO or MUGA To be completed at screening and at the EOT visit. ECHO or MUGA scans should also be performed in the case of an AE which may be related to cardiac dysfunction. The same modality used at screening should be used for all subsequent time points.
  • HBV surface antigen HbsAg
  • HBV core antibody HbcAb
  • HCV antibody HBV serology
  • reflex serum viral load testing will be performed.
  • test results from samples collected within 3 months of the patient’s first dose of study drug can be used as baseline if all prior serology data are negative for any viral particles.
  • Samples will be analyzed locally.
  • J Hematology Complete blood count to include hemoglobin, hematocrit, platelets, white blood cells, neutrophils, lymphocytes, monocytes and eosinophils. Samples must be collected pre dose and at the end of infusion on Day 1 of each cycle through Cycle 4.
  • k Serum chemistry panel Should include albumin (total protein), alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, bicarbonate, bilirubin (total and direct), calcium (total), chloride, creatinine, glucose (nonfasted), lactate dehydrogenase, magnesium, phosphorus or phosphate, potassium, sodium, and uric acid. Samples will be analyzed locally.
  • Coagulation Prothrombin time or international normalized ratio and partial thromboplastin time. Samples will be analyzed locally.
  • Thyroid function tests Includes FT3, FT4, and TSH. Samples will be collected at screening, at every odd number cycle during study treatment (ie, baseline, Cycle 3, Cycle 5), EOT, the 28-day follow-up visit, and if clinically indicated. TSH within normal limits for institution; supplementation is acceptable to achieve a TSH within normal limits; in patients with abnormal TSH if FT4 and FT3 are normal and the patient is clinically euthyroid, the patient is eligible. Samples will be analyzed locally.
  • Pregnancy test A urine pregnancy test will be performed at screening and within 72 hours of receiving the first dose of study treatment in women of childbearing potential. Pregnancy tests will also be routinely performed at the start of every cycle and at the EOT. Additional pregnancy tests may be undertaken if requested by the IRB or if required by local regulations.
  • Cytokine release syndrome analyses Serum to be collected for evaluation of IL-1 , TNFa, IL-6, IL-10, IFNy, sCD25, ferritin, and hsCRP. Samples will be sent to a central laboratory for analysis.
  • p HZ5A7.V5 administration Administered Day 1 of each 21-day cycle as an IV infusion. For patients receiving combination therapy, HZ5A7.V5 should be administered 30 minutes after completion of the pembrolizumab infusion. Observational period of 4 hours after dosing for C1D1 only.
  • Pembrolizumab administration For combination patients, 200 mg to be administered on Day 1 of each 21-day cycle as an IV infusion until the patient meets criteria for treatment discontinuation as defined in Patient Discontinuation from Study Drugs. The infusion will be given over 30 minutes.
  • Bone or positron-emission tomography scan may be utilized if bone metastasis is suspected. RECIST guidelines are presented in below. For patients who cross over, imaging collected at the time of disease progression may be used to establish a new baseline for the patient. Any region not evaluated at the time of disease progression must be evaluated prior to initiation of combination treatment. All imaging required to establish a new baseline must be completed within 28 days of combination treatment initiation. s Adverse events: All AEs must be recorded from the time of study treatment initiation through 28 days after the last dose of study treatment. In addition, all late-onset irAEs must be recorded through 120 days after the last dose of study treatment. The SAE reporting period begins at the time the patient provides informed consent and continues through 28 days after the last dose. In addition, all late-onset serious irAEs must be recorded through 120 days after the last dose.
  • Table 14 Schedule of Sampling for Pharmacokinetic, Pharmacodynamic, Biomarker, Tumor Biopsies, and ECG Assessments
  • Table 14 Schedule of Sampling for Pharmacokinetic, Pharmacodynamic, Biomarker, Tumor Biopsies, and ECG Assessments (Continued)
  • a Intra-patient dose escalation For patients approved for intra-patient dose escalation, patients will be assessed per the current cycle according to Table 13. All blood sampling and ECG assessments should be collected using the sampling schedule starting with Cycle 1 and beyond in Table 14. Sampling for PK,
  • exploratory biomarker and receptor occupancy should be performed during the first cycle after intra-patient dose escalation as follows: Day 1 (predose, EOI, and 4 hr post-dose), Day 2 (optional), Day 4 (optional), Day 8, and Day 15.
