Antibodies or antigen-binding fragments that bind to LILRB1 and uses thereof

Abstract

The present disclosure relates to antibodies, or antigen-binding fragments thereof, that bind to LILRB1, and the use of such antibodies or antigen-binding fragments in the diagnosis, prevention or treatment of neoplastic diseases.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to antibodies or antigen-binding fragments that bind to LILRB1, polynucleotides, vectors, host cells, and pharmaceutical compositions thereof. The present disclosure also relates to uses of the antibodies or antigen-binding fragments. BACKGROUND

[0002] The statements in this section merely provide background information related to the present disclosure and can not necessarily constitute prior art.

[0003] Immune checkpoint molecules, including inhibitory immune checkpoint molecules and stimulatory immune checkpoint molecules, are defined as ligand-receptor pairs that exert inhibitory or stimulatory effects on immune responses. Most immune checkpoint molecules have been considered so far are expressed on cells of the adaptive immune system, particularly on T cells, and on cells of the innate immune system. They are essential for maintaining self-tolerance and modulating the length and strength of immune responses of effectors in different tissues to minimize tissue damage. Increasing evidence has shown that inhibitory immune checkpoint molecules or stimulatory immune checkpoint molecules are expressed in a considerable proportion of tumor types (Zhang Y, Zheng J, Adv Exp Med Biol, 2020; 1248:201-226). Current immune checkpoint blockade strategies have been promising cancer therapies. Upon appropriate stimulation, both effector cells of the innate immune system and the adaptive immune system have the ability to attack cancer cells (Barkal AA et al., Nat Immunol, Jan 2018; 19(1):76-84).

[0004] LILRB1 (leukocyte immunoglobulin-like receptor B1), also known as ILT2, is a member of the leukocyte immunoglobulin-like receptor (LIR) family. LILRB1 is a tyrosine-based immunoreceptor inhibitory motif-containing receptor and is widely expressed on human immune cells, including B cells, monocytes and macrophages, dendritic cells, and subsets of natural killer (NK) cells and T cells. Ligands of LILRB1, such as major histocompatibility complex (MHC) class I molecules, activate LILRB1 and transduce inhibitory signals, thereby suppressing immune responses (Chen H, Chen Y, et al. J Immunother Cancer, Aug 2020; 8(2):e000515). SUMMARY

[0005] The present disclosure provides a novel antibody or antigen-binding fragment thereof binding to LILRB1, which improves immune response or reduces immune suppression by blocking LILRB1 signaling pathway, so that the antibody or antigen-binding fragment thereof binding to LILRB1 can improve tumor diseases. The antibody or antigen-binding fragment thereof binding to LILRB1 comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, HCDR1 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence as shown in SEQ ID NO: 17, HCDR2 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence as shown in SEQ ID NO: 18, HCDR3 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence as shown in SEQ ID NO: 19; and LCDR1 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence as shown in SEQ ID NO: 20, LCDR2 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence as shown in SEQ ID NO: 21, and LCDR3 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence as shown in SEQ ID NO: 22.

[0006] In some embodiments, HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences as shown in SEQ ID NO: 17, 18 and 19, respectively; and LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences as shown in SEQ ID NO: 20, 21 and 22, respectively.

[0007] In some embodiments, the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 7-8, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 9 and 13, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 9 and 14, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 9 and 15, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 9 and 16, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 10 and 13, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 10 and 14, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 10 and 15, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 10 and 16, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 11 and 13, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 11 and 14, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 11 and 15, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 11 and 16, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 12 and 13, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 12 and 14, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 12 and 15, respectively; or the heavy chain variable region and the light chain variable region comprise the amino acid sequences set forth as SEQ ID NOs: 12 and 16, respectively.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances the killing activity of immune cells (e.g., NK cells and macrophages), such as NK cell-mediated destruction of an unintended cell, phagocytosis of an unintended cell by a macrophage.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances the destruction of a cell expressing an MHC class I molecule, optionally, the MHC class I molecule comprises HLA-G, b2M, HLA-A2, or a combination thereof.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances NK cell-mediated destruction of a cell expressing HLA-G.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances phagocytosis of MHC class I molecule-expressing cells opsonized by immune checkpoint-related antibodies (e.g., anti-SIRPa antibodies, anti-CD47 antibodies) by macrophages.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 does not or hardly binds to LILRBs (e.g., LILRB2, LILRB3, LILRB4, LILRB5, LILRB1 excluded) and LILRA (LILRA1, LILRA2, LILRA3, LILRA4, LILRA5) family.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 blocks the interaction of LILRB1 with its ligands, such as human HLA-G, human HLA-A2.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 comprises an effector molecule-binding fragment (e.g., Fc fragment).

[0015] Also provided is an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof that binds to LILRB1 provided above, an isolated vector comprising the polynucleotide, and a host cell comprising the isolated polynucleotide, or the isolated vector.

[0016] In another aspect, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof that binds to LILRB1 described above.

[0017] In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds to LILRB1, the isolated polynucleotide, the isolated vector, or the host cell, and a pharmaceutically acceptable carrier.

[0018] In another aspect, provided herein is use of the antibody or antigen-binding fragment thereof that binds to LILRB1, the isolated polynucleotide, the isolated vector, the host cell, or the pharmaceutical composition in the manufacture of a therapeutic agent for diagnosing, preventing, or treating a tumor disease.

[0019] In another aspect, provided herein is a combination comprising an antibody or antigen binding fragment thereof described above that binds to LILRB1, and a second therapeutic agent for treating a neoplastic disease in a subject, wherein the second therapeutic agent is selected from one or more of an inhibitor of an inhibitory molecule, an activator of a costimulatory molecule, chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, a vaccine, or a cellular immunotherapy.

[0020] In another aspect, provided herein is a method for treating a subject having a neoplastic disease, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen binding fragment thereof that binds to LILRB1, an isolated polynucleotide, an isolated vector, a host cell, or a pharmaceutical composition. BRIEF DESCRIPTION OF DRAWINGS

[0021] The following is a brief description of the drawings that are presented to illustrate exemplary embodiments disclosed herein and is not intended to be limiting to their scope.

[0022] Figure 1 82c and a human IgG4 isotype control (hIgG4 iso) binding to K562 / SHP-1 cells expressing human LILRB1 in a FACS-based binding assay. + LILRB1 + cells.

[0023] Figure 2 82c, 15G8, and isotype control (hIgG4 iso) effect on enhancing NK92 / CD16a cell killing of A375 / HLA-G cells. Cytotoxicity index values obtained under the indicated treatments are listed at the top of each bar.

[0024] Figure 3 15G8, 82c, and isotype control (hIgG4 iso) effect on enhancing macrophage phagocytosis of DLD-1 / β2M cells. Phagocytosis index values obtained under the indicated treatments are listed at the top of each bar.

[0025] Figure 4A and Figure 4B 82c, 82c-derived humanized variants, and a human IgG1 LALA isotype control (hIgG1 LALA iso) binding to K562 / SHP-1 cells expressing human LILRB1 in a FACS-based binding assay. + LILRB1 + cells.

[0026] Figure 5Activity of 82c, hu082.02, hu082.03, hu082.06, hlgG4 iso, and hlgGl LALA iso to block human HLA-G interaction with human LILRB1 in a FACS-based competition assay is shown. Blocking rates obtained under the indicated treatment are listed at the top of each column.

[0027] Figure 6 Activity of hu082.03 and hlgGl LALA iso to block human HLA-A2 interaction with human LILRB1 in a FACS-based competition assay is shown.

[0028] Figure 7 Principle of the LILRB1 / SHP-1 recruitment assay is shown.

[0029] Figure 8 Activity of hu082.03, 15G8, hlgG4 iso, and hlgGl LALA iso to block MHC class I induced LILRB1 downstream signaling in a LILRB1 / SHP-1 recruitment assay using Raji cells (A) or A375 / HLA-G cells (B) as stimulator cells is shown.

