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WO2024260384A1 - Anticorps ou fragments de liaison à l'antigène se liant à lilrb1 et leurs utilisations - Google Patents

Anticorps ou fragments de liaison à l'antigène se liant à lilrb1 et leurs utilisations Download PDF

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WO2024260384A1
WO2024260384A1 PCT/CN2024/100199 CN2024100199W WO2024260384A1 WO 2024260384 A1 WO2024260384 A1 WO 2024260384A1 CN 2024100199 W CN2024100199 W CN 2024100199W WO 2024260384 A1 WO2024260384 A1 WO 2024260384A1
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seq
amino acid
acid sequence
variable region
chain variable
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Xiaofeng NIU
Haixia JIANG
Dawei Sun
Chunnian WANG
Hongtao Lu
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Elpiscience Biopharma Ltd
Elpiscience Suzhou Biopharma Ltd
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Elpiscience Biopharma Ltd
Elpiscience Suzhou Biopharma Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates to the antibodies or antigen-binding fragments binding to LILRB1, polynucleotides, vectors, host cells, and pharmaceutical compositions thereof.
  • the present disclosure also relates to the uses of the antibodies or antigen-binding fragments.
  • Immune checkpoint molecules including inhibitory and stimulatory immune checkpoint molecules, are defined as ligand-receptor pairs that exert inhibitory or stimulatory effects on immune responses. Most of the immune checkpoint molecules that have been described so far are expressed on cells of the adaptive immune system, particularly on T cells, and of the innate immune system. They are crucial for maintaining the self-tolerance and modulating the length and magnitude of immune responses of effectors in different tissues to minimize the tissue damage. More and more evidences have shown that inhibitory or stimulatory immune checkpoint molecules are expressed on a sizeable fraction of tumor types (Zhang Y, Zheng J. Adv Exp Med Biol. 2020; 1248: 201-226. ) . Current immune checkpoint blockade strategies have been a promising cancer treatment. When properly stimulated, effector cells of both the innate immune system and adaptive immune system possess the ability to attack cancer cells (Barkal AA, et al., Nat Immunol. 2018 Jan; 19 (1) : 76-84. ) .
  • LILRB1 leukocyte immunoglobulin like receptor B1 , also known as ILT2, is a member of the leukocyte immunoglobulin-like receptor (LIR) family.
  • LILRB1 is an immunoreceptor tyrosine-based inhibitory motif-containing receptor, and widely expressed on human immune cells, including B cells, monocytes and macrophages, dendritic cells and subsets of natural killer (NK) cells and T cells.
  • the ligands of LILRB1, such as major histocompatibility complex (MHC) class I molecules activate LILRB1 and transduce a suppressive signal, which inhibits the immune responses (Chen H, Chen Y, et ai., J Immunother Cancer. 2020 Aug; 8 (2) : e000515. ) .
  • MHC major histocompatibility complex
  • the present disclosure provides a novel antibody binding to LILRB1 or antigen-binding fragment thereof, which could improve immune response or decrease immune inhibition by blocking LILRB1 signaling pathway, thus the antibody binding to LILRB1 or antigen-binding fragment thereof can ameliorate the neoplastic disease.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof 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,
  • the 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,
  • the 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,
  • the 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 S
  • HCDR1, HCDR2 and HCDR3 comprise amino acid sequences as shown in SEQ ID NOs: 17, 18 and 19, respectively;
  • LCDR1, LCDR2 and LCDR3 comprise amino acid sequences as shown in SEQ ID NOs: 20, 21 and 22, respectively.
  • the heavy chain variable region and the light chain variable region comprise the amino acid sequences as shown in SEQ ID NOs: 7-8, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences as shown in SEQ ID NOs: 9 and 13, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences as shown in SEQ ID NOs: 9 and 14, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences as shown in SEQ ID NOs: 9 and 15, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences as shown in SEQ ID NOs: 9 and 16, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences as shown in SEQ ID NOs: 10 and 13, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences as shown in SEQ ID NOs: 10 and 14, respectively; the heavy chain variable region and the light chain variable region comprise the amino acid sequences as shown in SEQ ID NOs: 10 and 15, respectively; the heavy
  • the antibody binding to LILRB1 or antigen-binding fragment thereof potentiates immune cells (e.g., NK cells and macrophage cells) killing activity, such as, NK cell-mediated destruction of unexpected cells, macrophage cells phagocytosis of unexpected cells.
  • immune cells e.g., NK cells and macrophage cells
  • the antibody binding to LILRB1 or antigen-binding fragment thereof potentiates destruction of MHC class I molecule expressing cells, optionally, MHC class I molecule includes HLA-G, ⁇ 2M, HLA-A2 or combination thereof.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof potentiates NK cell-mediated destruction of HLA-G expressing cells.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof potentiated macrophage cells phagocytosis of immune checkpoint related antibody (e.g., anti-SIRP ⁇ antibody, anti-CD47 antibody) opsonized MHC class I molecule expressing cells.
  • immune checkpoint related antibody e.g., anti-SIRP ⁇ antibody, anti-CD47 antibody
  • the antibody binding to LILRB1 or antigen-binding fragment thereof doesn’t bind to or barely binds to LILRB (e.g., LILRB2, LILRB3, LILRB4, LILRB5, LILRB1 excluded) and LILRA (LILRA1, LILRA2, LILRA3, LILRA4, LILRA5) family.
  • LILRB e.g., LILRB2, LILRB3, LILRB4, LILRB5, LILRB1 excluded
  • LILRA LILRA1, LILRA2, LILRA3, LILRA4, LILRA5 family.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof blocks the interaction of LILRB1 and ligands thereof (such as human HLA-G, human HLA-A2) .
  • the antibody binding to LILRB1 or antigen-binding fragment thereof comprises an effector molecule binding fragment (e.g., Fc fragment) .
  • An isolated polynucleotide encoding the antibody binding to LILRB1 or antigen-binding fragment thereof provided above, an isolated vector comprising the polynucleotide, and a host cell comprising the isolated polynucleotide, or the isolated vector are also provided.
