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WO2024207855A1 - 抗lilrb1抗体或其抗原结合片段、制备方法和用途 - Google Patents

抗lilrb1抗体或其抗原结合片段、制备方法和用途 Download PDF

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Publication number
WO2024207855A1
WO2024207855A1 PCT/CN2024/070594 CN2024070594W WO2024207855A1 WO 2024207855 A1 WO2024207855 A1 WO 2024207855A1 CN 2024070594 W CN2024070594 W CN 2024070594W WO 2024207855 A1 WO2024207855 A1 WO 2024207855A1
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Prior art keywords
amino acid
acid sequence
seq
variable region
chain variable
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PCT/CN2024/070594
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English (en)
French (fr)
Inventor
李竹石
朱燕
邓涛
赵真虎
龙杨洋
陈洪
王颖
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成都优洛生物科技有限公司
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Priority to CN202480000182.4A priority Critical patent/CN118019766A/zh
Publication of WO2024207855A1 publication Critical patent/WO2024207855A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present application belongs to the field of immunotechnology, and specifically relates to anti-LILRB1 antibodies or antigen-binding fragments thereof, preparation methods and applications.
  • LILRB1 Leukocyte immunoglobulin-like receptor subfamily B1
  • ILT2 Leukocyte immunoglobulin-like receptor subfamily B1
  • LILRB1 is an inhibitory receptor expressed in cells such as B cells, T cells, NK cells, dendritic cells, macrophages and other immune cells.
  • LILRB1 is a type I transmembrane glycoprotein with an extracellular Ig-like domain that binds to ligands and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). It can recruit downstream phosphatases such as SHP1, SHP2, and SHIP, and inhibit the activation of immune cells through a series of signal transduction pathways.
  • ITIMs immunoreceptor tyrosine-based inhibitory motifs
  • LILRB1 is a receptor for MHC class I antigens and recognizes a broad spectrum of HLA-A, HLA-B, HLA-C, and HLA-G, with a particular preference for binding to HLA-G.
  • a variety of cancer cells overexpress HLA-G for immune escape.
  • the interaction between LILRB1 and HLA-G has been shown to inhibit the function of immune cells, thereby promoting tumor immune escape. It is expected that blocking the binding of LILRB1 to HLA-G can restore the suppressed immune cell activity, thereby exerting an anti-tumor effect.
  • the present application provides an anti-LILRB1 antibody or an antigen-binding fragment thereof, a preparation method and use thereof.
  • the anti-LILRB1 antibody or an antigen-binding fragment thereof can inhibit the immune escape mechanism of tumor cells so that immune cells can exhibit their anti-cancer effects.
  • an anti-LILRB1 antibody or an antigen-binding fragment thereof comprising:
  • HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 7, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 1 or 7, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence of SEQ ID NO: 1 or 7 and having the same or similar function as the amino acid sequence of SEQ ID NO: 1 or 7;
  • HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 8, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 2 or 8, or an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence of SEQ ID NO: 2 or 8 and having the same or similar function as the amino acid sequence of SEQ ID NO: 2 or 8;
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, 9 or 119, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 3, 9 or 119, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 3, 9 or 119 and having the same or similar function as the amino acid sequence shown in SEQ ID NO: 3, 9 or 119; and/or
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO:4 or 10, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:4 or 10, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:4 or 10 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:4 or 10;
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:5 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:5;
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO:6 or 11, or an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:6 or 11, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:6 or 11 and has the same or similar function as the amino acid sequence shown in SEQ ID NO:6 or 11.
  • substitution, deletion or addition of one or more amino acids described in the present application is substitution, deletion or addition of 1 or 2 amino acids. In some embodiments, the substitution, deletion or addition of one or more amino acids is a conservative modification.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively;
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 2 and 119, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively; or
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 7, 8 and 9, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 10, 5 and 11, respectively.
  • the HCDR2 has an amino acid sequence shown in SEQ ID NO: 12.
  • the HCDR3 has an amino acid sequence as shown in SEQ ID NO: 13. In some embodiments of the present application, the HCDR3 has an amino acid sequence as shown in SEQ ID NO: 93.
  • the LCDR3 has an amino acid sequence shown in SEQ ID NO: 14.
  • the LCDR3 has an amino acid sequence shown in SEQ ID NO: 15.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 12 and 13, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 14, respectively.
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 12 and 93, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 14, respectively.
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 7, 8 and 9, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 10, 5 and 15, respectively.
  • the LILRB1 is mammalian LILRB1, and the antibody is preferably a mouse antibody, a monkey antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a human antibody.
  • the antibody is an IgG antibody, preferably an IgG1, IgG2, IgG3 or IgG4 antibody, more preferably an IgG4 antibody.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof provided herein further comprises a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region is the constant region of IgG1, IgG2, IgG3 or IgG4, and the light chain constant region is the constant region of an antibody ⁇ chain or ⁇ chain.
  • the anti-LILRB1 antibody or its antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a Fab fragment, a Fab' fragment, a F(ab') 2 fragment, a Fd fragment, a Fv fragment, a dAb fragment, an isolated CDR region, a scFv and a nanobody.
  • the anti-LILRB1 antibody is a monoclonal antibody.
  • the antibody is a murine IgG4 antibody.
  • the antibody is a humanized IgG4 antibody.
  • the second aspect of the present application provides a nucleic acid molecule, which encodes the antibody or antigen-binding fragment thereof, or the heavy chain variable region and/or light chain variable region thereof according to the first aspect.
  • the third aspect of the present application provides a recombinant vector, which comprises the nucleic acid molecule described in the second aspect.
  • the fourth aspect of the present application provides a recombinant cell, which comprises the nucleic acid molecule described in the second aspect or the recombinant vector comprising the nucleic acid molecule described in the third aspect.
  • the fifth aspect of the present application provides a method for preparing an anti-LILRB1 antibody or an antigen-binding fragment thereof, the method comprising culturing the recombinant cell described in the fourth aspect.
  • the recombinant cell is a recombinant CHO cell.
  • the present application provides use of the antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, or the recombinant cell described in the fourth aspect in preparing a drug;
  • the drug is a drug for preventing and/or treating cancer.
  • the present application relates to the antibody or antigen-binding fragment thereof described in the first aspect of the present application, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, or the recombinant cell described in the fourth aspect for preventing and/or treating a LILRB1-related disease.
  • the present application relates to a method for preventing and/or treating a LILRB1-related disease, comprising administering the anti-LILRB1 antibody or antigen-binding fragment thereof described in the first aspect of the present application, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, or the recombinant cell described in the fourth aspect to a subject in need thereof.
  • the seventh aspect of the present application provides a use of the anti-LILRB1 antibody or antigen-binding fragment thereof described in the first aspect, or the nucleic acid molecule described in the second aspect, in the preparation of a reagent for diagnosing a LILRB1-related disease.
  • the present application relates to the anti-LILRB1 antibody or antigen-binding fragment thereof described in the first aspect, or the nucleic acid molecule described in the second aspect, for diagnosing a LILRB1-related disease.
  • the disease is cancer.
  • the cancer is a cancer expressing MHC class I molecules, preferably a cancer expressing HLA-G.
  • the cancer is characterized by overexpression of MHC class I molecules; preferably overexpression of HLA-G.
  • the present application provides a chimeric antigen receptor (CAR), comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signal transduction domain, wherein the extracellular antigen binding domain comprises the anti-LILRB1 antibody or antigen-binding fragment thereof described herein.
  • CAR chimeric antigen receptor
  • the present application provides a kit comprising the anti-LILRB1 antibody or antigen-binding fragment thereof or chimeric antigen receptor described in the present application.
  • the anti-LILRB1 antibody or its antigen-binding fragment provided in the present application can inhibit the immune escape mechanism of tumor cells so that immune cells can exhibit their anti-cancer effects, has the activity of promoting NK92 cells and macrophages to kill tumor cells, can bind to LILRB1, and block the binding of LILRB1 to HLA-G.
  • FIG1 is a graph showing the reporter gene activity results of the positive control BND-22 of the present application.
  • Figure 2 is a graph showing the activity detection results of the mouse antibody reporter gene method; wherein Figure 2A is a graph showing the activity detection results of the positive control BND-22 reporter gene method, Figure 2B is a graph showing the activity detection results of the mouse antibody 6-B8-C3-G6 reporter gene method, and Figure 2C is a graph showing the activity detection results of the mouse antibody 40-H9-G4-D8 reporter gene method;
  • FIG3 is a graph showing the test results of the mouse antibody promoting NK92 cell killing experiment
  • FIG4 is a graph showing the test results of the mouse antibody-promoting macrophage killing experiment.
  • FIG5 is a diagram showing the activity detection results of the chimeric antibody reporter gene method
  • FIG6 is a diagram showing the test results of the chimeric antibody promoting NK92 cell killing experiment
  • FIG7 is a diagram showing the detection results of the chimeric antibody promoting primary NK cell killing experiment
  • FIG8 is a diagram showing the test results of chimeric antibodies promoting macrophage killing experiments.
  • FIG9A is a diagram showing the detection results of the humanized antibody-promoting macrophage killing experiment.
  • FIG9B is a diagram showing the detection results of the humanized antibody-promoting macrophage killing experiment.
  • FIG10A is a tumor growth curve of the in vivo efficacy study of humanized antibodies
  • FIG. 10B is a graph showing the trend of body weight changes in the in vivo efficacy study of humanized antibodies.
  • “about” means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation as practiced in the art. Alternatively, “about” can mean a range of up to 20% (i.e., ⁇ 20%), such as fluctuations within ⁇ 10%, ⁇ 5%, ⁇ 2%, ⁇ 1%, or ⁇ 0.5% of the specific value given. In addition, particularly for biological systems or methods, the term can mean When a specific value is given in the present application or claims, unless otherwise stated, the meaning of "about” should be considered to be within the acceptable error range of the specific value. In this article, unless otherwise stated, the value of each measured value, step parameter or condition is modified by "about” by default.
  • treatment means that after administration, the disease or its related symptoms can be suppressed, contained, alleviated, improved, slowed down, relieved or eliminated, the progression of the disease or its related symptoms can be delayed, postponed, slowed down, suspended or terminated, or the recurrence of the disease or its related symptoms can be prevented, controlled or reduced.
  • the therapeutically effective amount or the preventively effective amount can be determined by the clinician according to the individual condition of the subject, the severity of the disease, sex, age, weight, mode of administration, etc., by conventional methods or experience.
  • the term "subject" as used herein encompasses any vertebrate, e.g., mammals, such as humans, non-human primates, sheep, dogs, cats, horses, cows, chickens, pigs, mice, etc.
  • the subject in the present application is a human.
  • antibody refers to a polypeptide encoded by an immunoglobulin gene and capable of binding to an antigen.
  • complementarity determining region may refer to a portion of the variable region of an antibody that confers antigen binding specificity, and may refer to an amino acid sequence found in a hypervariable region of a heavy or light chain of an immunoglobulin.
  • the heavy chain may include three complementary determining regions CDR (HCDR1, HCDR2, and HCDR3; and the light chain may include three complementary determining regions CDR (LCDR1, LCDR2, and LCDR3).
  • the CDRs may provide contact residues that play an important role in the binding of an antibody to its antigen or antigenic epitope.
  • X represents an unspecified amino acid, and its scope is designated by the definition in the relevant expression.
  • Xn multiple Xs appearing successively are numbered respectively (ie, written as Xn ) and their scopes are defined respectively.
  • the percent identity (degree of homology) between sequences can be determined by comparing two sequences, for example, using freely available computer programs commonly used for this purpose on the World Wide Web (eg www.ncbi.nlm.nih.gov), such as BLASTp or BLASTn with default settings.
  • an anti-LILRB1 antibody or an antigen-binding fragment thereof comprising:
  • HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 7, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 1 or 7, or an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the amino acid sequence of SEQ ID NO: 1 or 7 and having the same or similar function as the amino acid sequence of SEQ ID NO: 1 or 7;
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2 or 8, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 2 or 8, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2 or 8 and having the same or similar function as the amino acid sequence shown in SEQ ID NO: 2 or 8;
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, 9 or 119, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 3, 9 or 119, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 3, 9 or 119 and having the same or similar function as the amino acid sequence shown in SEQ ID NO: 3, 9 or 119; and/or
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO:4 or 10, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:4 or 10, or an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids of the amino acid sequence shown in SEQ ID NO:4 or 10 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:4 or 10;
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:5 and having the same or similar function as the amino acid sequence shown in SEQ ID NO:5;
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO:6 or 11, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:6 or 11, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:6 or 11, and has the same or similar function as the amino acid sequence shown in SEQ ID NO:6 or 11.
  • CDR sequences numbered according to any one or more of the AbM numbering rules, the Kabat numbering rules, the Chothia numbering rules and the Contact numbering rules (based on analysis of available complex crystal structures) also fall within the scope of protection of this application.
  • substitution, deletion or addition of one or more amino acids described in the present application refers to the substitution, deletion or addition of 1 or 2 amino acids.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof comprises: the heavy chain variable region comprising a complementarity determining region and the light chain variable region comprising a complementarity determining region.