[0030] Figure 9 Effect of 82c, hu082.02, hu082.03, hu082.06, hlgG4 iso, and hlgGl LALA iso on enhancing NK92 / CD16a cell killing of A375 / HLA-G cells is shown.

[0031] Figure 10 Effect of 82c, hu082.02, hu082.03, hu082.06, hlgG4 iso, and hlgGl LALA iso on enhancing macrophage phagocytosis of DLD-1 / β2M cells is shown. Phagocytosis index values obtained under the indicated treatment are listed at the top of each column.

[0032] Figure 11 Effect of hu082.03, hlgG4 iso, and hlgGl LALA iso on enhancing macrophage phagocytosis of A375 / HLA-G cells is shown. Phagocytosis index values obtained under the indicated treatment are listed at the top of each column. DETAILED DESCRIPTION

[0033] The present disclosure will be explained in greater detail below. This specification is not intended to be a detailed catalog of all the different ways or all the features that can be added to the present application. For example, features stated in relation to one embodiment can be incorporated into other embodiments, and features stated in relation to a particular embodiment can be deleted from that embodiment. In addition, many variations and additions of the described embodiments can be considered by one of skill in the art in light of the present disclosure, yet within the scope of the application. Accordingly, the following description is intended to illustrate some particular embodiments of the application, and is not intended to be exhaustive or to limit the application to the precise forms disclosed. Rather, the following description is intended to describe some particular embodiments of the application, and is intended not to limit the scope of the application to any one embodiment or group of embodiments.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, the preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used.

[0035] Antibodies or antigen-binding fragments thereof that bind to LILRB1 An antibody or antigen-binding fragment thereof that binds to LILRB1 provided herein comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, HCDR1 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 17, HCDR2 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 18, HCDR3 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 19; and LCDR1 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 20, LCDR2 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 21, LCDR3 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 22. CDRs are defined using the Kabat definition except that HCDR1 is defined using a combination of the Kabat and IMGT systems. The term “an antibody or antigen-binding fragment thereof that binds to LILRB1” with respect to the present disclosure as used herein includes an isolated, recombinant, or synthetic antibody, antibody conjugate, or antibody derivative.

[0036] The term "antibody" described in the present disclosure refers to an immunoglobulin, which is a four polypeptide chain structure consisting of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and the order of arrangement of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, the immunoglobulin can be classified into five types, or called isotypes of immunoglobulin, i.e., IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The Ig of the same type can be classified into different subtypes according to the difference in the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bond. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. The light chain is classified into κ chain or λ chain according to the difference in the constant region. Each of the five types of Ig can have κ chain or λ chain.

[0037] The term "antigen binding fragment" refers to an antigen binding fragment of an antibody and antibody analogs, which generally comprises at least part of the antigen binding region or variable region (e.g., one or more CDRs) of a parental antibody. The antibody fragment retains at least some of the binding specificity of the parental antibody. Typically, the antibody fragment retains at least 10% of the parental binding activity when expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parental antibody to the target. Examples of antigen binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multi-specific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol 23: 1126-1136.

[0038] A "heavy chain variable region" or "VH" with respect to an antibody refers to the fragment of the heavy chain that contains the three CDRs inserted between flanking stretches called framework regions, which are generally more highly conserved than the CDRs and form a scaffold that supports the CDRs. Similarly, a "light chain variable region" or "VL" with respect to an antibody refers to the fragment of the light chain that contains the three CDRs inserted between flanking stretches called framework regions, which are generally more highly conserved than the CDRs and form a scaffold that supports the CDRs.

[0039] The percent identity of two amino acid sequences is determined by dividing the number of identical residues by the total number of amino acid residues and multiplying the quotient by 100 to obtain a percentage. Gaps are not counted when evaluating identity. Thus, two copies of a perfectly identical sequence have 100% identity, but a sequence with a deletion, addition, or substitution can have a lower degree of identity. Those skilled in the art will recognize that there are several computer programs that can be used to determine the identity of sequences, such as those using algorithms such as BLAST. BLAST nucleotide searches are performed using the NBLAST program, and BLAST protein searches are performed using the BLASTP program, and the default parameters for each program are used.

[0040] Kabat and IMGT are well known to those skilled in the art, see, e.g., Kabat E A, Wu TT, Perry HM, et al. Sequence of Proteins of Immunological Interest. 1991. Lefranc MP, et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. 2003 Jan;27(l):55-77.

[0041] The CDRs of the antibody or antigen-binding fragment thereof that binds to LILRB1 can contain mutations, which can be selected from insertions, deletions, and / or substitutions; substitutions are preferably conservative amino acid substitutions.

[0042] The term "conservative amino acid" herein refers generally to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity). Conservative substitutions can be naturally occurring or can be introduced, e.g., using mutagenesis (e.g., Hutchinson et al., 1978, J. Biol. Chem. 253:6551). For example, the amino acids glycine, alanine, valine, leucine, and isoleucine can generally be substituted for one another (amino acids with aliphatic side chains). Of these possible substitutions, a preference is given to substituting glycine and alanine for one another (because they have relatively short side chains), and valine, leucine, and isoleucine for one another (because they are hydrophobic, larger aliphatic side chains). Other amino acids that can generally be substituted for one another include, but are not limited to, phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); and asparagine and glutamine (amino acids with amide side chains).

[0043] In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 17, 18, and 19, respectively, with no more than 3, 2, 1 amino acid deletions, insertions, and / or substitutions; and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 20, 21, and 22, respectively, with no more than 3, 2, 1 amino acid deletions, insertions, and / or substitutions.

[0044] In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 17, 18, and 19, respectively, with no more than 3, 2, 1 amino acid conservative substitutions; and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 20, 21, and 22, respectively, with no more than 3, 2, 1 amino acid conservative substitutions.

[0045] In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 17, 18, and 19, respectively; and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 20, 21, and 22, respectively.

[0046] In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 7; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 8; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 9; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 13; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 9; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 14; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 9; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 15; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 9; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 16; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 10; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 11; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 12; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth as SEQ ID NO: 11.at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 13; a heavy chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 10; and a light chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 14; a heavy chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 10; and a light chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 15; a heavy chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 10; and a light chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 16; a heavy chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 11; and a light chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 13; a heavy chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 11; and a light chain variable region that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 14;the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 11 and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 16; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 12 and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 13; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 12 and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 14; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 12 and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 15; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 12 and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 16; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 11 and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 15; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 11 and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 16;and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence as set forth in SEQ ID NO: 16.

[0047] The heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof that binds to LILRB1 can also contain mutations, which can be selected from insertions, deletions, and / or substitutions; substitutions are preferably conservative amino acid substitutions.

[0048] In some embodiments, the heavy chain variable region and the light chain variable region contain one or more mutations in the framework region, optionally the mutations do not significantly affect a desired activity of the heavy chain variable region or the light chain variable region. In some embodiments, the heavy chain variable region and the light chain variable region contain one or more conservative amino acids.

[0049] In some embodiments, the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 8; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 13; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 14; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 15; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 16; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 13; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 14; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 15; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 16; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 11, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 13; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 11, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 14; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 11, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 15; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 11, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 16; the heavy chain variable region comprises or has the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises or has the amino acid sequence of SEQ ID NO: 13;the heavy chain variable region comprises or has an amino acid sequence as set forth in SEQ ID NO: 12, and the light chain variable region comprises or has an amino acid sequence as set forth in SEQ ID NO: 14; the heavy chain variable region comprises or has an amino acid sequence as set forth in SEQ ID NO: 12, and the light chain variable region comprises or has an amino acid sequence as set forth in SEQ ID NO: 15; or the heavy chain variable region comprises or has an amino acid sequence as set forth in SEQ ID NO: 12, and the light chain variable region comprises or has an amino acid sequence as set forth in SEQ ID NO: 16.