  • the present disclosure provides a kit comprising the antibody binding to LILRB1 or antigen-binding fragment thereof described above.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody binding to LILRB1 or antigen-binding fragment thereof, the isolated polynucleotide, the isolated vector, or the host cell, and a pharmaceutically acceptable carrier.
  • neoplastic disease provides herein is the use of the antibody binding to LILRB1 or antigen-binding fragment thereof, 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 neoplastic disease.
  • a combination comprising the antibody binding to LILRB1 or antigen-binding fragment thereof described above, and a second therapeutic agent for use in treating a neoplastic disease in a subject, wherein the second therapeutic agent is chosen from one or more of: an inhibitor of an inhibitory molecule, an activator of a costimulatory molecule, a chemotherapy, a targeted anticancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, a vaccine, or a cellular immunotherapy.
  • a method for treating a subject having a neoplastic disease comprising administrating to the subject a therapeutically effective amount of the antibody binding to LILRB1 or antigen-binding fragment thereof, the isolated polynucleotide, the isolated vector, the host cell or the pharmaceutical composition.
  • Figure 1 shows the binding of 82c and human IgG4 isotype control (hIgG4 iso) to human LILRB1-expressing K562/SHP-1 + LILRB1 + cells in a FACS-based binding assay.
  • Figure 2 shows the effect of 82c, 15G8 and isotype control (hIgG4 iso) on potentiating NK92/CD16a cells killing of A375/HLA-G cells.
  • the cytotoxicity index values obtained under the indicated treatments are listed at the top of each column.
  • Figure 3 shows the effect of 15G8, 82c and isotype control (hIgG4 iso) on potentiating macrophage phagocytosis of DLD-1/ ⁇ 2M cells.
  • the phagocytosis index values obtained under the indicated treatments are listed at the top of each column.
  • Figure 4A and 4B show the binding of 82c, 82c derived humanized variants and human IgG1LALA isotype control (hIgG1LALA iso) to human LILRB1-expressing K562/SHP-1 + LILRB1 + cells in a FACS-based binding assay.
  • Figure 5 shows the activity of 82c, hu082.02, hu082.03, hu082.06, hIgG4 iso and hIgG1LALA iso to block the interaction of human HLA-G with human LILRB1 in a FACS-based competition assay.
  • the blocking ratios obtained under the indicated treatments are listed at the top of each column.
  • Figure 6 shows the activity of hu082.03 and hIgG1LALA iso to block the interaction of human HLA-A2 with human LILRB1 in a FACS-based competition assay.
  • Figure 7 shows the principle of LILRB1/SHP-1 recruitment assay.
  • Figure 8 shows the activity of hu082.03, 15G8, hIgG4 iso and hIgG1LALA iso to block MHC class I-induced LILRB1 downstream signals in LILRB1/SHP-1 recruitment assays using Raji cells (A) or A375/HLA-G cells (B) as stimulator cells.
  • Figure 9 shows the effect of 82c, hu082.02, hu082.03, hu082.06, hIgG4 iso and hIgG1LALA iso on potentiating NK92/CD16a cells killing of A375/HLA-G cells.
  • Figure 10 shows the effect of 82c, hu082.02, hu082.03, hu082.06, hIgG4 iso and hIgG1LALA iso on potentiating macrophage phagocytosis of DLD-1/ ⁇ 2M cells.
  • the phagocytosis index values obtained under the indicated treatments are listed at the top of each column.
  • Figure 11 shows the effect of hu082.03, hIgG4 iso and hIgG1LALA iso on potentiating macrophage phagocytosis of A375/HLA-G cells.
  • the phagocytosis index values obtained under the indicated treatments are listed at the top of each column.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof 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,
  • the 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,
  • the 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,
  • the HCDR3 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%identical to the amino acid sequence as
  • the CDRs are defined using Kabat definition except HCDR1, which is defined using a combination of Kabat and IMGT systems.
  • antibody binding to LILRB1 or antigen-binding fragment thereof as it is used herein with respect to the disclosure includes an isolated, recombinant or synthetic antibody, antibody conjugate or antibody derivative.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the amino acid composition and order of arrangement of the immunoglobulin heavy chain constant regions are different, so their antigenicity is also different.
  • immunoglobulins can be classified into five types, or known as isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain and ⁇ chain, respectively.
  • IgG can be classified into IgG1, IgG2, IgG3 and IgG4.
  • the light chain is classified into ⁇ chain or ⁇ chain according to the difference in the constant region.
  • Each of the five types of Ig can have ⁇ chain or ⁇ chain.
  • antigen-binding fragment refers to antigen-binding fragment of an antibody and antibody analog, which usually comprises at least part of the antigen-binding region or variable region (for example one or more CDRs) of the parental antibody.
  • the antibody fragment retains at least some of the binding specificity of the parental antibody. Generally, when the activity is represented on a mole basis, the antibody fragment retains at least 10%of the parental binding activity. 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.
  • antigen-binding fragments include, but are not limited to: Fab, Fab’ , F (ab’ ) 2, Fv fragment, linear antibody, single-chain antibody, nanobody, domain antibody and multispecific antibody.
  • Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23: 1126-1136.
  • Heavy chain variable region or “VH” with regard to an antibody refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.
  • light chain variable region or “VL” with regard to an antibody refers to the fragment of the light chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.
  • the identity percentage of two amino acid sequences is determined by dividing the number of the same residues by the total number of the amino acid residues and multiplying the quotient by 100 to obtain a percentage. Gaps are excluded when assessing identity. Therefore, two copies of completely identical sequences have 100%identity, but sequences with deletion, addition or replacement may have a lower degree of identity.
  • a person 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 programs using algorithms such as BLAST. BLAST nucleotide search is performed using the NBLAST program, and BLAST protein search is performed using the BLASTP program, and default parameters of each program are used.
  • Kabat and IMGT are known to these skilled in the art, see, for example, Kabat E A, Wu T T, Perry H M, et al., Sequence of Proteins of Immunological Interest [J] . 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 (1) : 55-77.