  • the anti-LILRB1 antibody or its antigen-binding fragment comprises: HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2 and HCDR3 respectively have the amino acid sequences shown in SEQ ID NO: 1, 2 and 3 or 119, and the LCDR1, LCDR2 and LCDR3 respectively have the amino acid sequences shown in SEQ ID NO: 4, 5 and 6.
  • HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 7, 8 and 9, respectively, and said LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 10, 5 and 11, respectively.
  • the HCDR2 has an amino acid sequence shown in SEQ ID NO: 12.
  • the HCDR3 has an amino acid sequence shown in SEQ ID NO: 13.
  • the LCDR3 has an amino acid sequence shown in SEQ ID NO: 14.
  • the LCDR3 has an amino acid sequence shown in SEQ ID NO: 15.
  • amino acid sequence IYPGX 1 GNT shown in SEQ ID NO: 2 covers the sequence (IYPGDGNT) of the heavy chain CDR2 (HCDR2) of the antibody and the sequence of the mutated HCDR2 obtained by mutating X 1 in the HCDR2 to other amino acids.
  • amino acid sequence ASYX 3 SSWFPY shown in SEQ ID NO: 3 covers the heavy chain CDR3 (HCDR3) sequence (ASYDSSWFPY) of the antibody and the mutated HCDR3 sequence obtained by mutating X 3 in the HCDR3 to other amino acids.
  • amino acid sequence ASYX5X6SWFPY shown in SEQ ID NO: 119 covers the mutated HCDR3 sequence obtained by mutating X6 in the heavy chain CDR3 (HCDR3) sequence (ASYDSSWFPY) of the antibody to other amino acids and mutating X5 to other amino acids or not mutating X5 .
  • the amino acid sequence X 2 HFWGAPWT shown in SEQ ID NO: 6 covers the sequence (QHFWGAPWT) of the light chain CDR3 (LCDR3) of the antibody and the mutated LCDR3 sequence obtained by mutating X 2 in the LCDR3 to other amino acids.
  • amino acid sequence X 4 HFWGSPWT shown in SEQ ID NO: 11 covers the sequence (QHFWGSPWT) of the light chain CDR3 (LCDR3) of the antibody and the mutated LCDR3 sequence obtained by mutating X 4 in the LCDR3 to other amino acids.
  • the X 1 can be selected from I, N, F, V, S, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D; in some embodiments, the X 1 can be selected from D, S or A; preferably, the X 1 is D.
  • X2 can be selected from I, N, F, V, S, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D; in some embodiments, X2 can be selected from Q, S or A; preferably, X2 is Q.
  • X3 can be selected from I, N, F, V, S, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D; in some embodiments, X3 can be selected from D, T, S or A; preferably, X3 is D.
  • X4 can be selected from I, N, F, V, S, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D; in some embodiments, X4 can be selected from Q, S or A; preferably, X4 is Q.
  • X 5 can be selected from I, N, F, V, S, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D; in some embodiments, X 5 can be selected from D, T, S, G or A; preferably, X 5 is D.
  • X 6 can be selected from I, N, F, V, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D; in some embodiments, X 6 can be selected from A, V or T; preferably, X 6 is V.
  • the HCDR2 may comprise an amino acid sequence selected from SEQ ID NO: 12, 16-34.
  • the HCDR3 may comprise an amino acid sequence selected from SEQ ID NO: 13, 35-53, 92-94.
  • the LCDR3 may comprise an amino acid sequence selected from SEQ ID NO: 14, 15, 54-91 (preferably SEQ ID NO: 14, 54-72).
  • the anti-LILRB1 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein:
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 12 and 13, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 14, respectively;
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 10, 5 and 15, respectively;
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 12 and 13, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 58, respectively;
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 12 and 92, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 14, respectively;
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 12 and 42, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 14, respectively;
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 12 and 93, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 14, respectively;
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 12 and 94, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 14, respectively;
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 10, 5 and 77, respectively; or
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 7, 8 and 9, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 10, 5 and 79, respectively.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein:
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 12 and 93, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 14, respectively;
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 12 and 94, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 14, respectively;
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 7, 8 and 9, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 10, 5 and 77, respectively;
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 1, 12 and 92, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 14, respectively; or
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 1, 12 and 13, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 4, 5 and 14, respectively.
  • the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 1, 12 and 93, respectively, and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 4, 5 and 14, respectively.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof provided in the present application comprises a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NOs: 95, 102-106, 110, 112, or an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NOs: 95, 102-106, 110, 112, or an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NOs: 95, 102-106, 110, 112 and has the same or similar functions as the amino acid sequence shown in SEQ ID NOs: 95, 102-106, 110, 112.
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NOs: 96-101, 107-109, 111, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NOs: 96-101, 107-109, 111, or an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NOs: 96-101, 107-109, 111 and has the same or similar functions as the amino acid sequence shown in SEQ ID NOs: 96-101, 107-109, 111.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region:
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:95, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:96, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:95, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:98, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:95, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:99, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:95, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:100, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:95, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:101, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:102, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:96, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:103, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:96, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:104, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:96, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:105, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:96, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:102, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:98, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:103, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:98, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 104 or has at least 80%, 81%, 82%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO:98, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:105, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:98, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:106, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:107, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:106, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:108, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:106, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:109, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:110, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:111, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:110, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:109, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 112, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO: 107, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 112, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 108, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof comprises:
  • the anti-LILRB1 antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 104 and the light chain variable region shown in SEQ ID NO: 98.
  • the LILRB1 is mammalian LILRB1, and the mammals include humans and non-human primates.
  • the antibody is an IgG antibody, IgG1, IgG2, IgG3 or IgG4 antibody, more preferably an IgG4 antibody.
  • the antibody is a murine IgG4 antibody or a humanized IgG4 antibody.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof provided herein further comprises a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region is the constant region of IgG1, IgG2, IgG3 or IgG4, and the light chain constant region is the constant region of an antibody ⁇ chain or ⁇ chain.
  • the heavy chain constant region is the constant region of IgG4, whose amino acid sequence is as shown in SEQ ID NO: 113 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
  • the light chain constant region is the constant region of the antibody kappa chain, whose amino acid sequence is as shown in SEQ ID NO: 114 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein:
  • the heavy chain comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:104, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a heavy chain constant region having an amino acid sequence as set forth in SEQ ID NO:113, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto,
  • the light chain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:98, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain constant region having an amino acid sequence as shown in SEQ ID NO:114, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the heavy chain comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:105, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a heavy chain constant region having an amino acid sequence as set forth in SEQ ID NO:113, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto,
  • the light chain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:96, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain constant region having an amino acid sequence as shown in SEQ ID NO:114, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the heavy chain comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:106, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a heavy chain constant region having an amino acid sequence as set forth in SEQ ID NO:113, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto,
  • the light chain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 107, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain constant region having an amino acid sequence as shown in SEQ ID NO: 114, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the heavy chain comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:102, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a heavy chain constant region having an amino acid sequence as set forth in SEQ ID NO:113, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto,
  • the light chain comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:96, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:114, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. 99% identical amino acid sequence to a light chain constant region of the
  • the heavy chain comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:95, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a heavy chain constant region having an amino acid sequence as set forth in SEQ ID NO:113, or an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto,
  • the light chain comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 96, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain constant region having an amino acid sequence as shown in SEQ ID NO: 114, or an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein:
  • the heavy chain comprises a heavy chain variable region as shown in SEQ ID NO: 104 and a heavy chain constant region as shown in SEQ ID NO: 113
  • the light chain comprises a light chain variable region as shown in SEQ ID NO: 98 and a light chain constant region as shown in SEQ ID NO: 114;
  • the anti-LILRB1 antibody or its antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a dAb fragment, an isolated CDR region, a scFv and a nanobody.
  • the anti-LILRB1 antibody is a monoclonal antibody.
  • the anti-LILRB1 antibody or its antigen-binding fragment may also be conservatively modified.
  • Conservative modification refers to amino acid modification that does not significantly affect or change the binding properties of the antibody.
  • conservative amino acid substitution may be substitution with another amino acid of the same type (having similar chemical properties or functions).
  • amino acids can be divided into the following categories according to their side chain properties: (1) non-polar amino acids: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar amino acids: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic amino acids: Asp (D), Glu (E); (4) basic amino acids: Lys (K), Arg (R), His (H).
  • amino acids can be divided based on common side chain properties: (1) hydrophobic amino acids: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic amino acids: Cys, Ser, Thr, Asn, Gln; (3) acidic amino acids: Asp, Glu; (4) basic amino acids: His, Lys, Arg; (5) amino acids that affect chain orientation: Gly, Pro; (6) aromatic amino acids: Trp, Tyr, Phe.
  • the heavy chain constant region is the constant region of IgG1, IgG2, IgG3 or IgG4, and the light chain constant region is the constant region of an antibody ⁇ chain or ⁇ chain.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a dAb fragment, an isolated CDR region, a scFv and a nanobody.
  • the anti-LILRB1 antibody is a monoclonal antibody.
  • the antibody is a murine IgG4 antibody.
  • the antibody is a humanized IgG4 antibody.
  • the anti-LILRB1 antibodies or antigen-binding fragments thereof described herein may exhibit the following properties:
  • LILRB1 preferably HLA-G
  • binding to LILRB1 includes binding to LILRB1 of a mammal, including a human, a non-human primate (such as a cynomolgus monkey). In some embodiments, binding to LILRB1 includes binding to a cell expressing LILRB1.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof can cross-bind to human and cynomolgus monkey LILRB1, or cross-bind to a cell expressing human or cynomolgus monkey LILRB1.
  • enhancing the killing effect of immune cells on tumor cells includes enhancing the killing of tumor cells by NK cells and/or macrophages. The killing effect.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof blocks HLA-G binding to LILRB1 with an IC 50 of ⁇ 12.5 ⁇ g/mL, ⁇ 2.0 ⁇ g/mL, ⁇ 1.0 ⁇ g/mL, or ⁇ 0.5 ⁇ g/mL.
  • a nucleic acid molecule which encodes the anti-LILRB1 antibody or its antigen-binding fragment, or its heavy chain variable region and/or light chain variable region.
  • a recombinant vector comprising the nucleic acid molecule.
  • vector refers to a tool for expressing a target gene in a host cell, such as a plasmid vector, a cosmid vector, and a viral vector such as a phage vector, a lentiviral vector, an adenoviral vector, a retroviral vector, and an adeno-associated viral vector.
  • the recombinant vector can be constructed by or by manipulating a plasmid (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), a phage (e.g., ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1, M13, etc.) or a viral vector (e.g., SV40, etc.), which is commonly used in the art.
  • a plasmid e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, p
  • a nucleic acid molecule in a recombinant vector, can be operably linked to a promoter.
  • operably linked is intended to refer to a functional connection between a target nucleotide sequence and an expression control sequence (e.g., a promoter sequence).
  • an expression control sequence e.g., a promoter sequence.
  • a regulatory element can control transcription and/or translation of a target polynucleotide.
  • Recombinant vectors can be constructed as cloning vectors or expression vectors.
  • vectors commonly available in the relevant art for expressing foreign proteins in plants, animals or microbial cells can be used.
  • Various methods well known in the art can be used to construct recombinant vectors.
  • a recombinant vector can be constructed accordingly.
  • the vector when constructing a vector as an expression vector for a prokaryotic host, the vector generally includes a strong promoter for transcription (e.g., pL ⁇ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription/translation termination sequence.
  • a strong promoter for transcription e.g., pL ⁇ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.
  • an expression vector for a eukaryotic host includes a replication origin that can be operated in a eukaryotic cell, such as f1 replication origin, SV40 replication origin, pMB1 replication origin, adenovirus replication origin, AAV replication origin, and BBV replication origin, but is not limited thereto.
  • the expression vector generally includes a promoter derived from a mammalian cell genome (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, etc.), and a polyadenylation sequence as a transcription termination sequence.
  • Recombinant cells can be prepared by introducing a recombinant vector into a suitable host cell.
  • Any host cell known in the art can be used in the present disclosure, as long as it allows the recombinant vector to be cloned and expressed continuously in a stable manner.
  • Examples of prokaryotic host cells that can be used in the present disclosure can be selected from Escherichia coli (E.
  • Escherichia coli such as Escherichia coli JM109, Escherichia coli BL21, Escherichia coli RR1, Escherichia coli LE392, Escherichia coli B, Escherichia coli X1776, Escherichia coli W3110, Bacillus species such as Bacillus subtilis and Bacillus thuringiensis, and Enterobacteriaceae strains, such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas species.
  • the eukaryotic host cell that can be used for transformation can be selected from but not limited to Saccharomyces cerevisiae, insect cells and animal cells, such as Sp2/0, CHO (Chinese hamster ovary) K1, CHO DG44, CHO S, CHO DXB11, CHO GS-KO, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK etc.
  • Nucleic acid molecules or recombinant vectors carrying nucleic acid molecules can be introduced (transfected) into host cells using methods well known in the relevant art. For example, when the host cell is a prokaryotic cell, CaCl or electroporation can be used to carry out this transfection.
  • microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection or particle bombardment can be used but not limited to realize gene introduction.
  • the phenotype associated with the selection marker can be utilized according to methods well known in the art.
  • the selection marker is a gene that confers resistance to certain antibiotics
  • host cells can be grown in culture medium in the presence of antibiotics to select target transformants.
  • a recombinant cell comprising the nucleic acid molecule or a recombinant vector comprising the nucleic acid molecule.
  • the recombinant cell is a prokaryotic cell. In other embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof. Mammalian cells are, for example, Chinese hamster ovary cells (CHO), human kidney epithelial cell line 293 cells or 293T cells.
  • the recombinant cell is a recombinant CHO cell.
  • a method for preparing an anti-LILRB1 antibody or an antigen-binding fragment thereof comprising culturing the recombinant cell.
  • the method may also comprise isolating and/or purifying the antibody or fragment thereof from the cell culture after the expression or culturing step, for example, by purification using a Protein A affinity column.
  • the drug is a drug for preventing and/or treating cancer.
  • the anti-LILRB1 antibody or antigen-binding fragment thereof or the nucleic acid molecule in preparing a reagent for diagnosing a LILRB1-related disease.
  • the LILRB1-related disease is cancer.
  • Cancer cells that can be killed according to the present disclosure include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testicles, tongue, cervix, or uterus.
  • the cancer may specifically be of the following histological types, although not limited thereto: tumor, malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; mesenchymal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant neoplasm; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenoma in adenomatous polyp; adenocarcinoma, Familial polyposis coli; solid carcinoma; carcinoid tumor, malignant neoplasm; branching alveolar adenocarcinoma; papillary adenocarcinoma; chromopho
  • the tumor may include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing sarcoma, glioblastoma, neuroblastoma or leukemia.
  • the cancer is melanoma or colon cancer expressing HLA-G.
  • the cancer is a cancer expressing MHC class I molecules, preferably a cancer expressing HLA-G.
  • the cancer is characterized by overexpression of MHC class I molecules; preferably overexpression of HLA-G.
  • the dosage and administration frequency of the anti-LILRB1 antibodies or antigen-binding fragments thereof described herein can be determined by a clinician according to factors such as the age, weight, sex, general condition, and severity of the disease of the subject; they can be administered by any conventional route known in the art, such as parenteral, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal administration.
  • parenteral intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal administration.
  • Example 1 Construction of human LILRB1 stable cell line and cynomolgus monkey LILRB1 stable cell line
  • the nucleic acid sequence of LILRB1 can be inserted into an appropriate expression vector to achieve heterologous expression of the nucleic acid sequence.
  • mammalian LILRB1 can be selected, such as human LILRB1, monkey LILRB1, rabbit LILRB1, etc., and can be expressed according to commonly used LILRB1 and its nucleic acid sequence were synthesized based on known sequences published in nucleic acid and protein sequence databases such as NCBI, ENSMBL or UniProt.
  • human LILRB1 and cynomolgus monkey LILRB1 are taken as examples.
  • the human LILRB1 and cynomolgus monkey LILRB1 recombinant proteins used for mouse immunization and activity detection were purchased from ACRO Biosystems.
  • the coding region sequence information of human LILRB1 and cynomolgus monkey LILRB1 was retrieved from the UniProt database as follows:
  • the full genes encoding the above two proteins were synthesized and subcloned into the Lenti-CMV-puro lentiviral expression vector to construct the Lenti-CMV-human ILT2 overexpression vector and the Lenti-CMV-cyno ILT2 overexpression vector (constructed by GenScript).
  • the plasmid was extracted using a plasmid extraction kit (purchased from Novazon) for later use.
  • transfection reagent LVTransm purchased from Aikonde
  • expression vectors Li-CMV-human ILT2 overexpression vector and Lenti-CMV-cyno ILT2 overexpression vector
  • lentiviral packaging plasmid Lenti-packaging Mix from the refrigerator. After thawing at room temperature, mix them thoroughly by pipetting up and down.
  • Recombinant CHO cells capable of stably expressing LILRB1 on the cell surface were prepared as follows:
  • the LILRB1 antibody BND-22 from Biond Biologics was used as a positive control antibody.
  • the amino acid sequence of BND-22 is from WO2021028921A1, as shown below:
  • the light chain and heavy chain genes of BND-22 were synthesized and subcloned into the pcDNA3.4 expression vector respectively.
  • CHO-K1 cells were transfected with BIOHUB 78PEI transfection reagent (purchased from Shanghai Qifa). After culturing at 37°C and 5% CO2 , the cell culture was collected, centrifuged, and purified with a Protein A affinity column.
  • the nucleic acid sequence information of the coding regions of HLA-G and ⁇ 2M was obtained from the UniProt database, and the genes were synthesized after codon optimization and subcloned into the Lenti-CMV-puro lentiviral expression vector.
  • Lentivirus was prepared (see, for example, Neha Tandon et al., Bio Protoc. 2018 Nov 5; 8(21): e3073), and A375 melanoma cells were infected respectively.
  • the positive control antibody BND-22 was diluted to 800 ⁇ g/mL, and then 3-fold serial dilution was performed, and 100 ⁇ L was added to each well of the 96-well plate, so that the final concentration of the antibody in the 96-well plate was 400 ⁇ g/mL, 133.33 ⁇ g/mL, 44.44 ⁇ g/mL, 14.81 ⁇ g/mL, 4.94 ⁇ g/mL, 1.65 ⁇ g/mL, 0.55 ⁇ g/mL, 0.18 ⁇ g/mL, and 0.06 ⁇ g/mL, respectively. The plates were incubated at 37°C and 5% CO2 overnight.
  • mice Twenty Balb/c mice (SPF grade) were divided into two groups for immunization. The first group of mice was immunized with human LILRB1 recombinant protein (purchased from ACRO The second group of mice was immunized with 75 ⁇ g of human LILRB1 recombinant protein and 25 ⁇ g of cynomolgus monkey LILRB1 recombinant protein (purchased from ACRO Biosystems) emulsified with Freund's adjuvant and then injected subcutaneously at multiple points. Each group of mice was immunized 3 times with an interval of 14 days. Mice with good immune titer detection were taken for shock immunization. After 3 days, the mice were killed by cervical dislocation.
  • mice The spleen of the mice was obtained under sterile conditions, and a single cell suspension of B cells was prepared. It was mixed with SP2/0 myeloma cells at a ratio of 1:1, and cell fusion was performed using a BTX cell electrofusion instrument. After electrofusion, all cells were immediately suspended in DMEM medium containing 20% FBS and HAT and inoculated into 96-well plates. About 10 days after fusion, the HT medium was changed to culture for two days, and the supernatant was taken to detect specific antibodies.
  • the clones that can cross-bind with human and cynomolgus monkey LILRB1 proteins and the recombinant cell lines obtained in Example 1 were preferred.
  • monoclonal clones (6-B8-C3-G6, 40-H9-G4-D8) that can secrete antibodies that specifically bind to human and cynomolgus monkey LILRB1 proteins and recombinant cell lines were obtained.
  • the antibody sequences are shown in Table 1.
  • the hybridomas of monoclonal 6-B8-C3-G6 and 40-H9-G4-D8 obtained in the above two rounds of subclone screening were amplified and cultured, and then the mouse antibodies were purified by protein A column affinity chromatography.
  • the purified mouse antibodies and the positive control BND-22 were detected by the reporter gene method according to the steps described in Example 2, and the IC 50 values were calculated.
  • Table 2 and Figure 2 show the activity detection results of the reporter gene method. It can be seen that both the mouse antibody and the positive control can dose-dependently activate the fluorescence signal of the reporter gene cell line.
  • the activity of the mouse antibody 6-B8-C3-G6 is better than that of the positive control BND-22, and the activity of the mouse antibody 40-H9-G4-D8 is equivalent to that of BND-22.
  • Figure 2A is a graph showing the activity detection results of the positive control BND-22 reporter gene method
  • Figure 2B is a graph showing the activity detection results of the mouse antibody 6-B8-C3-G6 reporter gene method
  • Figure 2C is a graph showing the activity detection results of the mouse antibody 40-H9-G4-D8 reporter gene method.
  • Example 5 Experiment to detect the effect of mouse antibodies on NK92 cell killing
  • mice antibodies 6-B8-C3-G6, 40-H9-G4-D8 and the positive control BND-22 in promoting NK92 cells to kill tumor cells was detected according to the following steps.
  • NK92 cells in the logarithmic proliferation phase were obtained, washed twice with PBS, and then the density was adjusted to 3 ⁇ 10 6 /mL with RPMI 1640 medium. 50 ⁇ L and 1.5 ⁇ 10 5 cells were inoculated into each well of a 96-well plate.
  • A375-HLA-G-Luciferase The constructed A375 cells stably transfected with HLA-G and luciferase (A375-HLA-G-Luciferase) were used as target cells.
  • A375-HLA-G-Luciferase cells in the logarithmic growth phase were taken, washed twice with 1 ⁇ PBS, and then the density was adjusted to 4 ⁇ 10 5 /mL with RPMI 1640 medium. 50 ⁇ L, 2 ⁇ 10 4 cells were inoculated into each well of a 96-well plate, so that the effector-target ratio was 7.5:1, and the final antibody concentrations in each well were 100 ⁇ g/mL, 20 ⁇ g/mL, 4 ⁇ g/mL, and 0 ⁇ g/mL, respectively.
  • the cells were co-cultured at 37°C and 5% CO 2 for 5 h.
  • Figure 3 shows the detection results of NK92 cell killing experiment. It can be seen that both mouse antibodies and positive controls can enhance the killing of A375 target cells by NK92 cells.
  • the activity of mouse antibodies 6-B8-C3-G6 and 40-H9-G4-D8 in enhancing killing is better than that of the positive control BND-22, especially under low concentration conditions.
  • Example 6 Experiment to detect the effect of mouse antibodies on promoting macrophage killing
  • mice antibodies 6-B8-C3-G6, 40-H9-G4-D8 and the positive control BND-22 in promoting primary macrophages to kill tumor cells was detected according to the following steps.
  • PBMC cells isolated from healthy subjects were taken, and the cell density was adjusted to 1 ⁇ 10 7 /mL using RPMI 1640 medium containing 1000U/mL GM-CSF and 10% FBS, and cultured overnight at 37°C and 5% CO 2.
  • Non-adherent suspended cells were removed, and cultured for 5 days using RPMI 1640 medium containing 1000U/mL GM-CSF and 10% FBS.
  • RPMI 1640 medium containing 20ng/mL IFN- ⁇ , 50ng/mL LPS, and 10% FBS was added for induction culture for 3 days to obtain M1 macrophages.
  • the induced macrophages were centrifuged at 500 g for 5 min, washed twice with 1 ⁇ PBS, and then the cell density was adjusted to 2 ⁇ 10 6 /mL with RPMI 1640 medium containing 10% FBS. 50 ⁇ L and 1 ⁇ 10 5 cells were inoculated into each well of a 96-well plate.
  • Figure 4 shows the detection results of the macrophage killing experiment. It can be seen that both mouse antibodies and positive controls can dose-dependently enhance the killing of A375 target cells by primary macrophages, and the killing enhancement activity of mouse antibodies 6-B8-C3-G6 and 40-H9-G4-D8 is comparable to that of the positive control BND-22.
  • the amino acid sequences of the light chain and heavy chain variable regions of the above-mentioned mouse antibodies 6-B8-C3-G6 and 40-H9-G4-D8 were obtained by hybridoma sequencing.
  • the heavy chain variable region was subcloned into the pcDNA3.4-hIgG4 expression vector, and the light chain variable region was subcloned into the pcDNA3.4-hIgKc expression vector to obtain the heavy chain expression vector and the light chain expression vector.
  • the DNA/LVTransm complex obtained above was added to 100 mL of 293F cells (cell density 1 ⁇ 10 6 /mL), and gently shaken to mix thoroughly.
  • the cells were cultured continuously for 7 days at 37°C, 5% CO 2 , and 130 rpm.
  • the culture supernatant was collected by centrifugation, filtered with a 0.45 ⁇ m filter membrane, and the chimeric antibodies were purified by protein A affinity chromatography.
  • the obtained chimeric antibodies were named 6-B8-C3-G6-chi and 40-H9-G4-D8-chi, respectively.
  • Table 3 and Figure 5 show the activity detection results of the reporter gene method. It can be seen that both the chimeric antibody and the positive control can dose-dependently activate the fluorescence signal of the reporter gene cell line, and the activity of the chimeric antibodies 6-B8-C3-G6-chi and 40-H9-G4-D8-chi is better than that of the positive control BND-22.
  • Example 9 Experiment to detect chimeric antibodies promoting NK92 cell killing
  • the chimeric antibodies 6-B8-C3-G6-chi, 40-H9-G4-D8-chi and the positive control BND-22 were tested according to the steps described in Example 5.