[0050] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 encompasses (unless otherwise indicated or otherwise suggested by context) a monoclonal antibody, a polyclonal antibody, a murine antibody, a hamster antibody, a goat antibody, a rabbit antibody, a chimeric antibody, a primatized antibody, a humanized antibody, a (fully) human antibody, a multimeric antibody, a heterodimeric antibody, a hemidiabody, a bivalent antibody, a trivalent antibody, or a tetravalent antibody, a bispecific antibody, a single-chain antibody (e.g., scFv, scFab, and scFabAC), a di-scFv, a diabody, a triabody, or a tetrabody, a single-domain antibody, and a modified Fab fragment. In certain embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 is monovalent.

[0051] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 comprises an effector molecule binding fragment, such as an Fc fragment, which communicates with the immune system when the antibody binds its target, the Fc fragment can be of any class (e.g., IgG, IgE, IgM, IgD, or IgA) or subclass of immunoglobulin molecule, preferably the Fc fragment is an IgG molecule. In certain embodiments, the Fc fragment is a human IgG, e.g., IgGl, IgG2, IgG3, or IgG4, optionally with one or more mutations compared to a wild-type human IgG molecule. An exemplary Fc fragment is human IgG4 with a Serine 228 Proline mutation, which means that the serine at position 228 (defined by Kabat) is mutated to proline.

[0052] In some embodiments, the Fc fragment is derived from human IgGl or IgG4. In some embodiments, the Fc fragment is derived from human IgGl with mutations Leu234Ala, Leu235Ala, or a combination thereof.

[0053] The antibodies or antigen-binding fragments thereof that bind to LILRB1 are chimeric or humanized. Typically, a chimeric antibody includes the heavy chain variable region and / or light chain variable region (including CDRs and framework residues) of one species (typically mouse) fused to the constant region of another species (typically human). Humanized antibodies typically include the heavy and / or light chain CDRs from a murine antibody grafted into a non-human primate antibody variable region framework or a human antibody variable region framework, typically further containing a human constant region. See, e.g., Riechmann et al. (1988) Nature 332:323-327.

[0054] Methods of making all of the antibodies or antigen-binding fragments described above are well known to those of skill in the art. See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81(21):6851-5; Sharon et al. (1984) Nature 309(5966):364-7.

[0055] In certain embodiments, the antibodies or antigen-binding fragments thereof that bind to LILRB1 are produced by the selective lymphocyte antibody method (SLAM) (Babcook et al., 1996, Proc. Natl. Acad. Sci, 93, 7843-7848; de Wildt et al., 1997, J. Immunol. Methods, 207:61-67 and in Lagerkvist et al., 1995, BioTechniques 18:862-869), which enables isolation from any kind of cells producing high affinity antibodies during the in vivo immune response. The above method relies on the isolation of individual antibody-producing cells, followed by clonal expansion of the cells, followed by screening of those clones producing anti-LILRB1 antibodies, followed by identification of the sequences of their variable heavy (VH) and light (VL) genes. Thus, B cells positive for antibodies to LILRB1 are isolated. The B cells can be from humans, mice, rats, hamsters, rabbits, goats, or other mammalian species. The antibody genes in these B cells can be cloned and expressed in host cells (e.g., E. coli), for example, by traditional recombinant DNA techniques. The antibody-expressing cells can be purified by traditional methods. If the antibodies are from a non-human source, they can be humanized by traditional methods, such as by mutagenizing their genes. The humanized antibodies can then be expressed in host cells and purified.

[0056] Monoclonal antibodies can be made by any method known in the art, such as the hybridoma technique (Kohler and Milstein, Nature, 1975, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today, 1983, 4, 72), and the EBV-hybridoma technique (Cole et al., “Monoclonal Antibodies and Cancer Therapy”, pp. 77-96, Alan R. Liss, Inc., 1985). Methods for producing and manufacturing recombinant antibodies are well known in the art (see, e.g., Simmons et al., 2002, Journal of Immunological Methods, 263, 133-147).

[0057] Antibodies or antigen-binding fragments thereof that bind to LILRB1 of the present disclosure can also be generated using various phage display methods known in the art, including methods disclosed by Brinkman et al., 1995, J. Immunol. Methods, 182:41-50; Ames et al., 1995, J. Immunol. Methods, 184, 177-186; Kettleborough et al., 1994, Eur. J. Immunol, 24, 952-958.

[0058] Further, transgenic (e.g., genetically engineered) mice, or other organisms (including other mammals) can be used to produce antibodies or antigen-binding fragments thereof that bind to LILRB1 (see, e.g., US 6,300,129). For example, it is known that mice can be used to produce large numbers of high affinity antibodies with human variable sequences (see, e.g., US 6,586,251), where the mouse is engineered to replace only the variable regions (V-segments, D-segments, and J-segments for heavy chains, and V-segments and J-segments for light chains) of the mouse immunoglobulin loci with corresponding human variable sequences.

[0059] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 described above can optionally improve immune response or reduce immune suppression by blocking LILRB1 -related inhibitory signaling axis. It can be appreciated that the blockade of LILRB1 signaling in immune cells can activate the activity of natural killer (NK) cells, T cells, or macrophages. In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 blocks the activation of LILRB1 on NK cells. In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 blocks the activation of LILRB1 on macrophages.

[0060] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances the killing activity (or cytotoxic activity) of immune cells, preferably, the immune cells are NK cells and / or macrophages.

[0061] The antibody or antigen-binding fragment thereof that binds to LILRB1 blocks the interaction of LILRB1 with its ligand, such as major histocompatibility complex (MHC) class I molecule. In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 blocks the interaction of LILRB1 with human leukocyte antigen-G (HLA-G). In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 blocks the interaction of LILRB1 with human leukocyte antigen HLA-A2.

[0062] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances the destruction of cells expressing MHC class I molecules, optionally, the MHC class I molecules include HLA-G, b2M (beta-2-microglobulin, a component of MHC class I), HLA-A2, or a combination thereof. In some embodiments, the cells expressing MHC class I molecules are undesirable or unintended, such as tumor cells, tumor cells expressing HLA-G.

[0063] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances the NK cell-mediated destruction of cells expressing HLA-G.

[0064] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances the macrophage-mediated phagocytosis of cells expressing MHC class I molecules. In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances the phagocytosis of MHC class I molecule-expressing cells (such as b2M-expressing cells, HLA-G-expressing cells) opsonized by immune checkpoint-related antibodies by macrophages.

[0065] In some embodiments, the immune checkpoint related antibody binds to an immune checkpoint molecule such as signal-regulatory-protein alpha (SIRPa), a CD47 molecule, a CD27 molecule, a CD40 molecule, a CD137 molecule, a programmed cell death protein 1 (PD-L1), a programmed death 1 receptor (PD-1), a tumor necrosis factor receptor superfamily member 4 (OX4), a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), or an inducible T-cell costimulator (ICOS).

[0066] In some embodiments, the immune checkpoint is signal SIRPa. In some embodiments, the immune checkpoint is CD47.

[0067] SIRPa is a member of the signal-regulatory-protein (SIRP) family, and SIRP family members are receptor-type transmembrane glycoproteins known to be involved in the negative regulation of receptor tyrosine kinase-coupled signaling processes. SIRPa recognizes the ubiquitously expressed “Don’t Eat Me” signal molecule CD47, and blockade of either CD47 or SIRPa can enhance the activity of SIRPa-expressing immune cells such as macrophages.

[0068] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances phagocytosis of a cell expressing MHC class I molecules by a macrophage opsonized against a SIRPa antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances phagocytosis of a cell expressing MHC class I molecules by a macrophage opsonized against a CD47 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances phagocytosis of a cell expressing b2M by a macrophage opsonized against a SIRPa antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances phagocytosis of a cell expressing HLA-G by a macrophage opsonized against a CD47 antibody or antigen-binding fragment.