  • the CDRs of the antibody binding to LILRB1 or antigen-binding fragment thereof could contain mutation, which may be selected from an insertion, a deletion, and/or a substitution; the substitution is preferably a substitution of conserved amino acids.
  • conserved amino acid generally refers 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) . conserveed substitutions may be naturally occurring or may be introduced for example using mutagenesis (e.g., Hutchinson et al., 1978, J. Biol. Chem. 253: 6551) .
  • the amino acids glycine, alanine, valine, leucine and isoleucine, for example, can often be substituted for one another (amino acids having aliphatic side chains) .
  • glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic) .
  • Other amino acids which may often be substituted for one another include but are not limited to, phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains) ; lysine, arginine and histidine (amino acids having basic side chains) ; aspartate and glutamate (amino acids having acidic side chains) ; and asparagine and glutamine (amino acids having amide side chains) .
  • the HCDR1, HCDR2 and HCDR3 comprise amino acid sequences as shown in SEQ ID NOs: 17, 18 and 19 with no more than 3, 2, 1 amino acid deletion, insertion and/or substitution, respectively;
  • LCDR1, LCDR2 and LCDR3 comprise amino acid sequences as shown in SEQ ID NOs: 20, 21 and 22 with no more than 3, 2, 1 amino acid deletion, insertion and/or substitution, respectively.
  • HCDR1, HCDR2 and HCDR3 comprise amino acid sequences as shown in SEQ ID NOs: 17, 18 and 19 with no more than 3, 2, 1 amino acid conservative substitution, respectively;
  • LCDR1, LCDR2 and LCDR3 comprise amino acid sequences as shown in SEQ ID NOs: 20, 21 and 22 with no more than 3, 2, 1 amino acid conservative substitution, respectively.
  • HCDR1, HCDR2 and HCDR3 comprise amino acid sequences as shown in SEQ ID NOs: 17, 18 and 19, respectively; and LCDR1, LCDR2 and LCDR3 comprise amino acid sequences as shown in SEQ ID NOs: 20, 21 and 22, respectively.
  • 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 as shown in 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 as shown in 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 as shown in 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 as shown in SEQ ID NO: 13; the heavy chain variable variable
  • the heavy chain variable region and the light chain variable region of the antibody binding to LILRB1 or antigen-binding fragment thereof also could contain mutation, which may be selected from an insertion, a deletion, and/or a substitution; the substitution is preferably a substitution of conserved amino acids.
  • the heavy chain variable region and the light chain variable region contains one or more mutation in framework region, optionally, the mutation do not significantly affect the 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 contains one or more conserved amino acids.
  • the heavy chain variable region comprises or has an amino acid sequence as shown in SEQ ID NO: 7, and the light chain variable region comprises or has an amino acid sequence as shown in SEQ ID NO: 8; the heavy chain variable region comprises or has an amino acid sequence as shown in SEQ ID NO: 9, and the light chain variable region comprises or has an amino acid sequence as shown in SEQ ID NO: 13; the heavy chain variable region comprises or has an amino acid sequence as shown in SEQ ID NO: 9, and the light chain variable region comprises or has an amino acid sequence as shown in SEQ ID NO: 14; the heavy chain variable region comprises or has an amino acid sequence as shown in SEQ ID NO: 9, and the light chain variable region comprises or has an amino acid sequence as shown in SEQ ID NO: 15; the heavy chain variable region comprises or has an amino acid sequence as shown in SEQ ID NO: 9, and the light chain variable region comprises or has an amino acid sequence as shown in SEQ ID NO: 16; the heavy chain variable region comprises or has an amino acid sequence as shown in SEQ ID NO: 10, and the light chain variable region comprises or has
  • the antibody binding to LILRB1 or antigen-binding fragment thereof encompasses (unless where otherwise indicated or where otherwise suggested by context) a monoclonal antibody, a polyclonal antibody, a murine antibody, hamster antibody, goat antibody, rabbit antibody, a chimeric antibody, a primatized antibody, a humanized antibody, a (fully) human antibody, a multimeric antibody, a heterodimeric antibody, a hemidimeric antibody, a bi-, tri-, or tetravalent antibody, a bispecific antibody, a single chain antibody (e.g., scFv, scFab, and scFabAC) , Bis-scFv, a diabody, triabody or tetrabody, single domain antibodies, and modified Fab fragments.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof is monovalent.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof comprises an effector molecule binding fragment, such as a Fc fragment that is to communicate to 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 IgG molecule.
  • the Fc fragment is human IgG, e.g., IgG1, IgG2, IgG3, or IgG4, optionally, with one or more mutation compared to wildtype human IgG molecules.
  • Exemplary Fc fragment is human IgG4 with Serine228Proline mutation, which means Serine at position 228 (defined with Kabat) is mutated to Proline.
  • the Fc fragment is derived from human IgG1 or IgG4. In some embodiments, the Fc fragment is derived from human IgG1 with mutation Leu234Ala, Leu235Ala, or combination thereof.
  • chimeric antibodies include the heavy and/or light chain variable regions, including both CDR and framework residues, of one species (typically mouse) fused to constant regions of another species (typically human) .
  • Humanized antibodies typically include heavy and/or light chain CDRs from a murine antibody grafted into a non-human primate or human antibody variable region framework, usually further comprising a human constant region. See, e.g., Riechmann et al. (1988) Nature 332: 323-327.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof is generated by Selected 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 the isolation from any species of cells producing high affinity antibodies during in vivo immune responses.
  • SAM Selected Lymphocyte Antibody Method
  • B cells that are positive for antibodies to LILRB1 are isolated.
  • the B cells may be from human, mouse, rat, hamster, rabbit, goat, or other mammalian species.
  • the antibody genes in these B cells may be cloned and expressed in a host cell (e.g., E. coli. ) , e.g., by conventional recombinant DNA technology.
  • the antibodies expressed cells may be purified by conventional means. If the antibodies are from a non-human source, they may be humanized by conventional methods, such as by mutagenesis of their genes.
  • the humanized antibodies may be subsequently expressed in a host cell and may be purified.