  • the final concentration of the antibody in each well is 20 ⁇ g/mL, 4 ⁇ g/mL, 0.8 ⁇ g/mL, 0.16 ⁇ g/mL, 0.032 ⁇ g/mL, 0.0064 ⁇ g/mL, 0.00128 ⁇ g/mL, and 0.00026 ⁇ g/mL, respectively.
  • Figure 6 shows the detection results of the NK92 cell killing experiment. It can be seen that both the chimeric antibody and the positive control can enhance the killing of A375 target cells by NK92 cells, and the chimeric antibodies 6-B8-C3-G6-chi and 40-H9-G4-D8-chi have better killing activity than the positive control BND-22.
  • Example 10 Experiment to detect chimeric antibodies promoting primary NK cell killing
  • A375-HLA-G-Luciferase cells in the logarithmic growth phase were digested with trypsin (purchased from Gibco) and collected, then centrifuged at 200 g for 5 min. The supernatant was discarded and washed once with detection buffer (RPMI 1640 medium). The cell density was adjusted to 4 ⁇ 10 5 /mL, and 25 ⁇ L and 1 ⁇ 10 4 cells were inoculated into each well of a 96-well plate.
  • Figure 7 shows the detection results of the primary NK cell killing experiment. It can be seen that both the chimeric antibody and the positive control can enhance the killing of A375 target cells by primary NK cells compared with the IgG4 isotype control.
  • the chimeric antibodies 6-B8-C3-G6-chi and 40-H9-G4-D8-chi have better killing activity than BND-22.
  • Example 11 Experiment to detect chimeric antibodies promoting macrophage killing
  • step (1) uses CD14+ magnetic beads (purchased from Miltenyi Biotec) to sort monocytes
  • step (4) The final concentration of antibodies in each well is 100 ⁇ g/mL, 20 ⁇ g/mL, and 4 ⁇ g/mL, respectively.
  • Figure 8 shows the detection results of the macrophage killing experiment.
  • both the chimeric antibody and the positive control can enhance the killing of primary macrophages to A375 target cells in a dose-dependent manner, and the chimeric antibodies 6-B8-C3-G6-chi and 40-H9-G4-D8-chi enhance the killing activity better than the positive control BND-22.
  • the chimeric antibodies 6-B8-C3-G6-chi and 40-H9-G4-D8-chi were humanized and designed based on the amino acid sequences of the heavy and light chains of their mouse antibodies, while keeping the CDR region sequences of the original antibodies unchanged. According to the results of germline alignment and antibody structure simulation, different human antibody templates were selected for the heavy and light chains, respectively. After humanization, the framework regions were back mutated to design candidate humanized antibody sequences. At the same time, the post-translational modification sites in the CDR region were eliminated by amino acid replacement.
  • the potential deamidation site QH of the light chain CDR3 of 40-H9-G4-D8-chi was replaced by SH
  • the potential isomerization site DS of the heavy chain CDR3 was replaced by DA, DV, TS or GT
  • the potential deamidation site QH of the light chain CDR3 of 6-B8-C3-G6-chi was replaced by SH or AH.
  • the heavy chain and light chain of the designed humanized antibody were gene synthesized separately, and the heavy chain was subcloned into the pcDNA3.4-hIgG4 expression vector, and the light chain was subcloned into the pcDNA3.4-hIgKc expression vector. After the constructed vector was verified by sequencing, it was transiently expressed in CHO cells.
  • the CDR sequences of representative humanized antibodies are shown in Table 4, and the variable region and constant region sequences are shown in Table 5.
  • Example 14 Experiment to detect the effect of humanized antibodies on promoting macrophage killing
  • FIG. 9A and 9B show the detection results of macrophage killing experiments. It can be seen that both humanized antibodies and positive controls can enhance the killing of A375 target cells by primary macrophages in a dose-dependent manner.
  • the humanized antibodies 40-H9-F1-M20, 6-B8-F2, 40-H9-F1-M17, 40-H9-F1, and 40-H9-F4-M19 have enhanced killing activity better than or equivalent to the positive control BND-22.
  • Example 15 In vivo efficacy of humanized antibodies in humanized mice
  • the humanized antibody 40-H9-F4-M19 and the positive control antibody BND-22 were studied for their in vivo efficacy in humanized mice.
  • B-Tg hLILRB1/hLILRB4 humanized C57BL/6 mice (purchased from Biocytogen) into which the human LILRB1/LILRB4 gene was transferred were used.
  • Female B-Tg hLILRB1/hLILRB4 mice were subcutaneously inoculated with 5E5/mouse mouse colon cancer MC38 cells overexpressing human HLA-G to establish a subcutaneous tumor-bearing model.
  • mice When the average tumor volume reached about 60 mm 3 , the mice were randomly assigned to three experimental groups, with 6 mice in each group, according to the tumor volume and body weight of the mice, and then the drug administration was started according to the scheme shown in Table 7. During the study, the survival and general condition of the mice were observed every day, the tumor volume and body weight were measured twice a week, and the tumor growth inhibition rate (TGI) was calculated.
  • TGI tumor growth inhibition rate
  • the tumor growth curve and body weight change trend graph to the end point of the study are shown in Figure 10A and Figure 10B, respectively.
  • the research results showed that the humanized antibody 40-H9-F4-M19 exhibited an anti-tumor effect in this mouse model, and its anti-tumor effect was higher than that of the same dose of BND-22. There was no significant decrease in body weight of animals in each group during the experiment.
  • X1 is I, N, F, V, S, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D;
  • X 2 is I, N, F, V, S, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D;
  • X 3 is I, N, F, V, S, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D;
  • X 4 is I, N, F, V, S, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D;
  • X 5 is I, N, F, V, S, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D;
  • X 6 is I, N, F, V, R, A, T, E, G, H, K, L, P, W, Y, C, M, Q or D.

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Abstract

一种抗LILRB1抗体或其抗原结合片段、制备方法和用途，所述抗LILRB1抗体或其抗原结合片段包含互补决定区的重链可变区HCDR1、HCDR2、HCDR3和/或互补决定区的轻链可变区LCDR1、LCDR2、LCDR3；所述抗LILRB1抗体或其抗原结合片段可以通过抑制肿瘤细胞的免疫逃逸机制从而使免疫细胞可以表现出其抗癌作用。

Description

抗LILRB1抗体或其抗原结合片段、制备方法和用途 技术领域
本申请属于免疫技术领域，具体涉及抗LILRB1抗体或其抗原结合片段、制备方法和应用。
背景技术
白细胞免疫球蛋白样受体亚家族B1(LILRB1；也称为ILT2、CD85j或LIR-1)是一种抑制性受体，其在细胞例如B细胞、T细胞、NK细胞、树突细胞、巨噬细胞和其他免疫细胞中表达。LILRB1结构为I型跨膜糖蛋白，具有结合配体和细胞内免疫受体酪氨酸抑制基序(ITIM)的胞外Ig样结构域，可以募集SHP1、SHP2、SHIP等下游磷酸酶，并经过一系列信号传导途径抑制免疫细胞的激活。
LILRB1是MHC I类抗原的受体，并且识别广谱的HLA-A、HLA-B、HLA-C和HLA-G，尤其倾向于结合HLA-G。同时，多种癌细胞过表达HLA-G以进行免疫逃逸。目前，LILRB1与HLA-G的相互作用已被证明可抑制免疫细胞的功能，从而促进肿瘤免疫逃逸。预期阻断LILRB1与HLA-G的结合可以恢复受抑制的免疫细胞活性，从而发挥抗肿瘤作用。
因此，需要开发与LILRB1结合并阻断LILRB1与HLA-G的药剂。
发明内容
针对现有技术存在的上述问题，本申请提供一种抗LILRB1抗体或其抗原结合片段、制备方法和用途，所述抗LILRB1抗体或其抗原结合片段可以通过抑制肿瘤细胞的免疫逃逸机制从而使免疫细胞可以表现出其抗癌作用。
本申请第一方面提供了一种抗LILRB1抗体或其抗原结合片段，其包含：
(a)包含下述互补决定区的重链可变区:
HCDR1，其包含SEQ ID NO:1或7所示的氨基酸序列，或与SEQ ID NO：1或7所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NO：1或7所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：1或7所示的氨基酸序列功能相同或相似；
HCDR2，其包含SEQ ID NO:2或8所示的氨基酸序列，或与SEQ ID NO：2或8所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NO：2或8所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：2或8所示的氨基酸序列功能相同或相似；