[0069] The antibody or antigen-binding fragment thereof that binds to LILRB1 specifically binds to LILRB1. In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 does not or does not substantially bind to one or more other LILRB family members such as LILRB2, LILRB3, LILRB4, and LILRB5.

[0070] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 does not or does not substantially bind to one or more LILRA family members, optionally, the LILRA family members contain LILRA1, LILRA2, LILRA3, LILRA4, and LILRA5.

[0071] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 does not or does not substantially bind to LILRB2, LILRB3, LILRB4, LILRB5, LILRA1, LILRA2, LILRA3, LILRA4, or LILRA5, or a combination thereof. Low cross-reactivity is advantageous to reduce adverse reactions of the antibody or antigen-binding fragment thereof that binds to LILRB1.

[0072] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 binds to LILRB1 with an EC 50 no more than 10 nM, 8 nM, or 5 nM, or an EC 50 no more than 2 μg / ml, 1 μg / ml, or 0.6 μg / ml.

[0073] In some embodiments, the antibody or antigen-binding fragment thereof that binds to LILRB1 has a binding affinity KD (affinity constant, KD = koff / kon, or KD = Kd / Ka) to LILRB1 of no more than 20 nM, 15 nM, or 10 nM.

[0074] EC 50 KD can be measured by methods well known in the art, such as FACS assay, competitive FACS. KD can be measured by methods well known in the art, such as Bio-Layer Interferometry (Octet), surface plasmon resonance (SPR) technology.

[0075] Polynucleotides, vectors, and host cells The present disclosure provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof that binds to LILRB1 described above.

[0076] A polynucleotide is a DNA, RNA, DNA / RNA hybrid, or modified nucleic acid sequence thereof. In some embodiments, a polynucleotide is a nucleic acid sequence of DNA. The encoding polynucleotide (DNA or RNA) can be a recombinant molecule or a synthetic molecule.

[0077] The present disclosure also relates to sequence variants of the polynucleotides described above. For example, the present disclosure includes nucleic acid sequences that are about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, 99.5%, 99.9%, or 100% identical to any of the polynucleotide sequences provided herein, including fragments thereof and complements thereof. The present disclosure also includes polynucleotides that differ from the polynucleotide sequences specifically provided herein due to the degeneracy of the genetic code.

[0078] The polynucleotide can further include regulatory sequences (e.g., a promoter sequence, an untranslated 5' region, and an untranslated 3' region) and / or vector sequences. For example, the polynucleotide constitutes a vector.

[0079] As used herein, the term "vector" refers to a polynucleotide that can be engineered to contain one or more cloned polynucleotides that can be propagated in a host cell. A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences that control the expression of a polypeptide of interest (such as, for example, a promoter and / or enhancer), and / or one or more selectable marker genes (such as, for example, an antibiotic resistance gene and a gene that can be used in a colorimetric assay, such as beta-galactosidase).

[0080] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which can involve the use of a non-pathogenic (defective), replication-competent virus, or a replication-defective virus can be used.

[0081] In an aspect, the present disclosure also provides an isolated vector comprising a polynucleotide as described herein. The provided isolated polynucleotide can be inserted into a vector using recombinant technology known in the art for further cloning (amplification of DNA) or for expression.

[0082] Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1 alpha), and a transcription termination sequence.

[0083] In some embodiments, the vectors provided herein comprise at least one promoter operably linked to a nucleic acid sequence (e.g., SV40, CMV, EF-1a), and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, and M13 phage, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

[0084] The present disclosure provides host cells comprising an isolated polynucleotide as described herein or an isolated vector as described herein.

[0085] As used herein, the term "host cell" refers to a cell that can be or has been a recipient of a vector or an isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate animal cells.

[0086] Vectors comprising a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof that binds to LILRB1 can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expression of the above-described polynucleotides (nucleic acid sequences of DNA, RNA, or DNA / RNA hybrid) in vectors herein are, for example, prokaryotic cells such as E. coli, or other microbial cells, or eukaryotic cells including, but not limited to, mammalian cells such as human cells, mouse cells, monkey cells, rabbit cells, goat cells, hamster cells, or rat cells, insect cells, avian cells, plant cells, and eukaryotic cells.

[0087] In some embodiments, the host cell can be, for example, (1) a bacterial cell such as E. coli; (2) fungal cells and Aspergillus cells, yeast cells such as Saccharomyces cerevisiae and K. lactis; (3) insect cell lines such as EXPRES SF® (cell line from Spodoptera frugiperda) cells (Protein Sciences Corp., Meriden, Conn., USA); (4) mammalian cells; or (5) plant cells.

[0088] Typical mammalian cells include COS 1 and COS 7 cells, Chinese hamster ovary (CHO) cells, NSO myeloma cells, NIH 3T3 cells, 293 cells, HEPG2 cells, HeLa cells, C127, 3T3, BHK, Bowes melanoma cells, L cells, MDCK, HEK293, WI38, murine ES cell lines (e.g., from strains 129 / SV, C57 / BL6, DBA-1, 129 / SVJ), K562, Jurkat cells, and BW5147. The present application thus provides cells expressing the antibodies of the present application, including but not limited to hybridoma cells, B cells, plasma cells, and mammalian and human host cells (e.g., adult embryonic stem cells) recombinantly modified to express the antibodies of the present application. Other useful mammalian cell lines are well known and can be readily obtained from the American Type Culture Collection (“ATCC”) (Manassas, Va., USA) and the National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository located at the Coriell Institute for Medical Research (Camden, N.J., USA). These cell types are merely representative, and this list is not meant to be an exhaustive list.

[0089] In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NSO, 293, and their derivatives.

[0090] The vectors can be introduced into the host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., using recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, bacteriophage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0091] Standard references setting forth the general principles of recombinant DNA technology known to those of skill in the art include Ausubel et al., Current Protocols In Molecular Biology, John Wiley & Sons, New York (1998 and 2001 supplements); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989); Kaufman et al., Eds., Handbook Of Molecular And Cellular Methods In Biology And Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991).

[0092] Standard reference works setting forth the general principles of immunology known to those of skill in the art include: Harlow and Lane, Antibodies: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999) and Roitt et al., Immunology, 3rd ed., Mosby-Year Book Europe Limited, London (1993). Standard reference works setting forth the general principles of medical physiology and pharmacology known to those of skill in the art include Fauci et al., eds., Harrison's Principles of Internal Medicine, 14th ed., McGraw-Hill Companies, Inc. (1998).

[0093] Kit The present disclosure provides a kit containing an antibody or antigen-binding fragment thereof described above that binds to LILRB1. If desired, such a kit can further include one or more of a variety of conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those in the art. Instructions, in pamphlet or label form, indicating the quantities of components to be administered, directions for administration, and / or directions for assembling the kit components, can also be included in the kit.

[0094] Pharmaceutical composition Provided herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described above that binds to LILRB1, an isolated polynucleotide, an isolated vector, or a host cell, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated in any manner known in the art.

[0095] The pharmaceutical composition can be formulated for parenteral (e.g., oral, intranasal, or by inhalation, ophthalmic artery, rectal, intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in unit dosage form, i.e., physically discrete units suitable for administration and uniform in content of active compound. The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0096] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, sterile diluent solutions (e.g., sterile water or saline), fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methyl parabens, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate, or phosphate), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers.