  • Monoclonal antibodies may be prepared by any method known in the art such as the hybridoma technique (Kohler &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) .
  • the methods for creating and manufacturing recombinant antibodies are well known in the art (see for example, Simmons et al., 2002, Journal of Immunological Methods, 263, 133-147) .
  • Antibody binding to LILRB1 or antigen-binding fragment thereof of the present disclosure may also be generated using various phage display methods known in the art which include those 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.
  • transgenic mice e.g., genetically engineered mice, or other organisms, including other mammals, may be used to produce the antibody binding to LILRB1 or antigen-binding fragment thereof (see for example US 6, 300, 129) .
  • mice engineered to replace only the variable regions of mouse immune loci (heavy chain V, D, and J segments, and light chain V and J segments) with corresponding human variable sequences can be used to produce large quantities of high affinity antibodies with human variable sequences (see, e.g., US 6, 586, 251) .
  • the antibody binding to LILRB1 or antigen-binding fragment thereof described above can improves immune response or decrease immune inhibition, optionally by blockade of the LILRB1-related inhibitory signaling axis. Understandably, blockade of LILRB1 signaling in immune cells is capable of activating the activity of natural killer NK cells, T cells or macrophages. In some embodiments, the antibody binding to LILRB1 or antigen-binding fragment thereof blocks the activation of LILRB1 on NK cells. In some embodiments, the antibody binding to LILRB1 or antigen-binding fragment thereof blocks the activation of LILRB1 on macrophages.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof potentiates immune cells killing activity (or cytotoxic activity) , preferably, the immune cells are NK cells, and/or macrophage cells.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof blocks the interaction of LILRB1 and ligands thereof, such as major histocompatibility complex (MHC) class I molecules.
  • MHC major histocompatibility complex
  • the antibody binding to LILRB1 or antigen-binding fragment thereof blocks the interaction of LILRB1 and human leukocyte antigen-G (HLA-G) .
  • the antibody binding to LILRB1 or antigen-binding fragment thereof blocks the interaction of LILRB1 and human leukocyte antigen HLA-A2.
  • MHC class I molecule includes HLA-G, ⁇ 2M (Beta-2-microglobulin, a component of MHC class I) , HLA-A2, or combination thereof.
  • MHC class I molecule expressing cells are undesired or unexpected, such as tumor cells, HLA-G expressing tumor cells.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof potentiates NK cell-mediated destruction of HLA-G expressing cells.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof potentiates macrophage cell-mediated phagocytosis of MHC class I molecule expressing cells. In some embodiments, the antibody binding to LILRB1 or antigen-binding fragment thereof potentiated macrophage cells phagocytosis of immune checkpoint related antibody opsonized MHC class I molecule expressing cells, such as ⁇ 2M expressing cells, HLA-G expressing cells.
  • the immune checkpoint related antibody binds to immune checkpoint molecules, such as signal regulatory protein alpha (SIRP ⁇ ) , CD47 molecule, CD27 molecule, CD40 molecule, CD137 molecule, programmed cell death protein 1 (PD-L1) , Programmed Death 1 receptor (PD-1) , tumor necrosis factor receptor superfamily, member 4 (OX4) , cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) , or inducible T-cell costimulatory (ICOS) .
  • SIRP ⁇ signal regulatory protein alpha
  • PD-L1 programmed cell death protein 1
  • PD-1 Programmed Death 1 receptor
  • OF4 tumor necrosis factor receptor superfamily
  • CTLA-4 cytotoxic T-Lymphocyte-Associated protein 4
  • ICOS inducible T-cell costimulatory
  • the immune checkpoint is signal SIRP ⁇ . In some embodiments, the immune checkpoint is CD47.
  • SIRP ⁇ 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. SIRP ⁇ recognizes the ubiquitously expressed ‘Don’ t Eat Me’s ignal molecule CD47, and antibody blockade of either CD47 or SIRP ⁇ could enhance the activity of SIRP ⁇ expressing immune cells, such as macrophages.
  • SIRP signal-regulatory-protein
  • the antibody binding to LILRB1 or antigen-binding fragment thereof potentiated macrophage cells phagocytosis of anti-SIRP ⁇ antibody or antigen binding fragment opsonized MHC class I molecule expressing cells. In some embodiments, the antibody binding to LILRB1 or antigen-binding fragment thereof potentiated macrophage cells phagocytosis of anti-CD47 antibody or antigen binding fragment opsonized MHC class I molecule expressing cells. In some embodiments, the antibody binding to LILRB1 or antigen-binding fragment thereof potentiated macrophage cells phagocytosis of anti-SIRP ⁇ antibody or antigen binding fragment opsonized ⁇ 2M expressing cells. In some embodiments, the antibody binding to LILRB1 or antigen-binding fragment thereof potentiated macrophage cells phagocytosis of anti-CD47 antibody or antigen binding fragment opsonized HLA-G expressing cells.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof specifically binds to LILRB1.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof doesn’ t bind to or barely binds to one or more other LILRB family members, such as LILRB2, LILRB3, LILRB4 and LILRB5.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof doesn’ t bind to or barely binds to one or more LILRA family members, optionally, LILRA family members contain LILRA1, LILRA2, LILRA3, LILRA4, and LILRA5.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof doesn’ t bind to or barely binds to LILRB2, LILRB3, LILRB4, LILRB5, LILRA1, LILRA2, LILRA3, LILRA4, or LILRA5, or combination thereof.
  • the antibody binding to LILRB1 or antigen-binding fragment thereof binds to LILRB1 with EC 50 no more than 10 nM, 8 nM or 5 nM, or with EC 50 no more than 2 ⁇ g/ml, 1 ⁇ g/ml, or 0.6 ⁇ g/ml.
  • the EC 50 could be measured by the well-known method in the art, such as FACS assay, competitive FACS.
  • the KD could be measured by the well-known method in the art, such as Bio-Layer Interferometry (Octet) , surface plasmon resonance (SPR) technique.
  • the present disclosure provides an isolated polynucleotide encoding an antibody binding to LILRB1 or antigen-binding fragment thereof describe above.