  • PK should be performed at Day 1 predose and at the EOI only. Collection of blood for circulating tumor biomarker assay does not need to be repeated starting with Cycle 1 atthe time of intra-patient dose escalation.
  • Crossover Patients who meet crossover criteria (see below) and provide consent to cross over will be assessed using the assessment schedule Table 13 and Table 14.
  • Screening crossover assessments will include all screening assessments except ECHO or MUGA, HBV/HCV/HIV tests, and thyroid function test, unless clinically indicated. Assessments performed during Parts la or 2a within 28 days of Day 1 may be used in lieu of the screening assessment.
  • c EOT visit Should occur within 1 week after the patient has been discontinued from the study treatment and should include complete tumor assessments if not completed within 6 weeks of the EOT visit.
  • d PK blood collection For combination patients, samples may be used for both HZ5A7. V5 and pembrolizumab evaluations, where required. Predose blood collection should be within 1 hr of infusion.
  • PK sampling should be performed at Cycle 3 on Day 1 (predose, EOI, and 4 hr post-dose), Day 2 (optional), Day 4 (optional), Day 8, and Day 15.
  • e ADA and nAb Samples collected will be used for the analysis of HZ5A7.V5 and pembrolizumab, where required.
  • f Archival tumor biopsy Sample is required for enrollment and should be taken within 1 year of enrollment. If an archival sample is unavailable, a fresh biopsy can be obtained during screening.
  • Paired tumor biopsy (optional): Consent required for patient participation in the collection of optional fresh tumor biopsy.
  • Biopsies may be performed, preferably during screening or at baseline (prior to first administration), approximately 2 weeks (on or between Days 15 to 21 of Cycle 1) following the first administration of study treatment or after the second administration of study drug (after Day 1 of Cycle 2), and for any clinical judgment (eg, pseudoprogression). Collection of a second on-treatment biopsy, or a biopsy at the time of disease progression and/or at EOT to confirmresponses may also be considered as clinically indicated.
  • Collection of a second on-treatment biopsy, or a biopsy at the time of disease progression and/or at EOT to confirm responses areoptional and may also be considered as clinically indicated.
  • tumor lesions/lymph nodes will be categorized measurable or non- measurable asfollows.
  • Measurable lesions are lesions that can be accurately measured in at least 1 dimension. [00449] Lesions with longest diameter twice the slice thickness and at least 10 mm or greater when assessed by CT or MRI (slice thickness/interval ⁇ 5 mm) are also measurable lesions. [00450] Lesions with longest diameter at least 20 mm when assessed by chest X-ray are also measurable lesions.
  • Malignant lymph nodes with the short axis 15 mm or greater when assessed by CT are also measurable lesions.
  • the shortest axis is used as the diameter for malignant lymph nodes, longest axis for all other measurable lesions. At baseline and in follow-up, only the short axis will be measured and followed.
  • Non-measurable disease includes lesions too small to be considered measurable (including nodes with short axis between 10 and 14.9 mm) and truly non-measurable disease such as pleural or pericardial effusions, ascites, inflammatory breast disease, leptomeningeal disease, lymphangitic involvement of skin or lung, clinical lesions that cannot be accurately measuredwith calipers, and abdominal masses identified by physical examination that are not measurable by reproducible imaging techniques.
  • Bone disease Bone disease is non-measurable by bone scan, PET scan, or plainfilms. However, these techniques can be used to confirm the presence or disappearance of bone lesions.
  • Soft tissue components that can be evaluated by CT or MRI and meet the definition of measurability at baseline (eg, lytic bone lesions or mixed lytic-blasticlesions, with identifiable soft issue components, that can be evaluated by cross-sectional imaging techniques such as CT or MRI can be consideredmeasurable lesions if the soft issue components meet the definition of measurability described above).
  • Cystic lesions Simple cysts should not be considered as malignant lesions and should not be recorded either as target or non-target disease. Cystic lesions thought to represent cystic metastases can be measurable lesions if they meet the specific definition above. If non-cystic lesions are also present, these are preferredas target lesions.