[0097] Formulations of the compositions can be prepared and enclosed in ampules, disposable syringes, or multiple-dose vials. Where appropriate, proper fluidity can be maintained, for example, by the use of coating such as lecithin, or surfactants. Absorption can be prolonged and / or facilitated by agents known in the art (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved through the use of implantable and microencapsulated delivery systems, which can include biodegradable biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0098] In some embodiments, the pharmaceutical composition is formulated as an injectable composition. The injectable pharmaceutical composition can be prepared in any conventional form, such as, for example, a liquid solution, suspension, emulsion, or a solid form suitable for producing a liquid solution, suspension, or emulsion. The injectable formulation can include sterile and / or non-pyrogenic solutions ready for injection, sterile dry soluble products ready for combination with a solvent prior to use (e.g., lyophilized powder, including tablets for subcutaneous injection), sterile suspensions ready for injection, sterile dry insoluble products ready for combination with a vehicle prior to use, and sterile and / or non-pyrogenic emulsions. The solution can be aqueous or non-aqueous.

[0099] In some embodiments, a sterile lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment thereof that binds to LILRB1 as disclosed herein in a suitable solvent. The solvent can contain other pharmacological components that improve the powder or excipients that stabilize the stability of the reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents.

[0100] The antibody or antigen-binding fragment, or pharmaceutical composition comprising the same, can be included alone in a container, pack, or dispenser, or as a part of a kit, together with a label and instructions for administration.

[0101] Methods of treatment The present disclosure provides use of an antibody or antigen-binding fragment thereof that binds to LILRB1, an isolated polynucleotide, an isolated vector, a host cell, a kit, or a pharmaceutical composition in the manufacture of a therapeutic agent for diagnosing, preventing, or treating a neoplastic disease.

[0102] The present disclosure provides use of an antibody or antigen-binding fragment thereof that binds to LILRB1, an isolated polynucleotide, an isolated vector, a host cell, or a pharmaceutical composition in the manufacture of a therapeutic agent for improving an immune response to a neoplastic disease.

[0103] The present disclosure provides a method for diagnosing, preventing, or treating a subject having a neoplastic disease, the method comprising administering to the subject a therapeutically effective amount of a therapeutic agent, such as an antibody or antigen-binding fragment thereof that binds to LILRB1, an isolated polynucleotide, an isolated vector, a host cell, a kit, or a pharmaceutical composition.

[0104] The term "therapeutically effective amount" refers to the amount of a composition or active agent disclosed herein effective to "treat" a disease or condition in a subject.

[0105] In some embodiments, the therapeutic agent can enhance an immune response to the disease, or can reduce immune suppression in vitro or in vivo.

[0106] Blocking LILRB1 signaling enhances phagocytosis by macrophages, restores cytotoxic function of NK cells, and it can be appreciated that an antibody or antigen-binding fragment thereof that binds to LILRB1 can reverse immune suppression and improve an immune response, such as a natural immune response, that can be helpful in treating a wide range of neoplastic diseases.

[0107] In some embodiments, the immune response of the subject is improved by enhancing immune cell killing activity, destruction of cells expressing MHC class I molecules, and / or phagocytosis of cells expressing MHC class I molecules by macrophages.

[0108] The present disclosure provides a combination comprising an antibody or antigen-binding fragment thereof that binds to LILRB1 as described in any one of claims 1-15, and a second therapeutic agent for treating a neoplastic disease in a subject, wherein the second therapeutic agent is selected from one or more of an inhibitor of an inhibitory molecule, an activator of a costimulatory molecule, chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, a vaccine, or a cellular immunotherapy.

[0109] In some embodiments, the second therapeutic agent is an antibody or antigen-binding fragment that binds to at least one immune checkpoint molecule (e.g., CD47, SIRPa) simultaneously or sequentially with the antibody or antigen-binding fragment. In some embodiments, the second therapeutic agent is an anti-SIRPa or anti-CD47 antibody.

[0110] In some embodiments, the tumor disease is a solid tumor or a liquid tumor. In some embodiments, the tumor disease is a solid tumor infiltrated by LILRB1-expressing tumor infiltrating lymphocytes. In some embodiments, the tumor disease comprises chronic myeloid leukemia, colorectal adenocarcinoma, breast cancer, carcinoid, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, hematological malignancy, melanoma or metastatic melanoma, non-small cell lung cancer, small cell lung cancer, bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the subject has a liquid tumor.

[0111] Without limitation, the method of treatment reduces the rate at which the volume of the tumor increases over time in the subject, reduces the risk of metastasis, or reduces the risk of additional metastases in the subject. In some embodiments, the treatment can arrest, slow, retard, or inhibit the progression of the cancer. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of the cancer in the subject.

[0112] A subject includes mammals including primates, rodents, canines, and swine, such as mice, rats, rabbits, cats, dogs, pigs, monkeys, chimpanzees, gorillas, and the like. In some embodiments, the subject includes mice, cynomolgus monkeys, and humans. In some embodiments, the subject is a human. The terms "patient" or "subject" are used interchangeably herein unless otherwise noted.

[0113] The daily dose of the therapeutic agent can be obtained from cell culture assays, animal studies, or clinical studies. A therapeutically effective amount of the therapeutic agent or active agent (such as an antibody binding to LILRB1 or its antigen-binding fragment) will be the amount by which the disease is treated in a subject, reducing the severity, frequency, and / or duration of one or more symptoms of the disease. Efficacy and administration can be determined by a healthcare professional or veterinary professional using methods known in the art and by observing one or more symptoms of the disease in the subject. Furthermore, it should be understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health condition, sex, and diet, the time of administration, route of administration, excretion rate, and the half-life in vivo of the antibody binding to LILRB1 or its antigen-binding fragment.

[0114] Example The invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0115] The materials used in the embodiments of this disclosure are known and commercially available.

[0116] Example 1: Reagent Generation 1.1 Reference Antibody Reference antibody 15G8 was generated according to patent WO 2021 / 028921 A1 (SEQ ID NO: 28 and 24). Hu5F9 (human IgG4 anti-CD47 antibody) was generated according to Jie Liu's paper (PLoS One [PLOS ONE]. Sep 21, 2015; 10(9):e0137345). The variable region sequences of 15G8 and Hu5F9 are shown in Table 1.

[0117] Table 1. Variable region sequences of 15G8 and Hu5F9

[0118] 1.2 Stable cell lines LakePharma produced a stable library of HEK293-LILRB1 cells expressing human LILRB1. The A375 / HLA-G monoclonal stable cell line was generated by Chempartner.

[0119] For K562 / SHP-1 + LILRB1 +Stable library generation, K562 cells were co-transfected with plasmids encoding non- tagged full-length human LILRB1 (c-terminally fused to a small beta-galactosidase fragment (ED) or with SHP-1 SH2 domain (protein tyrosine phosphatase 1 containing SH2 domain) with a complementary beta-galactosidase fragment (EA) and selectively cultured in medium containing 100 μg / mL hygromycin plus 2 μg / mL puromycin. For DLD-1 / beta2M stable library generation, DLD-1 cells were transfected with full-length beta-2-microglobulin (beta2M) expression plasmid and selectively cultured in medium containing 5 μg / mL puromycin.

[0120] 1.3 Recombinant proteins Human LILRB1 extracellular domain (ECD) recombinant protein with human Fc tag or 6xHis-tag (Q8NHL6, Gly24-His458) was purchased from R&D systems for immunization and hybridoma screening.

[0121] A series of recombinant proteins of LILRA / LILRB family members and potential ligands for LILRB1 were also purchased for in vitro assays. Human LILRA1 (NP_006854.1, Met1-Asn461), LILRA3 (AAH28208.1, Met1-Glu439), LILRA4 (P59901.2, Met1-Asn446), LILRA5 (NP_067073.1, Met1-Arg268), LILRB1 (ADJ55949.1, Met1-His458), LILRB2 (AAH36827.1, Met1-Val461), LILRB3 (AAI04994.1, Met1-Glu443), LILRB4 (AAI04994.1, Met1-Glu443), and LILRB5 (NP_006831.1, Met1-Gly458) ECD recombinant proteins with 6xHis-tag were purchased from Sino Biological. Human LILRA2 ECD (NP_001124389, Gly24-Asn449) recombinant protein with 6xHis-tag was purchased from R&D systems. Recombinant proteins of human HLA-A2 complex (MHC-HM431) and HLA-G complex tetramer (HLG-HM41CT) were purchased from Kactus.