  • the polynucleotide is nucleic acid sequence of DNA, RNA, DNA/RNA hybrids, or modifications thereof. In some embodiments, the polynucleotide is a nucleic acid sequence of DNA.
  • the encoding polynucleotide (DNA or RNA) may be recombinant or synthetic molecule.
  • the present disclosure also relates to sequence variants of the polynucleotide described above.
  • 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 thereto.
  • the present disclosure also includes polynucleotide that varies from the polynucleotide sequences specifically provided herein due to the degeneracy of the genetic code.
  • the polynucleotide may further include regulatory sequences (e.g., a promoter sequence, an untranslated 5’ region, and an untranslated 3’ region) and/or vector sequences.
  • regulatory sequences e.g., a promoter sequence, an untranslated 5’ region, and an untranslated 3’ region
  • vector sequences e.g., a vector.
  • vector refers to a polynucleotide that can be engineered to contain a cloned polynucleotide or 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 (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, ⁇ -galactosidase) .
  • regulatory sequences such as, for example, promoters and/or enhancers
  • selectable marker genes such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, ⁇ -galactosidase
  • a polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • vaccinia or other pox virus e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • the present disclosure also provides an isolated vector comprising the polynucleotide as described herein.
  • the provided isolated polynucleotide can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art.
  • the 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 ⁇ ) , and a transcription termination sequence.
  • a signal sequence e.g., a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1 ⁇ ) , and a transcription termination sequence.
  • the vector provided herein at least one promoter (e.g., SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence, and at least one selection marker.
  • promoter e.g., SV40, CMV, EF-1 ⁇
  • vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g.
  • SV40 lambda phage, and M13 phage, plasmid 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 etc.
  • the present disclosure provides hose cells comprising the isolated polynucleotide as described herein or the isolated vector as described herein.
  • host cell refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide.
  • Host cells may be prokaryotic cells or eukaryotic cells.
  • Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells.
  • Vectors comprising the polynucleotide sequence encoding the antibody binding to LILRB1 or antigen-binding fragment thereof can be introduced to a host cell for cloning or gene expression.
  • Suitable host cells for cloning or expressing the above-described polynucleotide (nucleic acid sequence of DNA, RNA or DNA/RNA hybrids) in the 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, mouse, monkey, rabbit, goat, hamster, or rat cells, insect cells, avian cells, plant cells and eukaryotic cells.
  • the host cells may be, for example, (1) bacterial cells, 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 (a cell line from Spodoptera frugiperda) cells (Protein Sciences Corp., Meriden, Conn., USA) ; (4) mammalian cells; or (5) plant cells.
  • bacterial cells such as E. coli
  • fungal cells and Aspergillus cells yeast cells, such as Saccharomyces cerevisiae, and K. lactis
  • insect cell lines such as (a cell line from Spodoptera frugiperda) cells (Protein Sciences Corp., Meriden, Conn., USA)
  • mammalian cells or (5) plant cells.
  • Typical mammalian cells include COS1 and COS7 cells, Chinese hamster ovary (CHO) cells, NS0 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.
  • CHO Chinese hamster ovary
  • the invention thus provides cells that express the antibodies of the present invention, including but not limited to hybridoma cells, B cells, plasma cells, as well as mammalian and human host cells recombinantly modified to express the antibodies of the present invention (e.g., adult embryonic stem cells) .
  • Other useful mammalian cell lines are well known and readily available from the American Type Culture Collection ( “ATCC” ) (Manassas, Va., USA) and the National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository at the Coriell Cell Repositories (Camden, N.J., USA) . These cell types are only representative, and this list is not meant to be an exhaustive list.
  • the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293 and their derivatives.
  • a vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) .
  • vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • 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, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999) , and Roitt et al., Immunology, 3d 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) .
  • kits that contain the antibody binding to LILRB1 or antigen-binding fragment thereof described above.
  • kits can further include, if desired, one or more of various 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 a person skilled in the art.
  • kit components such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers etc., as will be readily apparent to a person skilled in the art.
  • Instructions, either as inserts or a labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.
  • compositions that comprises the antibody binding to LILRB1 or antigen-binding fragment thereof, the isolated polynucleotide, the isolated vector, or the host cell described above, and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions may be formulated in any manner known in the art.
  • the pharmaceutical composition could be formulated for parenteral (e.g., orally, nasally, or by inhalation, ophthalmic, rectal, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .
  • parenteral e.g., orally, nasally, or by inhalation, ophthalmic, rectal, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal
  • dosage unit form i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage
  • the formulation of the pharmaceutical composition is compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) .
  • Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as 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
  • compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the antibody binding to LILRB1 or antigen-binding fragment thereof thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) .
  • an agent that delays absorption e.g., aluminum monostearate and gelatin
  • controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid) .
  • biodegradable, biocompatible polymers e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid
  • the pharmaceutical compositions are formulated into an injectable composition.
  • the injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion.
  • Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • a sterile, lyophilized powder is prepared by dissolving an antibody binding to LILRB1 or antigen-binding fragment thereof as disclosed herein in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder.
  • Excipients include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents.
  • the antibody or the antigen-binding fragment, or pharmaceutical compositions comprising them may be included in a container, package or dispenser alone or as part of a kit with labels and instructions for administration.
  • the present disclosure provides a use of the antibody binding to LILRB1 or antigen-binding fragment thereof, the isolated polynucleotide, the isolated vector, the host cell, the kit or the pharmaceutical composition in the manufacture of a therapeutic agent for diagnosing, preventing or treating a neoplastic disease.
  • the present disclosure provides a use of the antibody binding to LILRB1 or antigen-binding fragment thereof, the isolated polynucleotide, the isolated vector, the host cell, or the pharmaceutical composition in the manufacture of a therapeutic agent for improving immune response to a neoplastic disease.
  • the present disclosure provides a method for diagnosing, preventing, or treating a subject having a neoplastic disease, comprising administrating to the subject a therapeutically effective amount of therapeutic agent, such as the antibody binding to LILRB1 or antigen-binding fragment thereof, the isolated polynucleotide, the isolated vector, the host cell, the kit or the pharmaceutical composition.