HCDR3，其包含SEQ ID NO:3、9或119所示的氨基酸序列，或与SEQ ID NO：3、9或119所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NO：3、9或119所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：3、9或119所示的氨基酸序列功能相同或相似；和/或
(b)包含下述互补决定区的轻链可变区:
LCDR1，其包含SEQ ID NO:4或10所示的氨基酸序列，或与SEQ ID NO：4或10所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NO：4或10所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：4或10所示的氨基酸序列功能相同或相似；
LCDR2，其包含SEQ ID NO:5所示的氨基酸序列，或与SEQ ID NO：5所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NO：5所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：5所示的氨基酸序列功能相同或相似；
LCDR3，其包含SEQ ID NO:6或11所示的氨基酸序列，或与SEQ ID NO：6或11所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NO：6或11所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：6或11所示的氨基酸序列功能相同或相似。
本申请所述经取代、缺失或添加一个或多个氨基酸为经取代、缺失或添加1个或2个氨基酸。在一些实施方式中，所述一个或多个氨基酸的取代、缺失或添加为保守修饰。
在本申请的一些实施方式中，所述抗LILRB1抗体或其抗原结合片段包含：HCDR1、HCDR2和HCDR3、 LCDR1、LCDR2和LCDR3，所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、2和3所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和6所示的氨基酸序列；
在本申请的一些实施方式中，所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、2和119所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和6所示的氨基酸序列；或
在本申请的一些实施方式中，所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和11所示的氨基酸序列。
在本申请的一些实施方式中，所述HCDR2具有SEQ ID NO：12所示的氨基酸序列。
在本申请的一些实施方式中，所述HCDR3具有SEQ ID NO：13所示的氨基酸序列。在本申请的一些实施方式中，所述HCDR3具有SEQ ID NO：93所示的氨基酸序列。
在本申请的一些实施方式中，所述LCDR3具有SEQ ID NO：14所示的氨基酸序列。
在本申请的一些实施方式中，所述LCDR3具有SEQ ID NO：15所示的氨基酸序列。
在本申请的一些具体实施方式中，所述抗LILRB1抗体或其抗原结合片段包含：HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和13所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列。或者，所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和93所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列。
在本申请的一些具体实施方式中，所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和15所示的氨基酸序列。
在本申请的一些实施方式中，所述LILRB1为哺乳动物LILRB1，所述抗体优选为鼠源抗体、猴源抗体、兔源抗体、嵌合抗体、人源化抗体、人源抗体。
在本申请的一些实施方式中，所述抗体为IgG抗体。优选为IgG1、IgG2、IgG3或IgG4抗体，更优选为IgG4抗体。
本申请提供的抗LILRB1抗体或其抗原结合片段进一步包含重链恒定区和轻链恒定区。
在本申请的一些实施方式中，所述重链恒定区为IgG1、IgG2、IgG3或IgG4的恒定区，所述轻链恒定区为抗体κ链或λ链的恒定区。
在本申请的一些实施方式中，所述抗LILRB1抗体或其抗原结合片段选自单克隆抗体、多克隆抗体、Fab片段、Fab'片段、F(ab')2片段、Fd片段、Fv片段、dAb片段、分离的CDR区、scFv和纳米抗体。
在本申请的一些具体实施方式中，所述抗LILRB1抗体为单克隆抗体。
在本申请的一些具体实施方式中，所述抗体为鼠源IgG4抗体。或者，所述所述抗体为人源化IgG4抗体。
本申请第二方面提供了一种核酸分子，所述核酸分子编码第一方面所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
本申请第三方面提供了一种重组载体，所述重组载体包含第二方面所述的核酸分子。
本申请第四方面提供了一种重组细胞，所述重组细胞包含第二方面所述的核酸分子或第三方面所述的包含所述核酸分子的重组载体。
本申请第五方面提供了一种制备抗LILRB1抗体或其抗原结合片段的方法，所述方法包括培养第四方面所述的重组细胞。
在本申请的一些实施方式中，重组细胞为重组CHO细胞。
本申请第六方面提供了第一方面所述的抗体或其抗原结合片段、第二方面所述的核酸分子、第三方面所述的重组载体或第四方面所述的重组细胞在制备药物中的用途；
在本申请的一些实施方式中，所述药物为预防和/或治疗癌症的药物。
在一些实施方式中，本申请涉及用于预防和/或治疗LILRB1相关疾病的本申请第一方面所述的抗体或其抗原结合片段、第二方面所述的核酸分子、第三方面所述的重组载体或第四方面所述的重组细胞。或者，本申请涉及一种预防和/或治疗LILRB1相关疾病的方法，包括向有需要的受试者给予本申请第一方面所述的抗LILRB1抗体或其抗原结合片段、第二方面所述的核酸分子、第三方面所述的重组载体或第四方面所述的重组细胞。
本申请第七方面提供了第一方面所述的抗LILRB1抗体或其抗原结合片段、或第二方面所述的核酸分子在制备诊断LILRB1相关疾病的试剂中的用途。在一些实施方式中，本申请涉及用于诊断LILRB1相关疾病的本申请第一方面所述的抗LILRB1抗体或其抗原结合片段、或第二方面所述的核酸分子。
在本申请的一些实施方式中，所述疾病为癌症。
在本申请的一些实施方式中，所述癌症为表达MHC I类分子的癌症，优选为表达HLA-G的癌症。所述癌症的特征在于MHC I类分子的过表达；优选在于HLA-G的过表达。
在本申请的一些实施方式中，本申请提供了一种嵌合抗原受体(CAR)，包含胞外抗原结合结构域、跨膜结构域和胞内信号转导结构域，其中，所述胞外抗原结合结构域包含本文所述的抗LILRB1抗体或其抗原结合片段。
在本申请的一些实施方式中，本申请提供了一种试剂盒，包含本申请所述的抗LILRB1抗体或其抗原结合片段或者嵌合抗原受体。
本申请的有益效果：
本申请中提供的抗LILRB1抗体或其抗原结合片段可以通过抑制肿瘤细胞的免疫逃逸机制从而使免疫细胞可以表现出其抗癌作用，具有促进NK92细胞、巨噬细胞对肿瘤细胞杀伤的活性，可以与LILRB1结合，阻断LILRB1与HLA-G的结合。
附图说明
图1为本申请阳性对照BND-22的报告基因法活性结果图；
图2为鼠源抗体报告基因法的活性检测结果图；其中，图2A为阳性对照BND-22报告基因法的活性检测结果图，图2B为鼠源抗体6-B8-C3-G6报告基因法的活性检测结果图，图2C为鼠源抗体40-H9-G4-D8报告基因法的活性检测结果图；
图3为鼠源抗体促进NK92细胞杀伤实验的检测结果图；
图4为鼠源抗体促进巨噬细胞杀伤实验的检测结果图；
图5为嵌合抗体报告基因法的活性检测结果图；
图6为嵌合抗体促进NK92细胞杀伤实验的检测结果图；
图7为嵌合抗体促进原代NK细胞杀伤实验的检测结果图；
图8为嵌合抗体促进巨噬细胞杀伤实验的检测结果图；
图9A为人源化抗体促进巨噬细胞杀伤实验的检测结果图；
图9B为人源化抗体促进巨噬细胞杀伤实验的检测结果图；
图10A为人源化抗体体内药效研究的肿瘤生长曲线图；
图10B为人源化抗体体内药效研究的体重变化趋势图。
具体实施方式
为使本申请更加容易理解，下面将结合实施例来详细说明本申请，这些实施例仅起说明性作用，并不会限制本申请的应用范围。
除非另外定义，否则本文使用的各技术和科学术语具有与本公开所属领域的普通技术人员通常理解的含义相同的含义。参见如Singleton等，Dictionary of Microbiology and Molecular Biology 2nd ed.，J.Wiley&Sons(New York，NY 1994)；Sambrook等，Molecular Cloning，A Laboratory Manual，Cold Springs Harbor Press(Cold Springs Harbor，NY 1989)；Davis等，Basic Methods in Molecular Biology，Elsevier Science Publishing Inc.，New York,USA(2012)；Abbas等，Cellular and Molecular Immunology，Elsevier Science Health Science div(2009)；何维等，医学免疫学(第2版)，人民卫生出版社，2010年。
除非另有说明，本文使用的术语“包含、包括和含有(comprise、comprises和comprising)”或其等同物(contain、contains、containing、include、includes、including)为开放式表述，意味着除所列出的要素、组分和步骤外，还可涵盖其它未指明的要素、组分和步骤。
除非另有说明，本文使用的术语“任选的”或“任选地”表示其所修饰的对象或事件存在或不存在、或者发生或不发生。除非上下文另有明确指示，本文中的词语“或”意在包括“和”。
如本文所用，“约”表示在本领域普通技术人员判定的对特定值可以接受的误差范围内，其部分取决于如何测量或测定该值，即测量系统的限制。例如，“约”按照本领域实践可表示1倍或超过1倍标准偏差以内。或者，“约”可以表示多至20％(即±20％)的范围，例如在所给定的具体数值范围±10％范围、±5％范围、±2％范围内、±1％范围内或±0.5％范围内波动。此外，特别对于生物学系统或方法，该术语可以表 示多至一个数量级或多至某值的5倍。当本申请或权利要求中给出特定值时，除非另有说明，“约”的含义应认为是在该特定值的可接受的误差范围内。在本文中，除非另有说明，各测定值、步骤参数或条件的值默认均由“约”修饰。
在本文中，除非上下文另有明确规定，否则单数术语涵盖复数指代物，反之亦然。
除非另有说明，本文使用的术语“治疗”意味着在给药后可以抑制、遏制、减轻、改善、减缓、缓解或消除疾病或其相关症状，可以迟滞、延缓、减慢、中止或终止疾病或其相关症状的进展，或者可以阻止、控制或减少疾病或其相关症状的复发。对于疾病的预防和治疗来说，治疗有效量或预防有效量可由临床医师根据受试者的个人状况、疾病严重程度、性别、年龄、体重、给药方式等通过常规方法或经验确定。
除非另有说明，本文使用的术语“受试者”涵盖任何脊椎动物，例如哺乳动物，诸如人、非人灵长类动物、羊、犬、猫、马、牛、鸡、猪、鼠等。优选地，本申请中的受试者是人。
术语“抗体”是指由免疫球蛋白基因编码并且能够结合抗原的多肽。术语“互补决定区”可以指在抗体的可变区中赋予抗原结合特异性的部分，并且可以指在免疫球蛋白的重链或轻链的高可变区中发现的氨基酸序列。重链可以包括三个互补决定区CDR(HCDR1、HCDR2和HCDR3；以及轻链可以包括三个互补决定区CDR(LCDR1、LCDR2和LCDR3)。所述CDR可以提供在抗体与其抗原或抗原表位的结合中起重要作用的接触残基。
除非另有说明，本文使用的符号“X”代表未指明的氨基酸(Unspecified Amino Acid)，通过相关表述中的定义来指定其涵盖的范围，为了区别同一氨基酸序列中的多个“X”，对先后出现的多个X分别进行编号(即，撰写成Xn)并分别对其涵盖的范围进行定义。
在本文中，序列之间的同一性百分比(同源性程度)可以通过例如使用万维网(例如www.ncbi.nlm.nih.gov)上通常用于此目的的免费可用的计算机程序(例如具有默认设置的BLASTp或BLASTn)对两个序列进行比较来确定。
在一些实施方式中，提供了一种抗LILRB1抗体或其抗原结合片段，其包含：
(a)包含下述互补决定区的重链可变区:
HCDR1，其包含SEQ ID NO:1或7所示的氨基酸序列或与SEQ ID NO：1或7所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NO：1或7所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：1或7所示的氨基酸序列功能相同或相似；
HCDR2，其包含SEQ ID NO:2或8所示的氨基酸序列或与SEQ ID NO：2或8所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NO：2或8所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：2或8所示的氨基酸序列功能相同或相似；
HCDR3，其包含SEQ ID NO:3、9或119所示的氨基酸序列或与SEQ ID NO：3、9或119所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NO：3、9或119所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：3、9或119所示的氨基酸序列功能相同或相似；和/或
(b)包含下述互补决定区的轻链可变区:
LCDR1，其包含SEQ ID NO:4或10所示的氨基酸序列或与SEQ ID NO：4或10所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NO：4或10所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：4或10所示的氨基酸序列功能相同或相似；
LCDR2，其包含SEQ ID NO:5所示的氨基酸序列或与SEQ ID NO：5所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NO：5所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：5所示的氨基酸序列功能相同或相似；
LCDR3，其包含SEQ ID NO:6或11所示的氨基酸序列或与SEQ ID NO：6或11所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或SEQ ID NO：6或11所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与SEQ ID NO：6或11所示的氨基酸序列功能相同或相似。
上述CDR的范围已根据ImMunoGenTics(IMGT)编号规则进行了定义，但是本领域技术人员可以理解的是，根据AbM编号规则、Kabat编号规则、Chothia编号规则和Contact编号规则(基于对可用复杂晶体结构的分析)等中的任一种或多种进行编号的CDR序列也落入了本申请的保护范围内。
本申请所述经取代、缺失或添加一个或多个氨基酸为经取代、缺失或添加1个或2个氨基酸。
在本申请的一些实施方式中，所述抗LILRB1抗体或其抗原结合片段包含：所述包含互补决定区的重链可变区和所述包含互补决定区的轻链可变区。
在本申请的一些实施方式中，所述抗LILRB1抗体或其抗原结合片段包含：HCDR1、HCDR2和HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、2和3或119所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和6所示的氨基酸序列。
在本申请的一些实施方式中，HCDR1、HCDR2和HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和11所示的氨基酸序列。
在本申请的一些实施方式中，所述HCDR2具有SEQ ID NO：12所示的氨基酸序列。
在本申请的一些实施方式中，所述HCDR3具有SEQ ID NO：13所示的氨基酸序列。