[0122] Example 2: Hybridoma development and screening 2.1 Immunization and fusion Four mice from different strains (2 NZB / w and 2 C57BL / 6; SJL F1) were immunized with Fc-tagged human LILRB1 extracellular domain (ECD) recombinant protein using a rapid immunization strategy. The serum titers of the immunized mice were determined by enzyme-linked immunosorbent assay (ELISA) using 6xHis-tagged human LILRB1 ECD recombinant protein as antigen. When the serum titers reached high levels, a final boost was performed. Three days after the final boost, the pooled splenocytes and lymph node cells were harvested and fused with SP2 / 0 mouse myeloma cells. The fused cells were then seeded into 384-well plates for screening.

[0123] 2.2 Primary and secondary screening Ten to twelve days after fusion, the supernatants harvested from each well of the hybridoma cells were assayed by ELISA using 6xHis-tagged human LILRB1 ECD recombinant protein as antigen. The hybridoma cells in the positive wells were expanded into 24-well plates and the culture supernatants were subjected to secondary screening by fluorescence-activated cell sorting (FACS)-based binding assay using HEK293T-LILRB1 cells expressing human LILRB1 as target cells. Meanwhile, the culture supernatants were tested in ELISA using His-tagged LILRB1 / LILRB2 / LILRB3 / LILRB4 / LILRB5 ECD recombinant proteins as antigens to assess the binding specificity to LILRB1. The hybridoma cells secreting antibodies with the highest human LILRB1 -specific binding activity were then subcloned.

[0124] 2.3 Hybridoma subcloning and screening The selected hybridoma cells were limit-diluted into 96-well plates at a density of 1 cell / well to obtain monoclonal hybridoma cells. The supernatants harvested from these monoclonal cells were screened by the ELISA and FACS-based binding assay used in the primary and secondary screening. The antibodies secreted by the positive clones were then assayed for their activity in blocking the interaction of human LILRB1 with human leukocyte antigen-G (HLA-G) or human leukocyte antigen-A2 (HLA-A2) (refer to the method described in Section 3.2.4). As shown in Table 2, the monoclonal antibody secreted by clone 4F22G1 effectively blocked the interaction of human LILRB1 with human HLA-G or human HLA-A2.

[0125] Table 2. Blocking activity of clone 4F22G1

[0126] Example 3: Generation and characterization of chimeric antibodies 3.1 Generation of chimeric antibodies The heavy chain variable region and the light chain variable region of the monoclonal antibody secreted by clone 4F22G1 were sequenced. Based on the sequencing results, a human IgG4 chimeric antibody with Serine228Proline mutation in the Fc region was generated and named 82c, where the suffix "c" stands for chimeric. The standard one-letter amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, in which the CDRs are underlined and italicized.

[0127] VH: EVQLVESGGGLVKPGGSRKLSCAASGFTFS DYGMH WVRQAPEKGLEWVA YISSDSSIIFYTDTVK G RFTISRDNAKNTLFLQMTSLRSEDKAMYYCAR PTKWDVFVY WGQGTLVTVSA (SEQ ID NO: 7) VL: DIVMTQSQKFMSTSVGDRVSITC KASQNVRTAVA WYQQKPGQSPKALIY LASNRHT GIPDRFTGSGSGTDFTLTVNNVQSEDLADYFC LQHWNFPYT FGGGTKLEIK (SEQ ID NO: 8) 3.2 Characterization of the chimeric antibody 3.2.1 Binding activity The human LILRB1 binding activity of 82c was detected by FACS-based binding assay using K562 cells stably expressing human LILRB1 as target cells. 82c bound to membrane-bound human LILRB1 strongly in a dose-dependent manner (Figure 3A). Figure 1 ). The EC 50 and the top geometric mean fluorescence intensity (TOP MFI) were 2.40 nM and 12656, respectively, calculated using four-parameter nonlinear fitting by GraphPad Prism 9.0.

[0128] 3.2.2 Affinity detection The binding affinity and kinetics of 82c and benchmark antibody 15G8 to human LILRB1 were determined using bio-layer interferometry (Octet). The association and dissociation curves were fitted with a 1 : 1 binding model, and the association rate constant (Ka), dissociation rate constant (Kd), and equilibrium dissociation constant (KD) were calculated and summarized in Table 3. 82c (KD = 0.21 nM) showed higher human LILRB1 binding affinity than 15G8 (KD = 5.53 nM).

[0129] Table 3. Table 3. Human LILRB1 binding affinity of 82c and 15G8

[0130] 3.2.3 Binding selectivity The binding selectivity of 82c and benchmark antibody 15G8 to LILRA / LILRB family members was evaluated by ELISA using LILRA / LILRB ECD recombinant proteins as antigens. Briefly, anti-LILRB1 antibodies (50 μl, 100 nM) were incubated for 1 hour at 37°C in human LILRA / LILRB ECD recombinant protein-coated ELISA plates. After washing, horseradish peroxidase (HRP)-labeled detection antibodies (50 μl) were added and incubated for 1 hour at 37°C. Color development was performed by adding 100 μl / well of TMB (tetramethylbenzidine) solution. After incubation at room temperature (RT) for 10-15 minutes, the reaction was stopped by adding 50 μl of 1 N HCI. The plates were then immediately read at 450 nm for optical density (OD450) using a microplate reader, shown in Table 4. 82c did not bind to LILRA / LILRB family members other than human LILRB1, showing good binding specificity, while 15G8 cross-reacted with human LILRA2 and LILRA3.

[0131] Table 4. Binding of 82c and 15G8 to LILRA / LILRB family members

[0132] 3.2.4 Blocking activity Classical and non-classical MHC class I molecules are important ligands for LILRB1 in specific biological contexts. The blocking activity of 82c and benchmark antibody 15G8 to block the interaction of human LILRB1 with either the non-classical MHC class I molecule human HLA-G or the classical MHC class I molecule human HLA-A2 was evaluated by a FACS-based competition assay. Briefly, HEK293-LILRB1 cells expressing human LILRB1 were pre-incubated with 5, 1, 0.2, or 0.04 μg / mL of 82c or 15G8 for 30 minutes. Recombinant proteins of human HLA-G complex tetramer (1 μg / mL) or HLA-A2 complex (30 μg / mL) were then added and incubated for another 30 minutes. Blocking activity was determined by quantifying the blockade of HLA-G complex tetramer or HLA-A2 complex recombinant protein binding to HEK293-LILRB1 cells. As shown in Table 5, 82c and 15G8 effectively blocked the interaction of human LILRB1 with human HLA-G or human HLA-A2 in a dose-dependent manner.