  • a therapeutically effective amount of therapeutic agent such as the antibody binding to LILRB1 or antigen-binding fragment thereof, the isolated polynucleotide, the isolated vector, the host cell, the kit or the pharmaceutical composition.
  • terapéuticaally effective amount refers to an amount of composition or active agent as disclosed herein effective to “treat” a disease or disorder in a subject.
  • the therapeutic agent can enhance immune response to the disease, or could decrease immune inhibition in vitro or in vivo.
  • LILRB1 signaling augments macrophage phagocytosis, restores cytotoxic function of NK cells, understandably, antibody binding to LILRB1 or antigen-binding fragment thereof that binds to LILRB1 could reverse immune inhibitory and improve immune response (such as natural immune response) , which is helpful for treating extensive neoplastic diseases.
  • the immune response in the subject improves by potentiating immune cells killing activity, destruction of MHC class I molecule expressing cells, and/or macrophage cells phagocytosis of MHC class I molecule expressing cells.
  • the present disclosure provides a combination comprising the antibody binding to LILRB1 or antigen-binding fragment thereof of any of claims 1-15 and a second therapeutic agent for use in treating a neoplastic disease in a subject, wherein the second therapeutic agent is chosen from one or more of: an inhibitor of an inhibitory molecule, an activator of a costimulatory molecule, a chemotherapy, a targeted anticancer therapy, an oncolytic drug, a cytotoxic agent, an immune-based therapy, a cytokine, a vaccine, or a cellular immunotherapy.
  • the second therapeutic agent is an antibody or antigen-binding fragment binding to at least one immune checkpoint molecule (e.g., CD47, SIRP ⁇ ) simultaneously or at intervals.
  • the second therapeutic agent is an anti-SIRP ⁇ or an anti-CD47 antibody.
  • the neoplastic disease is solid tumor or liquid tumor. In some embodiments, the neoplastic disease is a solid tumor which is infiltrated by tumor-infiltrating lymphocytes which express LILRB1. In some embodiments, the neoplastic disease includes chronic myelogenous leukemia, colorectal adenocarcinoma, breast cancer, carcinoid cancer, 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, hematologic malignancy, melanoma or metastatic melanoma, non-small cell lung carcinoma, 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
  • the methods of treatment reduce the rate of the increase of volume of a tumor in a subject over time, reduce the risk of developing a metastasis, or reduce the risk of developing an additional metastasis in a subject.
  • the treatment can halt, slow, retard, or inhibit progression of a cancer.
  • the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.
  • the subject comprises mammals including primate, rodent, canine and swine, such as mice, rats, rabbits, cats, dogs, pig, monkey, chimpanzee, gorilla, and the like.
  • the subject comprises mouse, cynomolgus, and human.
  • the subject is human. Except when noted, the term “patient” or “subject” are used herein interchangeably.
  • the daily dosage of the therapeutic agent could obtain from cell culture assays, animal studies or clinical research.
  • a therapeutically effective amount of therapeutic agent or active agent (such as, antibody binding to LILRB1 or antigen-binding fragment thereof) will be an amount that treats the disease in a subject, decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject.
  • the effectiveness and dosing can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject.
  • the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody binding to LILRB1 or antigen-binding fragment thereof in vivo.
  • the reference antibody 15G8 was generated according to patent WO 2021/028921 A1 (SEQ ID NO:28 and 24) .
  • Hu5F9 ahuman IgG4 anti-CD47 antibody
  • Jie Liu’s paper PoS One. 2015 Sep 21; 10 (9) : e0137345.
  • the variable region sequences of 15G8 and Hu5F9 are shown in Table 1.
  • Human LILRB1-expressing HEK293-LILRB1 stable pool was generated by LakePharma.
  • A375/HLA-G monoclonal stable cell line was generated by Chempartner.
  • K562 cells were co-transfected with the plasmids encoding non-tagged full length human LILRB1 with a small ⁇ -galactosidase fragment (ED) fused to its c-terminal, or the SH2-domain of SHP-1 (SH2 domain-containing protein tyrosine phosphatase 1) with the complementing ⁇ -galactosidase fragment (EA) , and selectively cultured in medium containing 100 ⁇ g/mL hygromycin plus 2 ⁇ g/mL puromycin.
  • DLD-1 cells were transfected with full length beta-2-microglobulin ( ⁇ 2M) expression plasmid and selectively cultured in medium containing 5 ⁇ g/mL puromycin.
  • Human LILRB1 extracellular domain (ECD) recombinant proteins (Q8NHL6, Gly24-His458) with human Fc tag or 6xHis-tag were purchased from R&D systems for immunization and hybridoma screening.
  • LILRA1 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.
  • 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 quick immunization strategy.
  • ECD extracellular domain
  • serum titer of the immunized mice was detected by enzyme linked immunosorbent assay (ELISA) .
  • ELISA enzyme linked immunosorbent assay
  • Final boost was conducted when the serum titers reached high levels.
  • 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.
  • 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. Then the hybridoma cells secreting antibodies with top human LILRB1 specific binding activity were subcloned.
  • the selected hybridoma cells were limited diluted into 96-well plates at the density of 1 cell/well to obtain monoclonal hybridoma cells.
  • Supernatants harvested from these monoclonal cells were screened by ELISA and the FACS-based binding assay used in primary and secondary screening.
  • Antibodies secreted by the positive clones were then assayed for the activity to block the interaction of human LILRB1 with human leukocyte antigen-G (HLA-G) or human leukocyte antigen-A2 (HLA-A2) (refer to the methods describe in section 3.2.4) .
  • HLA-G human leukocyte antigen-G
  • HLA-A2 human leukocyte antigen-A2
  • Example 3 Chimeric antibody generation and characterization
  • the heavy chain and light chain variable regions of clone 4F22G1 secreted monoclonal antibody was sequenced. According to the sequencing results, a human IgG4 chimeric antibody with Serine228Proline mutation at Fc region was generated and named as 82c, where the suffix “c” stands for chimeric.