在本申请的一些实施方式中，所述LCDR3具有SEQ ID NO：14所示的氨基酸序列。
在本申请的一些实施方式中，所述LCDR3具有SEQ ID NO：15所示的氨基酸序列。
在申请中，以SEQ ID NO：2示出的氨基酸序列IYPGX1GNT涵盖了抗体的重链CDR2(HCDR2)的序列(IYPGDGNT)以及将所述HCDR2中的X1突变为其它氨基酸所得到的经突变的HCDR2的序列。
本申请中，以SEQ ID NO：3示出的氨基酸序列ASYX3SSWFPY涵盖了抗体的重链CDR3(HCDR3)的序列(ASYDSSWFPY)以及将所述HCDR3中的X3突变为其它氨基酸所得到的经突变的HCDR3的序列。
本申请中，以SEQ ID NO：119示出的氨基酸序列ASYX5X6SWFPY涵盖了将抗体的重链CDR3(HCDR3)的序列(ASYDSSWFPY)中的X6突变为其它氨基酸，同时将X5突变为其它氨基酸或不进行突变所得到的经突变的HCDR3的序列。
在本申请中，以SEQ ID NO：6示出的氨基酸序列X2HFWGAPWT涵盖了抗体的轻链CDR3(LCDR3)的序列(QHFWGAPWT)以及将所述LCDR3中的X2突变为其它氨基酸所得到的经突变的LCDR3的序列。
在本申请中，以SEQ ID NO：11示出的氨基酸序列X4HFWGSPWT涵盖了抗体的轻链CDR3(LCDR3)的序列(QHFWGSPWT)以及将所述LCDR3中的X4突变为其它氨基酸所得到的经突变的LCDR3的序列。
在本申请中，所述X1可选自I、N、F、V、S、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D；在一些实施方式中，所述X1可选自D、S或A；优选地，所述X1为D。
在本申请中，所述X2可选自I、N、F、V、S、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D；在一些实施方式中，所述X2可选自Q、S或A；优选地，所述X2为Q。
在本申请中，所述X3可选自I、N、F、V、S、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D；在一些实施方式中，所述X3可选自D、T、S或A；优选地，所述X3为D。
在本申请中，X4可选自I、N、F、V、S、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D；在一些实施方式中，所述X4可选自Q、S或A；优选地，所述X4为Q。
在本申请中，X5可选自I、N、F、V、S、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D；在一些实施方式中，所述X5可选自D、T、S、G或A；优选地，所述X5为D。
在本申请中，X6可选自I、N、F、V、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D；在一些实施方式中，所述X6可选自A、V或T；优选地，所述X6为V。
在本申请的一些具体实施方式中，所述HCDR2可包含选自SEQ ID NO：12、16-34所示的氨基酸序列。
在本申请的一些具体实施方式中，所述HCDR3可包含选自SEQ ID NO：13、35-53、92-94所示的氨基酸序列。
在本申请的一些具体实施方式中，所述LCDR3可包含选自SEQ ID NO：14、15、54-91(优选SEQ ID NO：14、54-72)所示的氨基酸序列。
在本申请的一些具体实施方式中，所述抗LILRB1抗体或其抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中，
(1)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和13所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
(2)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和15所示的氨基酸序列；
(3)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和13所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和58所示的氨基酸序列；
(4)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和92所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
(5)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和42所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
(6)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和93所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
(7)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和94所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
(8)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和77所示的氨基酸序列；或者
(9)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和79所示的氨基酸序列。
在优选的实施方式中，所述抗LILRB1抗体或其抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中：
(1)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和93所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
(2)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和94所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
(3)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和77所示的氨基酸序列；
(4)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和92所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；或者
(5)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和13所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列。
在更为优选的实施方式中，所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和93所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列。
本申请提供的抗LILRB1抗体或其抗原结合片段包含重链可变区和轻链可变区。
在本申请的一些实施方式中，所述重链可变区包括SEQ ID NOs：95、102-106、110、112所示的氨基酸序列或与SEQ ID NOs：95、102-106、110、112所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NOs：95、102-106、110、112所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NOs：95、102-106、110、112所示的氨基酸序列功能相同或相似。在本申请的一些实施方式中，所述轻链可变区包括SEQ ID NOs：96-101、107-109、111所示的氨基酸序列或与SEQ ID NOs：96-101、107-109、111所示的氨基酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或SEQ ID NOs：96-101、107-109、111所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NOs：96-101、107-109、111所示的氨基酸序列功能相同或相似。
在本申请的一些实施方式中，抗LILRB1抗体或其抗原结合片段包含重链可变区和轻链可变区：
(1)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：96所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(2)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：97所示的氨基酸序列或与其具有至少80％、81％、 82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(3)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：98所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(4)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：99所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(5)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：100所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(6)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：101所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(7)所述重链可变区包含SEQ ID NO：102所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：96所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(8)所述重链可变区包含SEQ ID NO：103所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：96所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(9)所述重链可变区包含SEQ ID NO：104所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：96所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(10)所述重链可变区包含SEQ ID NO：105所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：96所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(11)所述重链可变区包含SEQ ID NO：102所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：98所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(12)所述重链可变区包含SEQ ID NO：103所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：98所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(13)所述重链可变区包含SEQ ID NO：104所示的氨基酸序列或与其具有至少80％、81％、82％、 83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：98所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(14)所述重链可变区包含SEQ ID NO：105所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：98所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(15)所述重链可变区包含SEQ ID NO：106所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：107所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(16)所述重链可变区包含SEQ ID NO：106所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：108所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(17)所述重链可变区包含SEQ ID NO：106所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：109所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(18)所述重链可变区包含SEQ ID NO：110所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：111所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(19)所述重链可变区包含SEQ ID NO：110所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：109所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(20)所述重链可变区包含SEQ ID NO：112所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：107所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
(21)所述重链可变区包含SEQ ID NO：112所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：108所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。
在优选的实施方式中，抗LILRB1抗体或其抗原结合片段包含：
(1)SEQ ID NO：104所示的重链可变区，SEQ ID NO：98所示的轻链可变区；
(2)SEQ ID NO：105所示的重链可变区，SEQ ID NO：96所示的轻链可变区；
(3)SEQ ID NO：106所示的重链可变区，SEQ ID NO：107所示的轻链可变区；
(4)SEQ ID NO：102所示的重链可变区，SEQ ID NO：96所示的轻链可变区；或者
(5)SEQ ID NO：95所示的重链可变区，SEQ ID NO：96所示的轻链可变区。
在更优选的实施方式中，抗LILRB1抗体或其抗原结合片段包含SEQ ID NO：104所示的重链可变区，SEQ ID NO：98所示的轻链可变区。
在本申请的一些实施方式中，所述LILRB1为哺乳动物LILRB1，所述哺乳动物包括人和非人灵长类 动物，如食蟹猴；所述抗体优选为鼠源抗体、猴源抗体、兔源抗体、嵌合抗体、人源化抗体或人源抗体。
在本申请的一些实施方式中，所述抗体为IgG抗体。为IgG1、IgG2、IgG3或IgG4抗体，更优选为IgG4抗体。
在本申请的一些具体实施方式中，所述抗体为鼠源IgG4抗体或人源化IgG4抗体。
本申请提供的抗LILRB1抗体或其抗原结合片段进一步包含重链恒定区和轻链恒定区。
在本申请的一些实施方式中，所述重链恒定区为IgG1、IgG2、IgG3或IgG4的恒定区，所述轻链恒定区为抗体κ链或λ链的恒定区。