  • Target lesions should be selected on the basis of size (longest lesions) and suitability for accurate repeated measurements. Record the longest diameter for each lesion, except in the case of pathological lymph nodes for which the short axis should be recorded. The sum of thediameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions at baseline will be the basis for comparison with assessments performed on study.
  • Measurements for target lesions that become small should continue to be recorded. If a target lesion becomes too small to measure, 0 mm should be recorded if the lesion is considered to have disappeared; otherwise, a default valueof 5 mm should be recorded. NOTE: When nodal lesions decrease to ⁇ 10 mm (normal), the actual measurement shouldstill be recorded.
  • Non-measurable disease is non-target. All measurable lesions not identified as target lesions are also included as non-target disease. Measurements are not required but rather assessments will be expressed as Absent, Indeterminate, Present/Not Increased, or Increased. [00466] Multiple non-target lesions in 1 organ may be recorded as a single item in the eCRF(eg, ‘multiple enlarged pelvic lymph nodes’ or ‘multiple liver metastases’).
  • Target Disease Complete Response (CR): Complete disappearance of all target lesions with the exception of nodal disease. All target nodes must decrease to normal size (short axis ⁇ 10 mm). All target lesions must be assessed.
  • Partial Response > 30% decrease under baseline of the sum of diameters of all target- measurable lesions. The short diameter is used in the sum for target nodes, whilethe longest diameter is used in the sum for all other target lesions. All target lesions must be assessed.
  • Stable Disease Does not qualify for CR, PR, or PD. All target lesions must be assessed. SD can follow PR only in the rare case that the sum increases by ⁇ 2 0% from the nadir, but enough that a previously documented 30% decrease no longer holds.
  • Progressive Disease 20% increase in the sum of diameters of target measurable lesions above the smallest sum observed (over baseline if no decrease in the sum is observed duringtherapy), with a minimum absolute increase of 5 mm.
  • Indeterminate PD has not been documented, and one of the following conditions has been met: 1 or more target measurable lesions have not been assessed; Assessment methods used were inconsistent with those used at baseline; 1 or more target lesions cannot be measured accurately (eg, poorly visibleunless due to being too small to measure); or 1 or more target lesions were excised or irradiated and have not reappeared or increased.
  • CR Disappearance of all non-target lesions and normalization of tumor marker levels. All lymph nodes must be ‘normal’ in size ( ⁇ 10 mm short axis).
  • Non-CR/non-PD Persistence of any non-target lesions and/or tumor marker level above the normal limits.
  • PD Unequivocal progression of pre-existing lesions.
  • the overall tumor burden must increase sufficiently to merit discontinuation of therapy.
  • progression due to unequivocal increase in non-target disease should be rare.
  • any new unequivocal malignant lesion indicates PD. If a new lesion is equivocal, for example due to its small size, continued assessment will clarify the etiology. If repeat assessments confirm the lesion, then progression should be recorded on the date of the initial assessment. A lesion identified in an area not previously scanned will be considered a new lesion.
  • CR determination depends on a residual lesion that decreased in size but did not disappear completely, it is recommended the residual lesion be investigated with biopsy or fine needle aspirate. If no disease is identified, objective status is CR. [00477] If PD determination depends on a lesion with an increase possibly due to necrosis, the lesion may be investigated with biopsy or fine needle aspirate to clarify status.
  • CR complete response
  • PD progressive disease
  • PR partial response
  • SD stable disease.
  • Table 16 Objective Response Status at each Evaluation for Patients with Non-target Disease Only
  • Cross over to a combination treatment regimen with pembrolizumab may be considered for patients enrolled in Part la and Part 2a who experience unequivocal disease progression during monotherapy, if they meet the following conditions.
  • the candidate for crossover combination treatment with pembrolizumab tolerated the current monotherapy dose level well, with highest toxicity observed being Grade 2 or below except those events which are clearly and incontrovertibly due to underlying disease/disease progression or extraneous cause. Isolated Grade 3 laboratory abnormalities that are not associated with clinical sequelae may be allowed.
  • the patient meets all inclusion and exclusion criteria, including Part lb specific criteria except for tumor types, prior to initiating combination therapy with pembrolizumab.
  • the patient may receive combination treatment with pembrolizumab as long as the washout period between the combination treatment with pembrolizumab is > 21 days after the prior administration of HZ5A7.V5.