  • the standard single-letter amino acid sequences of heavy chain variable region (VH) and the light chain variable region (VL) are as shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, of which the CDRs are underlined and in italic.
  • Human LILRB1 binding activity of 82c was detected by a FACS-based binding assay using K562 cells stably expressing human LILRB1 as target cells. 82c strongly bound to membrane-bound human LILRB1 in a dose dependent manner ( Figure 1) .
  • the EC 50 and TOP geometric mean fluorescence intensity (TOP MFI) were 2.40 nM and 12656, respectively, which were calculated by GraphPad Prism 9.0 using Four-Parameter nonlinear fitting.
  • the binding affinity and kinetics of 82c and benchmark antibody 15G8 to human LILRB1 were determined using Bio-Layer Interferometry (Octet) .
  • the binding selectivity of 82c and benchmark antibody 15G8 to LILRA/LILRB family members was assessed by ELISA using LILRA/LILRB ECD recombinant proteins as antigens. Briefly, anti-LILRB1 antibodies (50 ⁇ l, 100 nM) were incubated in human LILRA/LILRB ECD recombinant proteins-coated ELISA plate for 1 hour at 37°C. After washing, horseradish peroxidase (HRP) labeled detection antibody (50 ⁇ l) was added and incubated for 1 hour at 37°C. Color development was conducted by the addition of 100 ⁇ l/well of TMB (Tetramethylbenzidine) solution.
  • HRP horseradish peroxidase
  • the classical and non-classical MHC-class I molecules are important ligands of LILRB1 in particular biological contexts.
  • the activity of 82c and benchmark antibody 15G8 to block the interaction of human LILRB1 with the non-classical MHC-class I molecule human HLA-G or the classical MHC-class I molecule human HLA-A2 was assessed by FACS-based competition assays. Briefly human LILRB1-expressing HEK293-LILRB1 cells were preincubated with 5, 1, 0.2, or 0.04 ⁇ g/mL of 82c or 15G8 for 30 minutes.
  • HLA-G complex tetramer (1 ⁇ g/mL) or HLA-A2 complex (30 ⁇ g/mL) was added for another 30 minutes of incubation.
  • the blocking activity was determined by quantitating the blockade of HLA-G complex tetramer or HLA-A2 complex recombinant protein binding to HEK293-LILRB1 cells.
  • 82c and 15G8 potently blocked the interaction of human LILRB1 with human HLA-G or human HLA-A2 in a dose dependent manner.
  • NK92/CD16a cells as effector cells and HLA-G-expressing A375/HLA-G cells as target cells.
  • NK92/CD16a cells was preincubated with the test antibodies for 20 minutes at RT and then co-cultured with CellTrace violet labeled A375/HLA-G cells at the E/T (effector cells/target cells) ratio of 4: 1 for 4 hours at 37°C.
  • Propidium Iodide (PI) is a DNA binding dye and does not permeate viable cell membranes.
  • the killing of the target cells by effector cells in the presence of the test antibodies can be detected by PI staining.
  • the specific cytotoxicity induced by each tested antibody was determined by quantitating the percentage of PI labeled cells (dead cells) in total cells (CellTrace violet + cells) .
  • 82c and 15G8 effectively potentiated NK92/CD16a cells killing of A375/HLA-G cells at 100 nM and 2 nM.
  • 82c showed better efficacy than 15G8 at 2 nM. This result suggests that blocking LILRB1 with 082c can effectively potentiate NK cell-mediated destruction of HLA-G expressing tumor cells.
  • opsonized DLD-1/ ⁇ 2M cells was assessed by a FACS-based phagocytosis assay. Briefly, human monocyte derived macrophages (hMDMs) from healthy donors were labeled with CellTrace Far Red and then co-cultured with CellTrace Violet labeled DLD-1/ ⁇ 2M cells in the presence of the antibodies as tested for 2 hours at 37°C.
  • hMDMs human monocyte derived macrophages
  • CellTrace Far Red and CellTrace Violet double positive cells were macrophages that had engulfed target tumor cells. Therefore, the percentage of this population of macrophages in total macrophages (CellTrace Far Red + ) was calculated as phagocytosis index to quantitate the activity of each sample to potentiate macrophage phagocytosis.
  • 82c effectively potentiated hMDM phagocytosis of anti-SIRP ⁇ antibody opsonized DLD-1/ ⁇ 2M cells with better potency than 15G8 at 10 nM. Both of 82c and 15G8 showed no effect against none-opsonized DLD-1/ ⁇ 2M cells. This result suggests that blocking LILRB1 with 82c can synergize with CD47/SIRP ⁇ “don’ t eat me” signal inhibitors in enhancing macrophage phagocytosis of tumor cells.
  • Complementarity-determining region (CDR) grafting method was used for humanization of 82c. Briefly, IGHV3-21*05 (IMGT allele name, 81.6%homology) and IGKV1-17*02 (62.1%homology) were selected as humanization templates for heavy chain and light chain, respectively, based on their homology to the original mouse antibody sequences. CDRs were defined using Kabat definition except heavy chain CDR1, which was defined using a combination of Kabat and IMGT systems.
  • the CDRs and different combinations of canonical residues from 82c were grafted onto the templates and 16 variants (human IgG1LALA, human IgG1 with mutations of Leu234Ala together with Leu235Ala in Fc region) were produced for further characterization. They were designated as hu082.01 to hu082.16, where the prefix “hu” indicates “humanized” , and the number in the suffix denotes the serial number.
  • the VH and VL of hu082.01 to hu082.16 were shown below.
  • Human LILRB1 binding activity of 82c and 82c derived humanized variants was assessed by a 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 membrane-bound human LILRB1.
  • the EC 50 and TOP MFI values calculated by GraphPad Prism 9.0 using Four-Parameter nonlinear fitting are summarized in Table 6.
  • a single dose affinity ranking test of 82c derived humanized variants was carried out using surface plasmon resonance (SPR) technique.