在本申请的具体实施方式中，所述重链恒定区为IgG4的恒定区，其氨基酸序列如SEQ ID NO：113所示或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；所述轻链恒定区为抗体κ链的恒定区，其氨基酸序列如SEQ ID NO：114所示或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性。
在本申请的一些实施方式中，所述抗LILRB1抗体或其抗原结合片段包含重链和轻链，其中，
(1)所述重链包含具有SEQ ID NO：104所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链可变区和具有SEQ ID NO：113所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链恒定区，
所述轻链包含具有SEQ ID NO：98所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链可变区和具有SEQ ID NO：114所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链恒定区；
(2)所述重链包含具有SEQ ID NO：105所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链可变区和具有SEQ ID NO：113所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链恒定区，
所述轻链包含具有SEQ ID NO：96所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链可变区和具有SEQ ID NO：114所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链恒定区；
(3)所述重链包含具有SEQ ID NO：106所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链可变区和具有SEQ ID NO：113所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链恒定区，
所述轻链包含具有SEQ ID NO：107所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链可变区和具有SEQ ID NO：114所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链恒定区；
(4)所述重链包含具有SEQ ID NO：102所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链可变区和具有SEQ ID NO：113所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链恒定区，
所述轻链包含具有SEQ ID NO：96所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链可变区和具有SEQ ID NO：114所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或 99％同一性的氨基酸序列的轻链恒定区；或者
(5)所述重链包含具有SEQ ID NO：95所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链可变区和具有SEQ ID NO：113所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链恒定区，
所述轻链包含具有SEQ ID NO：96所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链可变区和具有SEQ ID NO：114所示的氨基酸序列或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链恒定区。
在本申请的一些实施方式中，所述抗LILRB1抗体或其抗原结合片段包含重链和轻链，其中，
(1)所述重链包含SEQ ID NO：104所示的重链可变区和SEQ ID NO：113所示的重链恒定区，所述轻链包含SEQ ID NO：98所示的轻链可变区和SEQ ID NO：114所示的轻链恒定区；
(2)SEQ ID NO：105所示的重链可变区和SEQ ID NO：113所示的重链恒定区，所述轻链包含SEQ ID NO：96所示的轻链可变区和SEQ ID NO：114所示的轻链恒定区；
(3)SEQ ID NO：106所示的重链可变区和SEQ ID NO：113所示的重链恒定区，所述轻链包含SEQ ID NO：107所示的轻链可变区和SEQ ID NO：114所示的轻链恒定区；
(4)SEQ ID NO：102所示的重链可变区和SEQ ID NO：113所示的重链恒定区，所述轻链包含SEQ ID NO：96所示的轻链可变区和SEQ ID NO：114所示的轻链恒定区；或者
(5)SEQ ID NO：95所示的重链可变区和SEQ ID NO：113所示的重链恒定区，所述轻链包含SEQ ID NO：96所示的轻链可变区和SEQ ID NO：114所示的轻链恒定区。
在本申请的一些实施方式中，所述抗LILRB1抗体或其抗原结合片段选自单克隆抗体、多克隆抗体、Fab片段、Fab'片段、F(ab')2片段、Fd片段、Fv片段、dAb片段、分离的CDR区、scFv和纳米抗体。
在本申请的一些具体实施方式中，所述抗LILRB1抗体为单克隆抗体。
在本申请中，还可对抗LILRB1抗体或其抗原结合片段进行保守修饰。保守修饰是指不会明显影响或改变抗体结合特性的氨基酸修饰。例如，保守氨基酸取代可为采用同类(具有类似的化学性质或者功能)的另一种氨基酸进行取代。作为示例，可根据氨基酸的侧链性质将其分为：(1)非极性氨基酸：Ala(A)、Val(V)、Leu(L)、Ile(I)、Pro(P)、Phe(F)、Trp(W)、Met(M)；(2)不带电荷的极性氨基酸：Gly(G)、Ser(S)、Thr(T)、Cys(C)、Tyr(Y)、Asn(N)、Gln(Q)；(3)酸性氨基酸：Asp(D)、Glu(E)；(4)碱性氨基酸：Lys(K)、Arg(R)、His(H)。或者，可基于共同的侧链特性将氨基酸分为：(1)疏水氨基酸：Met、Ala、Val、Leu、Ile；(2)中性亲水氨基酸：Cys、Ser、Thr、Asn、Gln；(3)酸性氨基酸：Asp、Glu；(4)碱性氨基酸：His、Lys、Arg；(5)影响链取向的氨基酸：Gly、Pro；(6)芳香族氨基酸：Trp、Tyr、Phe。
在本申请的一些实施方式中，所述重链恒定区为IgG1、IgG2、IgG3或IgG4的恒定区，所述轻链恒定区为抗体κ链或λ链的恒定区。
在本申请的一些实施方式中，所述抗LILRB1抗体或其抗原结合片段选自单克隆抗体、Fab片段、Fab'片段、F(ab')2片段、Fd片段、Fv片段、dAb片段、分离的CDR区、scFv和纳米抗体。
在本申请的一些具体实施方式中，所述抗LILRB1抗体为单克隆抗体。
在本申请的一些具体实施方式中，所述抗体为鼠源IgG4抗体。或者，所述所述抗体为人源化IgG4抗体。
作为非限制性示例，本申请所述的抗LILRB1抗体或其抗原结合片段可展现出下述性质：
(1)与LILRB1结合；和/或
(2)阻断LILRB1与MHC I类分子(优选HLA-G)的结合；和/或
(3)增强免疫细胞对肿瘤细胞的杀伤作用。
在一些实施方式中，与LILRB1结合包括与哺乳动物的LILRB1结合，所述哺乳动物包括人、非人灵长类动物(如食蟹猴)。在一些实施方式中，与LILRB1结合包括与表达LILRB1的细胞结合。所述抗LILRB1抗体或其抗原结合片段能够与人和食蟹猴LILRB1交叉结合，或与表达人或食蟹猴LILRB1的细胞交叉结合。
在一些实施方式中，增强免疫细胞对肿瘤细胞的杀伤作用包括增强NK细胞和/或巨噬细胞对肿瘤细胞 的杀伤作用。
在一些实施方式中，所述抗LILRB1抗体或其抗原结合片段阻断HLA-G结合LILRB1的IC50＜12.5μg/mL、＜2.0μg/mL、＜1.0μg/mL或＜0.5μg/mL。
在本申请的一些具体实施方式中，提供了一种核酸分子，所述核酸分子编码所述抗LILRB1抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
在本申请的一些实施方式中，提供了一种重组载体，所述重组载体包含所述核酸分子。
术语“载体”是指用于在宿主细胞中表达靶基因的工具，例如质粒载体、粘粒载体和病毒载体如噬菌体载体、慢病毒载体、腺病毒载体、逆转录病毒载体和腺相关病毒载体。重组载体可以由以下构建或通过操纵以下来构建：质粒(例如，pSC101、pGV1106、pACYC177、ColE1、pKT230、pME290、pBR322、pUC8/9、pUC6、pBD9、pHC79、pIJ61、pLAFR1、pHV14、pGEX系列、pET系列、pUC19等)、噬菌体(例如，λgt4λB、λ-Charon、λΔz1、M13等)或病毒载体(例如，SV40等)，这是本领域中常用的。
在重组载体中，核酸分子可以与启动子可操作地连接。术语“可操作地连接”旨在涉及目标核苷酸序列与表达调控序列(例如，启动子序列)之间的功能连接。当“可操作地连接”时，调控元件可以控制目标多核苷酸的转录和/或翻译。
重组载体通常可以构建为克隆载体或表达载体。对于重组表达载体，可以使用相关领域中通常可获得的用于在植物、动物或微生物细胞中表达外来蛋白质的载体。本领域中众所周知的各种方法可以用于构建重组载体。
为了在宿主例如原核或真核细胞中使用，可以相应地构建重组载体。例如，当构建载体作为用于原核宿主的表达载体时，所述载体通常包括用于转录的强启动子(例如，pLλ启动子、CMV启动子、trp启动子、lac启动子、tac启动子、T7启动子等)、用于启动翻译的核糖体结合位点和转录/翻译终止序列。另一方面，用于真核宿主的表达载体包括可在真核细胞中操作的复制起点，例如f1复制起点、SV40复制起点、pMB1复制起点、腺病毒复制起点、AAV复制起点和BBV复制起点，但不限于此。另外，表达载体通常包括源自哺乳动物细胞基因组的启动子(例如金属硫蛋白启动子)或源自哺乳动物病毒的启动子(例如腺病毒晚期启动子、牛痘病毒7.5K启动子、SV40启动子、巨细胞病毒启动子、HSV的tk启动子等)，以及作为转录终止序列的多聚腺苷酸化序列。可以通过将重组载体引入合适的宿主细胞来制备重组细胞。本领域已知的任何宿主细胞都可以用于本公开中，只要它允许重组载体以稳定的方式连续克隆和表达。可用于本公开的原核宿主细胞的实例可以选自大肠杆菌(E.coli)如大肠杆菌JM109、大肠杆菌BL21、大肠杆菌RR1、大肠杆菌LE392、大肠杆菌B、大肠杆菌X1776、大肠杆菌W3110，芽孢杆菌属菌种如枯草芽孢杆菌(Bacillus subtilis)和苏云金芽孢杆菌(Bacillus thuringiensis)，以及肠杆菌科菌株，例如鼠伤寒沙门氏菌(Salmonella typhimurium)、粘质沙雷氏菌(Serratia marcescens)和各种假单胞菌菌种。可用于转化的真核宿主细胞可以选自但不限于酿酒酵母(Saccharomyces cerevisiae)、昆虫细胞和动物细胞，例如Sp2/0、CHO(中国仓鼠卵巢)K1、CHO DG44、CHO S、CHO DXB11、CHO GS-KO、PER.C6、W138、BHK、COS-7、293、HepG2、Huh7、3T3、RIN、MDCK等。可以使用相关领域中众所周知的方法将核酸分子或携带核酸分子的重组载体引入(转染)到宿主细胞中。例如，当宿主细胞是原核细胞时，可以使用CaCl2或电穿孔方法进行这种转染。对于真核宿主细胞，可以使用但不限于显微注射、磷酸钙沉淀、电穿孔、脂质体介导的转染或粒子轰击来实现基因引入。
为了选择转化的宿主细胞，可以根据本领域中众所周知的方法利用与选择标志物相关的表型。例如，当所述选择标志物是赋予某些抗生素抗性的基因时，宿主细胞可以在存在抗生素的情况下在培养基中生长以选择目标转化体。
在本申请的一些实施方式中，提供了一种重组细胞，所述重组细胞包含所述核酸分子或包含所述核酸分子的重组载体。
在一些实施方式中，重组细胞为原核细胞。在另一些实施方式中，重组细胞为真核细胞。在一些实施方式中，重组细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其他细胞。哺乳动物细胞例如为中国仓鼠卵巢细胞(CHO)、人肾上皮细胞系293细胞或293T细胞。
在本申请的一些实施方式中，重组细胞为重组CHO细胞。
在本申请的一些实施方式中，提供了一种制备抗LILRB1抗体或其抗原结合片段的方法，所述方法包括培养所述重组细胞。所述方法还可以包括在表达或培养步骤之后从细胞培养物中分离和/或纯化所述抗体或其片段，例如采用Protein A亲和柱纯化。
在本申请的一些实施方式中，提供了所述抗LILRB1抗体或其抗原结合片段、或所述核酸分子、或所述重组载体、或所述重组细胞在制备药物中的用途。
在本申请的一些实施方式中，所述药物为预防和/治疗癌症的药物。
在本申请的一些实施方式中，提供了所述抗LILRB1抗体或其抗原结合片段或所述核酸分子在制备诊断LILRB1相关疾病的试剂中的用途。
在本申请的一些实施方式中，所述LILRB1相关疾病为癌症。
根据本公开可杀伤的癌细胞包括但不限于来自以下的癌细胞：膀胱、血液、骨、骨髓、脑、乳腺、结肠、食管、胃肠、牙龈、头、肾、肝、肺、鼻咽、颈、卵巢、前列腺、皮肤、胃、胰腺、睾丸、舌头、宫颈或子宫。
另外，癌症可具体地具有以下组织学类型，尽管不限于这些：肿瘤，恶性肿瘤；癌；未分化的癌；巨细胞和梭形细胞癌；小细胞癌；乳头状癌；鳞状细胞癌；淋巴上皮癌；基底细胞癌；皮母质癌；移行细胞癌；乳头状移行细胞癌；腺癌；胃泌素瘤，恶性肿瘤；胆管癌；肝细胞癌；混合型肝细胞癌和胆管癌；小梁腺癌；腺样囊性癌；腺瘤息肉中的腺瘤；腺癌，家族性结肠息肉病；实体癌；类癌肿瘤，恶性肿瘤；分支肺泡腺癌；乳头状腺癌；嫌色细胞癌；嗜酸性细胞癌；嗜酸性腺癌；嗜碱性细胞癌；透明细胞腺癌；颗粒细胞癌；滤泡腺癌；乳头状和滤泡腺癌；非包封硬化性癌；肾上腺皮质癌；内膜样癌；皮肤附属器癌；大汗腺腺癌；皮脂腺癌；耵聍腺癌；粘液表皮样癌；囊腺癌；乳头状囊腺癌；乳头状浆液性囊腺癌；粘液性囊腺癌；粘液腺癌；印戒细胞癌；浸润性导管癌；髓样癌；小叶癌；炎性癌；乳腺佩吉特病；腺泡细胞癌；腺鳞癌；腺癌伴鳞状上皮化生；胸腺瘤，恶性肿瘤；卵巢间质肿瘤，恶性肿瘤；泡膜细胞瘤，恶性肿瘤；粒层细胞瘤，恶性肿瘤；男性细胞瘤，恶性肿瘤；塞尔托利氏细胞癌；莱狄希细胞瘤，恶性肿瘤；脂质细胞瘤，恶性肿瘤；副神经节瘤，恶性肿瘤；乳腺外副神经节瘤，恶性肿瘤；嗜铬细胞瘤；血管球肉瘤(glomangiosarcoma)；恶性黑色素瘤；无色素性黑色素瘤；表面扩张黑色素瘤；巨大色素痣中的恶性黑色素瘤；上皮样细胞黑色素瘤；蓝色痣，恶性肿瘤；肉瘤；纤维肉瘤；纤维组织细胞瘤，恶性肿瘤；粘液肉瘤；脂肪肉瘤；平滑肌肉瘤；横纹肌肉瘤；胚胎性横纹肌肉瘤；腺泡状横纹肌肉瘤；间质肉瘤；混合肿瘤，恶性肿瘤；苗勒管混合肿瘤；肾母细胞瘤；肝母细胞瘤；癌肉瘤；间质瘤，恶性肿瘤；布伦纳肿瘤，恶性肿瘤；叶状肿瘤，恶性肿瘤；滑膜肉瘤；间皮瘤，恶性肿瘤；无性细胞瘤；胚胎性癌；畸胎瘤，恶性肿瘤；甲状腺肿样卵巢瘤，恶性肿瘤；绒毛膜癌；中肾瘤，恶性肿瘤；血管肉瘤；血管内皮瘤，恶性肿瘤；卡波济氏肉瘤；血管外皮细胞瘤，恶性肿瘤；淋巴管肉瘤；骨肉瘤；皮质旁成骨肉瘤；软骨肉瘤；软骨母细胞瘤，恶性肿瘤；间质性软骨肉瘤；骨巨细胞肿瘤；尤文氏肉瘤；牙源性肿瘤，恶性肿瘤；成釉细胞牙肉瘤；成釉细胞瘤，恶性肿瘤；成釉细胞纤维肉瘤；松果体瘤，恶性肿瘤；脊索瘤；胶质瘤，恶性肿瘤；室管膜瘤；星形细胞瘤；原浆型星形细胞瘤；纤维型星形细胞瘤；星形母细胞瘤；胶质母细胞瘤；少突神经胶质瘤；少突神经胶质母细胞瘤；原始神经外胚层瘤；小脑肉瘤；神经节母细胞瘤；神经母细胞瘤；视网膜母细胞瘤；嗅神经源性肿瘤；脑膜瘤，恶性肿瘤；神经纤维肉瘤；神经鞘瘤，恶性肿瘤；颗粒细胞瘤，恶性肿瘤；恶性淋巴瘤；霍奇金病；副肉芽肿；恶性淋巴瘤，小淋巴细胞；恶性淋巴瘤，大细胞，弥漫性；恶性淋巴瘤，滤泡状；蕈状真菌病；其他指定的非霍奇金淋巴瘤；恶性组织细胞增多症；多发性骨髓瘤；肥大细胞肉瘤；免疫增生性小肠疾病；白血病；淋巴性白血病；浆细胞白血病；红白血病；淋巴肉瘤细胞白血病；髓性白血病；嗜碱细胞性白血病；嗜酸粒细胞性白血病；单核细胞性白血病；肥大细胞白血病；巨核细胞白血病；髓系肉瘤和毛细胞白血病。在某些方面，肿瘤可包括骨肉瘤、血管肉瘤、横纹肌肉瘤、平滑肌肉瘤、尤文肉瘤、胶质母细胞瘤、神经母细胞瘤或白血病。在具体实施方式中，所述癌症为表达HLA-G的黑色素瘤、结肠癌。
在本申请的一些实施方式中，所述癌症为表达MHC I类分子的癌症，优选为表达HLA-G的癌症。所述癌症的特征在于MHC I类分子的过表达；优选在于HLA-G的过表达。