  • Patients may be treated at a lower or higher dose level of HZ5A7.V5, when combined with pembrolizumab, than the originally assigned monotherapy dose.
  • Hz5A7.v5 Heavy chain amino acid sequence with signal sequence underlined (SEQ ID NO: 125) MDMRVPAOLLGLLLLWLRGARCEVOLVESGGGLVQPGGSLRLSCAASGFTFSSYGMS WVRQAPGKGLEWVATISGGGSYTNYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV YYCARREWRYTLYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVI ⁇ FNWYVDGVEVHNAI ⁇ TI ⁇ PREEQYGSTYRVVSVLTVLHQDWLNGI ⁇ EYI ⁇ CI ⁇ V SNKALPAPIEKTISKAKGQPREPQVYTLPP
  • Hz5A7.v5 Heavy chain amino acid sequence without signal sequence (SEQ ID NO: 126) EVQLVESGGGL VQPGGSLRLSC AASGFTF S S YGMSWVRQ APGKGLEW VATISGGGS YT NYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARREWRYTLYAMDYWGQG TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW
  • Hz5A7.v5 Light chain amino acid sequence with signal sequence underlined (SEQ ID NO: 127) MDMRVPAOLLGLLLLWLRGARCDIOLTQSPSFLSASVGDRVTITCRASESVESYGSSFM HWYQQKPGKAPKLLIYLTSNLESGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQNNE DPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC
  • Hz5A7.v5 Light chain amino acid sequence without signal sequence (SEQ ID NO: 128) DIQLTQSPSFLSASVGDRVTITCRASESVESYGSSFMHWYQQKPGKAPKLLIYLTSNLES GVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQNNEDPFTFGQGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
  • HHHHHH Human ILT3 Domain 2 with stem amino acid sequence (aa 124-259) (SEQ ID NO:155)

Abstract

La présente divulgation concerne des utilisations d'agents de liaison, tels que des anticorps, qui se lient de manière spécifique à ILT3, y compris l'ILT3 humaine, pour traiter une maladie ou un trouble.
PCT/US2022/025327 2022-01-19 2022-04-19 Utilisations d'agents de liaison ilt3 WO2023140875A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
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US20150045369A1 (en) * 2012-04-10 2015-02-12 Vib Vzw Novel markers for detecting microsatellite instability in cancer and determining synthetic lethality with inhibition of the dna base excision repair pathway
US20190153093A1 (en) * 2017-11-17 2019-05-23 Merck Sharp & Dohme Corp. Antibodies specific for immunoglobulin-like transcript 3 (ilt3) and uses thereof
US20200031926A1 (en) * 2017-02-09 2020-01-30 Bluefin Biomedicine, Inc. Anti-ilt3 antibodies and antibody drug conjugates
US20200207856A1 (en) * 2017-12-22 2020-07-02 Jounce Therapeutics, Inc. Antibodies to lilrb2
US20210221887A1 (en) * 2019-12-19 2021-07-22 Ngm Biopharmaceuticals, Inc. Ilt3-binding agents and methods of use thereof
US20210355211A1 (en) * 2020-05-01 2021-11-18 Ngm Biopharmaceuticals, Inc. Ilt-binding agents and methods of use thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150045369A1 (en) * 2012-04-10 2015-02-12 Vib Vzw Novel markers for detecting microsatellite instability in cancer and determining synthetic lethality with inhibition of the dna base excision repair pathway
US20200031926A1 (en) * 2017-02-09 2020-01-30 Bluefin Biomedicine, Inc. Anti-ilt3 antibodies and antibody drug conjugates
US20190153093A1 (en) * 2017-11-17 2019-05-23 Merck Sharp & Dohme Corp. Antibodies specific for immunoglobulin-like transcript 3 (ilt3) and uses thereof
US20200207856A1 (en) * 2017-12-22 2020-07-02 Jounce Therapeutics, Inc. Antibodies to lilrb2
US20210221887A1 (en) * 2019-12-19 2021-07-22 Ngm Biopharmaceuticals, Inc. Ilt3-binding agents and methods of use thereof
US20210355211A1 (en) * 2020-05-01 2021-11-18 Ngm Biopharmaceuticals, Inc. Ilt-binding agents and methods of use thereof

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