  • SPR surface plasmon resonance
  • the association and dissociation curves were fitted with the 1: 1 binding model, and the Ka, Kd and KD values were calculated and summarized in Table 7. All the tested variants performed comparably in the ranking test.
  • hu082.03 The activity of hu082.03 to block the interaction of human LILRB1 with human HLA-A2 was assessed by a FACS-based competition assay (refer to the method described in section 3.2.4, the final concentration of human HLA-A2 complex recombinant protein was adjusted to 10 ⁇ g/mL) .
  • hu082.03 potently blocked the binding human HLA-A2 complex recombinant protein to HEK293-LILRB1 cells in a dose dependent manner ( Figure 6) .
  • the IC 50 and TOP blocking ratio were 1.45 nM and 99.4%, respectively, which were calculated by GraphPad Prism 9.0 using Four-Parameter nonlinear fitting.
  • MHC class I molecules-induced LILRB1 signaling is transmitted by phosphorylation of the immunoreceptor tyrosine-based inhibition motifs present on the cytoplasmic tail of LILRB1.
  • SHP-1 and SHP-2 SH2 domain-containing protein tyrosine phosphatase 2
  • SHP-1 and SHP-2 SH2 domain-containing protein tyrosine phosphatase 2
  • full length human LILRB1 was engineered with a small ⁇ -galactosidase fragment (ED) fused to its c-terminal, and the SH2-domain of SHP-1 was engineered with the complementing ⁇ -galactosidase fragment (EA) .
  • ED small ⁇ -galactosidase fragment
  • EA complementing ⁇ -galactosidase fragment
  • LILRB1/SHP-1 recruitment assay K562/SHP-1 + LILRB1 + cells were co-cultured with A375/HLA-G cells or Raji cells in the presence of the test antibodies overnight at 37°C.
  • Gal-screen substrate Applied Biosystems
  • Chemiluminescence was then measured using a plate reader to quantitate the reporter activity.
  • hu082.03 and 15G8 effectively disrupted classical MHC-class I molecules (Raji) -induced recruitment of SHP-1 to LILRB1 intracellular tail with comparable activity.
  • the TOP blocking ratios were 94.23%and 84.69%, respectively.
  • NK cells killing of tumor cells was assessed by an in vitro NK killing assay (refer to the method describe in section 3.2.5) .
  • three tested humanized variants were confirmed to retain similar activity to their parental antibody 82c in potentiating NK92/CD16a killing of A375/HLA-G cells.
  • the EC 50 values 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 TOP cytotoxicity values were 57.01%, 55.78%, 57.02%, and 56.74%, respectively.
  • the EC 50 and TOP cytotoxicity values were calculated by GraphPad Prism 9.0 using Four-Parameter nonlinear fitting.
  • Hu082.03 was also tested in a FACS-based phagocytosis assay using HLA-G-expressing A375/HLA-G cells as target cells. Briefly hMDMs from healthy donors (donor 1 and donor 2) were labeled with CellTrace Far Red and then co-cultured with CellTrace Violet labeled A375/HLA-G cells in the presence of the antibodies as tested for 2 hours at 37°C. The percentage of CellTrace Far Red and CellTrace Violet double macrophages in total macrophages (CellTrace Far Red + ) was calculated as phagocytosis index to quantitate the activity of each sample to potentiate macrophage phagocytosis.
  • hu082.03 effectively potentiated hMDM phagocytosis of Hu5F9 (ahuman IgG4 anti-CD47 antibody) opsonized A375/HLA-G cells in a dose dependent manner.
  • Hu082.03 showed no effect against none-opsonized A375/HLA-G cells.

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Abstract

L'invention concerne un anticorps se liant à LILRB1 ou un fragment de liaison à l'antigène de celui-ci et l'utilisation d'un tel anticorps ou d'un tel fragment de liaison à l'antigène dans le diagnostic, la prévention ou le traitement d'une maladie néoplasique.
PCT/CN2024/100199 2023-06-20 2024-06-19 Anticorps ou fragments de liaison à l'antigène se liant à lilrb1 et leurs utilisations Pending WO2024260384A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021074157A1 (fr) * 2019-10-14 2021-04-22 Innate Pharma Traitement du cancer avec des inhibiteurs d'ilt-2
US20210277112A1 (en) * 2019-08-12 2021-09-09 Biond Biologics Ltd. Antibodies Against ILT2 and Use Thereof
WO2022026360A2 (fr) * 2020-07-28 2022-02-03 The Board Of Regents Of The University Of Texas System Anticorps monoclonaux contre lilrb1 pour une utilisation diagnostique et thérapeutique
CN114945594A (zh) * 2019-12-23 2022-08-26 株式会社Lg化学 抗lilrb1抗体及其用途
CN115867353A (zh) * 2020-07-28 2023-03-28 株式会社Lg化学 抗lilrb1抗体及其用途
CN115975033A (zh) * 2022-10-10 2023-04-18 中国科学院微生物研究所 一种lilrb1单克隆抗体及其应用
CN117321083A (zh) * 2021-03-11 2023-12-29 大学保健网 Lilrb1和lilrb2结合分子及其用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210277112A1 (en) * 2019-08-12 2021-09-09 Biond Biologics Ltd. Antibodies Against ILT2 and Use Thereof
WO2021074157A1 (fr) * 2019-10-14 2021-04-22 Innate Pharma Traitement du cancer avec des inhibiteurs d'ilt-2
CN114945594A (zh) * 2019-12-23 2022-08-26 株式会社Lg化学 抗lilrb1抗体及其用途
WO2022026360A2 (fr) * 2020-07-28 2022-02-03 The Board Of Regents Of The University Of Texas System Anticorps monoclonaux contre lilrb1 pour une utilisation diagnostique et thérapeutique
CN115867353A (zh) * 2020-07-28 2023-03-28 株式会社Lg化学 抗lilrb1抗体及其用途
CN117321083A (zh) * 2021-03-11 2023-12-29 大学保健网 Lilrb1和lilrb2结合分子及其用途
CN115975033A (zh) * 2022-10-10 2023-04-18 中国科学院微生物研究所 一种lilrb1单克隆抗体及其应用

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