[0133] Table 5. Blocking activity of 82c and 15G8

[0134] 3.2.5 NK92 / CD16a killing assay The effect of 82c and 15G8 on enhancing NK cell killing of tumor cells was evaluated by an in vitro NK killing assay using NK92 / CD16a cells as effector cells and A375 / HLA-G cells expressing HLA-G as target cells. Briefly, NK92 / CD16a cells were pre-incubated with test antibodies for 20 min at room temperature, then co-cultured with CellTrace violet-labeled A375 / HLA-G cells at an E / T (effector cell / target cell) ratio of 4: 1 for 4 h at 37°C. Propidium iodide (PI) is a DNA-binding dye and does not penetrate the membrane of living cells. Therefore, killing of target cells by effector cells in the presence of test antibodies can be detected by PI staining. Specific cytotoxicity induced by each test antibody was determined by quantifying the percentage of PI-labeled cells (dead cells) among total cells (CellTrace violet-labeled cells) by flow cytometry. + As shown in Figure 6, 82c and 15G8 effectively enhanced the killing of A375 / HLA-G cells by NK92 / CD16a cells at 100 nM and 2 nM. And at 2 nM, 82c showed better therapeutic effect than 15G8. This result indicates that blocking LILRB1 with 082c can effectively enhance NK cell-mediated destruction of tumor cells expressing HLA-G. Figure 2

[0135] 3.2.6 Phagocytosis assay of macrophages The effect of 82c and benchmark antibody 15G8 on enhancing phagocytosis of DLD-1 / β2M cells by macrophages opsonized with human IgG4 anti-SIRPa chimeric antibody 025c (VH as set forth in SEQ ID NO: 5, and VL as set forth in SEQ ID NO: 6) was evaluated by a FACS-based phagocytosis assay. Briefly, human monocyte-derived macrophages (hMDM) from a healthy donor were labeled with CellTrace Far Red, then co-cultured with CellTrace Violet-labeled DLD-1 / β2M cells in the presence of test antibodies for 2 h at 37°C. CellTrace Far Red and CellTrace Violet double-positive cells are macrophages that have phagocytosed target tumor cells. Therefore, the percentage of this macrophage population among total macrophages (CellTrace Far Red + ​as phagocytosis index to quantify the activity of each sample to enhance macrophage phagocytosis. As shown in Figure 3 Figure 82c effectively enhanced hMDM phagocytosis of SIRPa- opsonized DLD-1 / 2M cells, with potency superior to 15G8 at 10 nM. Neither 82c nor 15G8 had an effect on unopsonized DLD-1 / 2M cells. This result indicates that blocking LILRB1 with 82c can synergize with CD47 / SIRPa“do not eat me” signal inhibitors to enhance macrophage phagocytosis of tumor cells.

[0136] Example 4: Humanization of Antibodies 4.1 Humanization Design 82c was humanized using the complementarity determining region (CDR) grafting method. Briefly, IGHV3-21*05 (IMGT allele name, 81.6% homology) and IGKV1-17*02 (62.1% homology) were chosen as the humanization templates for the heavy and light chains, respectively, based on their homology to the original mouse antibody sequences. CDRs were defined using the Kabat definition except for the heavy chain CDR1 which was defined using a combination of the Kabat and IMGT systems. To graft, different combinations of CDRs and canonical residues from 82c were grafted onto the templates, resulting in 16 variants (human IgG1 LALA, human IgG1 with Leu234Ala and Leu235Ala mutations in the Fc region) for further characterization. These variants were designated as hu082.01 to hu082.16, where the prefix “hu” stands for “humanized” and the number in the suffix represents the serial number. The VH and VL of hu082.01 to hu082.16 are shown below.

[0137]

[0138] 4.2 Characterization of Humanized Variants 4.2.1 Binding Activity The human LILRB1 binding activity of 82c and 82c-derived humanized variants was evaluated by FACS-based binding assay using K562 cells stably expressing human LILRB1 as target cells. As shown in Figure 4A and Figure 4B All humanized variants were confirmed to retain similar activity to their parental antibody 82c in binding to membrane-bound human LILRB1. Table 6 summarizes the EC 50 and TOP MFI values calculated by GraphPad Prism 9.0 using four-parameter nonlinear fitting.

[0139] Table 6. Table 6. Binding activity of 82c and 82c-derived humanized variants

[0140] 4.2.2 Affinity Testing Single-dose affinity ranking tests were performed on the 82c-derived humanized variants using surface plasmon resonance (SPR) technology. Association and dissociation curves were fitted using a 1:1 binding model, and the Ka, Kd, ​​and KD values ​​were calculated and summarized in Table 7. All tested variants performed comparably in the ranking tests.

[0141] The binding affinity and kinetics of hu082.02, hu082.03, and hu082.06 to human LILRB1 were further determined using SPR technology through full-dose testing. As shown in Table 8, the humanized variants of the three tested variants were confirmed to retain antigen-binding affinity similar to their parent antibody 82c.

[0142] Table 7. Binding affinity ranking of 82c and humanized variants derived from 82c

[0143] Table 8. Binding kinetics of 82c, hu082.02, hu082.03 and hu082.06 with human LILRB1

[0144] 4.2.3 Blocking activity The activity of hu082.02, hu082.03, and hu082.06 in blocking the interaction between human LILRB1 and human HLA-G tetramer was assessed by FACS-based competitive assays (refer to the method described in Section 3.2.4). The three tested humanized variants showed comparable activity to their parent antibody 82c in blocking the binding of the recombinant human HLA-G complex tetramer to HEK293-LILRB1 cells. Figure 5 ).

[0145] The activity of hu082.03 in blocking the interaction between human LILRB1 and human HLA-A2 was assessed by FACS-based competitive assay (the final concentration of the recombinant human HLA-A2 complex was adjusted to 10 µg / mL, following the method described in Section 3.2.4). hu082.03 effectively blocked the binding of the recombinant human HLA-A2 complex to HEK293-LILRB1 cells in a dose-dependent manner. Figure 6 IC 50 The highest blocking rates were 1.45 nM and 99.4%, respectively, calculated using a four-parameter nonlinear fitting method with GraphPad Prism 9.0.

[0146] Following binding to LILRB1, MHC class I molecule-induced LILRB1 signaling is transmitted via phosphorylation of a tyrosine-based immunoreceptor inhibitory motif located on the cytoplasmic tail region of LILRB1. Subsequent binding and activation of SHP-1 and SHP-2 (protein tyrosine phosphatase 2 containing the SH2 domain) blocks the tyrosine phosphorylation-dependent signaling pathway. Based on this signal transduction mechanism, a reporter gene assay, namely the LILRB1 / SHP-1 recruitment assay, was developed to assess the neutralizing activity of anti-LILRB1 antibodies against MHC class I molecule-induced LILRB1 signaling. Figure 7 In short, full-length human LILRB1 was engineered with a small β-galactosidase fragment (ED) fused to its C-terminus, and the SH2 domain of SHP-1 was engineered with a complementary β-galactosidase fragment (EA). These constructs were stably expressed in human K562 cells, generating K562 / SHP-1. + LILRB1 + Cells. By co-culturing with cells expressing human HLA-G or classical MHC class I molecules, ligand binding leads to phosphorylation of the LILRB1-ED fusion protein, inducing the recruitment of SHP-1-EA, thereby compelling the production of an active β-galactosidase. This active enzyme then hydrolyzes the substrate to produce chemiluminescence as a measure of reporter gene activity in MHC class I molecule-induced LILRB1 signaling.

[0147] In the LILRB1 / SHP-1 recruitment assay, K562 / SHP-1 was detected in the presence of the test antibody. + LILRB1 + Cells were co-cultured overnight at 37°C with A375 / HLA-G cells or Raji cells. The next day, Gal selection substrate (Applied Biosystems) was added, and the cells were incubated at 28°C for approximately 60 minutes. Chemiluminescence was then measured using a microplate reader to quantify reporter gene activity. Figure 8 As shown in Figure A, hu082.03 and 15G8 effectively interfered with the recruitment of SHP-1 to the intracellular tail region of LILRB1 induced by classical MHC class I molecules (Raji), with comparable activities. The highest blocking rates were 94.23% and 84.69%, respectively. However, 15G8 was less active than hu082.03 in blocking HLA-G (A375 / HLA-G)-induced SHP-1 recruitment to the intracellular tail region of LILRB1. Figure 8 B). The highest blocking rates were 100% and 66.2%, respectively. The ICs shown in Table 9... 50 The values ​​and maximum blocking rates were calculated using a four-parameter nonlinear fit with GraphPad Prism 9.0.

[0148] Table 9. Blocking activity of hu082.03 in LILRB1 / SHP-1 recruitment assay

[0149] 4.2.4 NK92 / CD16a killing assay The effect of hu082.02, hu082.03 and hu082.06 on enhancing NK cell killing of tumor cells was assessed by in vitro NK killing assays (refer to the method described in section 3.2.5). As shown in Figure 4.2.4, it was confirmed that the three tested humanized variants retained similar activity to their parental antibody 82c in enhancing NK92 / CD16a killing of A375 / HLA-G cells. Figure 9 EC50values of 82c, hu082.02, hu082.03 and hu082.06 were 0.90 nM, 1.31 nM, 1.38 nM and 1.30 nM, respectively. The highest cytotoxicity values were 57.01%, 55.78%, 57.02% and 56.74%, respectively. EC50and highest cytotoxicity values were calculated by GraphPad Prism 9.0 using four-parameter nonlinear fitting. 50 50

[0150] 4.2.5 Phagocytosis assay of macrophages The effect of hu082.02, hu082.03 and hu082.06 on enhancing hMDM phagocytosis of 025c opsonized DLD-1 / β2M cells was assessed by FACS-based phagocytosis assay (refer to the method described in section 3.2.6). As shown in Figure 4.2.5, it was confirmed that the three tested humanized variants retained similar activity to their parental antibody 82c in enhancing hMDM phagocytosis of 025c opsonized DLD-1 / β2M cells. Figure 10

[0151] Hu082.03 was also tested in FACS-based phagocytosis assay using A375 / HLA-G cells expressing HLA-G as target cells. Briefly, hMDM from healthy donors (Donor 1 and Donor 2) were labeled with CellTrace FarRed and then co-cultured with CellTrace Violet labeled A375 / HLA-G cells in the presence of test antibodies at 37°C for 2 hours. The percentage of CellTrace Far Red and CellTrace Violet double macrophages in total macrophages (CellTrace Far Red + ) was calculated as phagocytosis index to quantify the activity of each sample in enhancing macrophage phagocytosis. As shown in Figure 4.2.6, it was confirmed that hu082.03 retained similar activity to its parental antibody 82c in enhancing hMDM phagocytosis of A375 / HLA-G cells.​​​Figure 11 As shown, hu082.03 potently and dose-dependently enhanced hMDM phagocytosis of Hu5F9 (human IgG4 anti-CD47 antibody) opsonized A375 / HLA-G cells. Hu082.03 had no effect on unopsonized A375 / HLA-G cells.

[0152] While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are or can be presently unforeseen can arise to applicants or others skilled in the art. Therefore, the appended claims as filed and as they can be amended during the pendency of this application (including the submission of any subsequent continuation, continuation-in-part, division, or equivalent application) are intended to cover all such alternatives, modifications variations, improvements, and substantial equivalents falls within the scope of the disclosure.

Claims

1. An antibody or antigen-binding fragment thereof that binds to LILRB1, comprising: a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, which HCDR1 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 17, which HCDR2 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 18, which HCDR3 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, which LCDR1 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 20, which LCDR2 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 21, which LCDR3 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO:

22.

2. The antibody or antigen-binding fragment thereof that binds to LILRB1 of claim 1, wherein the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 17, 18, and 19, respectively; and the LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively.

3. The antibody or antigen-binding fragment thereof that binds to LILRB1 of claim 1 or 2, wherein the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 7; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 8; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 9; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 13; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 9; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 14; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 9; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 15; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 9; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 16; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 10; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 13; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 10; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 15; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 10; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 15; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 10; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 16; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 11; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 13; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 11; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 14; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 11; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 16; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 11; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 16; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 12; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 13; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 12; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 14; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 12; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 15; the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO: 12; and the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence of SEQ ID NO:

16.

4. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-3, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 11, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 15; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 11, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 16; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 13; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 14; the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 15; or the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 12, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:

16.

5. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances killing activity of an immune cell, optionally, the immune cell comprises an NK cell and a macrophage.

6. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances destruction of a cell expressing an MHC class I molecule, optionally, the MHC class I molecule comprises HLA-G, b2M, HLA-A2, or a combination thereof.

7. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances NK cell-mediated destruction of a cell expressing HLA-G.

8. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof that binds to LILRB1 enhances phagocytosis of a macrophage of a cell expressing an MHC class I molecule opsonized by an immune checkpoint-related antibody.

9. The antibody or antigen-binding fragment thereof that binds to LILRB1 of claim 8, wherein the immune checkpoint-related antibody binds to an immune checkpoint molecule, optionally, the immune checkpoint comprises SIRPa, CD47, or a combination thereof.

10. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof that binds to LILRB1 improves an immune response or reduces immune suppression.

11. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof that binds to LILRB1 does not or does not substantially bind to LILRB2, LILRB3, LILRB4, LILRB5, LILRA1, LILRA2, LILRA3, LILRA4, or LILRA5, or a combination thereof.

12. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof that binds to LILRB1 blocks the interaction of LILRB1 with its ligand, optionally, the ligand comprises human HLA-G, human HLA-A2, or a combination thereof.

13. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof that binds to LILRB1 binds to LILRB1 with an EC 50 no more than 10 nM, 8 nM, or 5 nM, or an EC 50 no more than 2 pg / ml, 1 pg / ml, or 0.6 pg / ml.

14. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-13, wherein the antibody or antigen-binding fragment thereof that binds to LILRB1 has a binding affinity KD (affinity constant) to LILRB1 of no more than 20 nM, 15 nM, or 10 nM.

15. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-14, wherein the antibody or antigen-binding fragment thereof that binds to LILRB1 comprises an effector molecule-binding fragment, optionally, the effector molecule-binding fragment is an Fc fragment.

16. The antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-15, wherein the Fc fragment is derived from human IgGl or IgG4, optionally, the Fc fragment is derived from human IgGl comprising the mutations Leu234Ala, Leu235Ala, or a combination thereof.

17. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-16.

18. An isolated vector comprising the isolated polynucleotide of claim 17.

19. A host cell comprising the isolated polynucleotide of claim 17 or the isolated vector of claim 18.

20. A kit comprising the antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-16.

21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-16, the isolated polynucleotide of claim 17, the isolated vector of claim 18, or the host cell of claim 19, and a pharmaceutically acceptable carrier.

22. Use of the antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-16, the isolated polynucleotide of claim 17, the isolated vector of claim 18, the host cell of claim 19, the kit of claim 20, or the pharmaceutical composition of claim 21 in the manufacture of a therapeutic agent for the diagnosis, prevention, or treatment of a neoplastic disease.

23. The use of claim 22, wherein the neoplastic disease is a solid tumor.

24. The use of claim 22 or 23, wherein the neoplastic disease comprises chronic myeloid leukemia, colorectal adenocarcinoma, breast cancer, carcinoid, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, hematological malignancy, melanoma or metastatic melanoma, non-small cell lung cancer, small cell lung cancer, bladder cancer, or metastatic hormone-refractory prostate cancer.

25. A combination comprising the antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-16, and a second therapeutic agent for treating a neoplastic disease in a subject, wherein the second therapeutic agent is selected from one or more of the following: an inhibitor of an inhibitory molecule, an activator of a costimulatory molecule, chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, a vaccine, or a cellular immunotherapy.

26. The combination of claim 25, wherein the second therapeutic agent is an antibody or antigen-binding fragment that binds to at least one immune checkpoint molecule, optionally CD47, SIRPa.

27. A method for treating a subject having a neoplastic disease, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof that binds to LILRB1 of any one of claims 1-16, the isolated polynucleotide of claim 17, the isolated vector of claim 18, the host cell of claim 19, the kit of claim 20, or the pharmaceutical composition of claim 21.

28. The method of claim 27, wherein the immune response of the subject is improved by enhancing: (1) immune cell killing activity; (2) destruction of cells expressing MHC class I molecules; and / or (3) phagocytosis of cells expressing MHC class I molecules by macrophages.

29. The method of claim 28, wherein the method further comprises administering an antibody that binds to at least one immune checkpoint molecule, optionally CD47, SIRPa.

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