本申请所述的抗LILRB1抗体或其抗原结合片段的剂量和给药频率可由临床医师根据如下因素确定，例如受试者的年龄、体重、性别、一般状况、疾病严重程度；它们可通过本领域已知的任何常规途径给予，例如胃肠外、静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内施用。
实施例
下面将结合实施例对本申请的方案进行进一步的说明，但是本领域技术人员将能够理解的是，本申请的保护范围并不仅限于这些实施例。
除非另有特别说明，下述实施例中涉及的试剂、材料或仪器为可商购得到的。
实施例1：人LILRB1稳转细胞株和食蟹猴LILRB1稳转细胞株构建
(1)过表达慢病毒载体构建
可将LILRB1的核酸序列插入适当的表达载体中，由此来实现上述核酸序列的异源表达。对于LILRB1而言，可以选用来自哺乳动物的LILRB1，例如人LILRB1、猴LILRB1、兔LILRB1等，并且可根据常用的 核酸序列和蛋白质序列的数据库例如NCBI、ENSMBL或UniProt公开的已知序列来合成LILRB1及其核酸序列。
在此，以人LILRB1和食蟹猴LILRB1为例。用于小鼠免疫和活性检测的人LILRB1、食蟹猴LILRB1重组蛋白购自ACRO Biosystems。
从UniProt数据库中检索人LILRB1、食蟹猴LILRB1的编码区序列信息，如下所示：
人LILRB1全长序列，SEQ NO ID:115：
食蟹猴LILRB1全长序列，SEQ NO ID:116：
基于上述信息，分别合成编码以上两种蛋白的全基因，并分别亚克隆至Lenti-CMV-puro慢病毒表达载体，构建Lenti-CMV-human ILT2过表达载体和Lenti-CMV-cyno ILT2过表达载体(由金斯瑞构建)。使用质粒大提试剂盒(购自诺唯赞)抽提质粒备用。
(2)慢病毒包装
S1.准备15cm细胞培养皿，以5×106个细胞/皿接种293T细胞(吉妮欧生物)，加入完全培养基(补充有10％FBS的DMEM高糖培养基)，置于37℃、5％CO2培养箱过夜培养。
S2.从冰箱中取出转染试剂LVTransm(购自爱康得)、表达载体(Lenti-CMV-human ILT2过表达载体和Lenti-CMV-cyno ILT2过表达载体)以及慢病毒包装质粒Lenti-packaging Mix，室温解冻后，用移液枪上下吹打完全混匀。
S3.按照以下流程制备转染复合物：
S3-1.取1×PBS缓冲液温热至室温，然后取2mL PBS缓冲液加入至6孔板的一个孔，然后向该孔中分别加入
(A)10μg Lenti-CMV-human ILT2过表达载体、30μL Lenti-packaging Mix；和
(B)10μg Lenti-CMV-cyno ILT2过表达载体、30μL Lenti-packaging Mix；
S3-2.移液枪上下吹打充分混匀后，加入50μL LVTransm，立即用移液器上下吹打混匀，室温下静置10-15分钟。
S3-3.将上述转染复合物逐滴加入到S1的15cm细胞培养皿中，轻轻晃动培养皿，充分混匀。将培养皿置于37℃、5％CO2培养箱培养6～8小时后，将含有转染试剂的培养基去掉，更换为新鲜的完全培养基，将培养皿重新放回培养箱中继续培养。
S4.连续培养24小时后，收集培养皿中含有病毒的培养基上清至50mL离心管中；向培养皿内重新加入25mL左右新鲜的完全培养基，继续培养24小时；
S5.收集培养皿中含有病毒的培养基上清至50mL离心管中；向培养皿内重新加入25mL左右新鲜的完全培养基，继续培养24小时；
S6.收集培养皿中含有病毒的培养基上清，并与前面收集的两次培养基上清混合；
S7.用0.45μm的PES材质的滤膜将上述的培养基上清过滤后，将滤液转至无菌离心管中，50000×g、4℃ 下离心2小时。离心结束后，在生物安全柜中，小心地将离心管中的液体吸去，加入1mL PBS缓冲液将沉淀重悬，将得到的Lenti-CMV-human ILT2过表达慢病毒和Lenti-CMV-cyno ILT2过表达慢病毒置于-80℃保存。
(3)筛选CHO-S-human LILRB1稳转细胞株和CHO-S-cyno LILRB1稳转细胞株
按照如下过程制备能够在细胞表面稳定表达LILRB1的重组CHO细胞：
S1.从液氮中复苏CHO-S细胞，并使用CHO Grow CD1无血清培养基在37℃、5％CO2培养箱中进行培养。连续传代5次，使细胞处于对数生长期。
S2.取新的6孔板，按照5×106个细胞/孔的密度将上述对数生长期的CHO-S细胞接种至6孔板中，并加入3mL CHO Grow CD1无血清培养基和10mL制备得到的Lenti-CMV-human ILT2过表达慢病毒和Lenti-CMV-cyno ILT2，用移液器轻轻混匀；
S3.将6孔板置于离心机中，800×g室温离心1小时；
S4.离心结束后，将6孔板取出，置于37℃、5％CO2培养箱中继续培养24小时；
S5.培养24h后，将6孔板中的培养基更换为新鲜的CHO Grow CD1无血清培养基，继续培养24小时；
S6.将6孔板中的培养基更换为含有10μg/mL Puromycin的CHO Grow CD1无血清培养基，进行重组细胞株的筛选，筛选得到CHO-S-human LILRB1稳转细胞株和CHO-S-cyno LILRB1稳转细胞株。
实施例2：用于功能活性评价的报告基因法建立
以Biond Biologics公司的LILRB1抗体BND-22作为阳性对照抗体，BND-22的氨基酸序列来自WO2021028921A1，如下所示：
BND-22重链序列，本申请SEQ NO ID:117：
BND-22轻链序列，本申请SEQ NO ID:118：
将BND-22的轻链和重链基因合成后分别亚克隆至pcDNA3.4表达载体，采用BIOHUB 78PEI转染试剂(购自上海起发)转染CHO-K1细胞，在37℃、5％CO2下培养后，收集细胞培养物，离心，并用Protein A亲和柱纯化。
从UniProt数据库中获得HLA-G和β2M的编码区核酸序列信息，密码子优化后进行基因合成，分别亚克隆至Lenti-CMV-puro慢病毒表达载体中。制备慢病毒(可参见例如Neha Tandon等，Bio Protoc.2018 Nov 5；8(21):e3073中的记载)，分别感染A375黑色素瘤细胞，使用2μg/mL puromycin抗生素筛选稳定过表达HLA-G的重组细胞株，进一步感染CD3抗体膜型表达载体Lenti-EF1a-Muromonab进行稳转株的筛选，获得A375-HLA-G-Muromonab细胞作为报告基因法的靶细胞。使用Jurkat-LILRB1 Reporter报告基因细胞(购自吉满生物)作为效应细胞，与A375-HLA-G-Muromonab细胞建立LILRB1抗体的报告基因法。按照如下步骤验证阳性对照BND-22对报告基因细胞株的激活作用。
(1)取对数增殖期Jurkat-LILRB1 Reporter报告基因细胞与A375-HLA-G-Muromonab细胞，用1×PBS清洗两次后按照效靶比5:1混匀，以RPMI 1640培养基调整密度至1.2×106/mL，每孔接种100μL，1.2×105个细胞。
(2)将阳性对照抗体BND-22稀释至800μg/mL，随后进行3倍梯度稀释，96孔板内每孔加入100μL，使96孔板内抗体终浓度依次为400μg/mL、133.33μg/mL、44.44μg/mL、14.81μg/mL、4.94μg/mL、1.65μg/mL、0.55μg/mL、0.18μg/mL、0.06μg/mL。置于37℃、5％CO2条件下孵育过夜。
(3)每孔加入20μL One-Glo发光底物(购自Promega)，室温避光放置5min，使用Tecan M1000型多功能酶标仪读取Luciferase荧光数值。数据如图1(阳性对照BND-22的报告基因法活性结果图)所示，提示阳性对照BND-22能够阻断HLA-G结合LILRB1从而剂量依赖性激活下游信号通路，该功能实验体系可以用于候选抗体的活性评价。
实施例3：杂交瘤筛选
取20只Balb/c小鼠(SPF级)，分为二组进行免疫，第一组小鼠用人LILRB1重组蛋白(购自ACRO  Biosystems)100μg与弗氏佐剂乳化后皮下多点注射免疫。第二组小鼠用人LILRB1重组蛋白75μg和食蟹猴LILRB1重组蛋白(购自ACRO Biosystems)25μg与弗氏佐剂乳化后皮下多点注射免疫。每组小鼠均免疫3次，间隔时间14天。取免疫效价检测较好的小鼠冲击免疫，3天后对小鼠进行颈部脱臼处死，无菌条件下获得小鼠脾脏，制备B细胞单细胞悬液，并以1:1的比例与SP2/0骨髓瘤细胞混合，使用BTX细胞电融合仪进行细胞融合。电融合后，所有细胞立即悬浮在含有20％FBS和HAT的DMEM培养基中，接种至96孔板。融合后约10天改为HT培养基培养两天后，取上清检测特异性抗体。
从96孔板的每个孔中取出100μL培养液上清，同时向每个孔内补充新鲜的HT培养基。将取出的培养液上清通过ELISA法检测与人LILRB1、食蟹猴LILBR1的结合(HRP Anti-Mouse IgG H&L购自博奥龙，TMB显色液购自湖州英创)，同时通过流式细胞法检测与实施例1得到的重组细胞株(CHO-S-human LILRB1稳转细胞株和CHO-S-cyno LILRB1稳转细胞株)的交叉结合情况，优选能够与人、食蟹猴LILRB1蛋白及实施例1得到重组细胞株(CHO-S-human LILRB1稳转细胞株和CHO-S-cyno LILRB1稳转细胞株)交叉结合的克隆。通过两轮亚克隆获得可分泌与人、食蟹猴LILRB1蛋白及重组细胞株具有特异性结合的抗体的单克隆(6-B8-C3-G6、40-H9-G4-D8)，抗体序列见表1。
表1抗体CDR序列
实施例4：通过报告基因法对鼠源抗体的活性进行的检测
对上述两轮亚克隆筛选得到的单克隆6-B8-C3-G6、40-H9-G4-D8的杂交瘤进行扩增培养，然后通过蛋白A柱亲和层析进行鼠源抗体的纯化，将纯化后的鼠源抗体以及阳性对照BND-22按照实施例2所述步骤进行报告基因法检测，计算IC50值。
表2和图2(鼠源抗体报告基因法的活性检测结果图)示出了报告基因法的活性检测结果，可以看出，鼠源抗体和阳性对照均可剂量依赖性激活报告基因细胞株的荧光信号，鼠源抗体6-B8-C3-G6活性优于阳性对照BND-22，鼠源抗体40-H9-G4-D8活性与BND-22相当。
在图2中，图2A为阳性对照BND-22报告基因法的活性检测结果图，图2B为鼠源抗体6-B8-C3-G6报告基因法的活性检测结果图，图2C为鼠源抗体40-H9-G4-D8报告基因法的活性检测结果图。
表2鼠源抗体报告基因法的活性检测结果
实施例5：检测鼠源抗体促进NK92细胞杀伤的实验
按照如下步骤检测鼠源抗体6-B8-C3-G6、40-H9-G4-D8以及阳性对照BND-22促进NK92细胞对肿瘤细胞杀伤的活性。
(1)取对数增殖期NK92细胞，PBS清洗两次后用RPMI 1640培养基调整密度至3×106/mL，96孔板内每孔接种50μL、1.5×105个细胞。
(2)取阳性对照BND-22、鼠源抗体6-B8-C3-G6、40-H9-G4-D8稀释至200μg/mL，随后进行5倍梯度稀释至抗体浓度依次为200μg/mL、40μg/mL、8μg/mL，96孔板内每孔加入100μL。置于37℃、5％CO2条件下孵育2h。
(3)以构建的稳转HLA-G以及萤光素酶的A375细胞(A375-HLA-G-Luciferase)作为靶细胞。取对数增殖期A375-HLA-G-Luciferase细胞，1×PBS清洗两次后用RPMI 1640培养基调整密度至4×105/mL， 96孔板内每孔接种50μL、2×104个细胞，使效靶比为7.5:1，每孔抗体终浓度依次为100μg/mL、20μg/mL、4μg/mL、0μg/mL。置于37℃、5％CO2条件下共培养5h。
(4)每孔加入20μL One-Glo发光底物，室温避光放置5min，使用Tecan M1000型多功能酶标仪读取Luciferase荧光数值，并计算各孔NK92细胞对靶细胞的杀伤率。
图3(鼠源抗体促进NK92细胞杀伤实验的检测结果图)示出了NK92细胞杀伤实验的检测结果，可以看出，鼠源抗体和阳性对照均可增强NK92细胞对A375靶细胞的杀伤，鼠源抗体6-B8-C3-G6、40-H9-G4-D8增强杀伤的活性优于阳性对照BND-22，特别是在低浓度条件下。
实施例6：检测鼠源抗体促进巨噬细胞杀伤的实验
按照如下步骤检测鼠源抗体6-B8-C3-G6、40-H9-G4-D8以及阳性对照BND-22促进原代巨噬细胞对肿瘤细胞杀伤的活性。
(1)取分离自健康人的PBMC细胞，使用含有1000U/mL GM-CSF、10％FBS的RPMI 1640培养基调整细胞密度至1×107/mL，于37℃、5％CO2条件下培养过夜。去除未贴壁的悬浮细胞，使用含有1000U/mL GM-CSF、10％FBS的RPMI 1640培养基继续培养5天。培养结束后，加入含有20ng/mL IFN-γ、50ng/mL LPS、10％FBS的RPMI 1640培养基诱导培养3天，以获得M1型巨噬细胞。
(2)取诱导后的巨噬细胞500g离心5min，1×PBS清洗两次后用含有10％FBS的RPMI 1640培养基调整细胞密度至2×106/mL，96孔板内每孔接种50μL、1×105个细胞。
(3)取阳性对照BND-22、鼠源抗体6-B8-C3-G6、40-H9-G4-D8稀释至40μg/mL，置于37℃、5％CO2条件下孵育1h。
(4)取对数增殖期A375-HLA-G-Luciferase细胞，1×PBS清洗两次后调整细胞密度至4×105/mL，96孔板内每孔接种50μL、2×104个细胞，使效靶比为5:1，96孔板内抗体终浓度为20μg/mL。同时设置仅含靶细胞的孔以及仅含靶细胞和效应细胞的孔作为对照。置于37℃、5％CO2条件下共培养6h。
(5)每孔加入20μL One-Glo发光底物，室温避光放置5min，使用Tecan M1000型多功能酶标仪读取Luciferase荧光数值，并计算各孔巨噬细胞对靶细胞的杀伤率。
图4(鼠源抗体促进巨噬细胞杀伤实验的检测结果图，图4中，TC：仅靶细胞；EC+TC：仅效应细胞和靶细胞)示出了巨噬细胞杀伤实验的检测结果，可以看出，鼠源抗体和阳性对照均可剂量依赖性增强原代巨噬细胞对A375靶细胞的杀伤，鼠源抗体6-B8-C3-G6、40-H9-G4-D8增强杀伤的活性与阳性对照BND-22相当。
实施例7：嵌合抗体的构建与表达
对上述鼠源抗体6-B8-C3-G6、40-H9-G4-D8经杂交瘤测序获得轻链和重链可变区氨基酸序列。将重链可变区亚克隆至pcDNA3.4-hIgG4表达载体，轻链可变区亚克隆至pcDNA3.4-hIgKc表达载体，得到重链表达载体和轻链表达载体。
取2mL PBS缓冲液加入至6孔板中，然后分别加入40μg重链表达载体和80μg轻链表达载体，混匀后加入360μL LVTransm转染试剂(购自爱康得)并充分混匀，室温静置10分钟。
将上述得到的DNA/LVTransm复合物加入到100mL的293F细胞(细胞密度1×106/mL)中，轻轻晃动充分混匀。将细胞置于37℃、5％CO2、130rpm条件下连续培养7天，离心收集培养基上清，用0.45μm滤膜过滤后，使用蛋白A亲和层析纯化嵌合抗体，得到的嵌合抗体分别命名为6-B8-C3-G6-chi、40-H9-G4-D8-chi。
实施例8：通过报告基因法对嵌合抗体的活性进行的检测
按照实施例4所述步骤检测嵌合抗体6-B8-C3-G6-chi、40-H9-G4-D8-chi以及阳性对照BND-22的报告基因法活性。
表3和图5(嵌合抗体报告基因法的活性检测结果图)示出了报告基因法的活性检测结果，可以看出，嵌合抗体和阳性对照均可剂量依赖性激活报告基因细胞株的荧光信号，嵌合抗体6-B8-C3-G6-chi、40-H9-G4-D8-chi活性优于阳性对照BND-22。
表3嵌合抗体报告基因法的活性检测结果
实施例9：检测嵌合抗体促进NK92细胞杀伤的实验
按照实施例5所述步骤检测嵌合抗体6-B8-C3-G6-chi、40-H9-G4-D8-chi以及阳性对照BND-22促进 NK92细胞对肿瘤细胞杀伤的活性。其中，步骤(3)每孔抗体终浓度依次为20μg/mL、4μg/mL、0.8μg/mL、0.16μg/mL、0.032μg/mL、0.0064μg/mL、0.00128μg/mL、0.00026μg/mL。
图6(嵌合抗体促进NK92细胞杀伤实验的检测结果图)示出了NK92细胞杀伤实验的检测结果，可以看出，嵌合抗体和阳性对照均可增强NK92细胞对A375靶细胞的杀伤，嵌合抗体6-B8-C3-G6-chi、40-H9-G4-D8-chi增强杀伤的活性优于阳性对照BND-22。
实施例10：检测嵌合抗体促进原代NK细胞杀伤的实验
按照如下步骤检测嵌合抗体6-B8-C3-G6-chi、40-H9-G4-D8-chi以及阳性对照BND-22促进原代NK细胞对肿瘤细胞杀伤的活性。
(1)取对数增殖期A375-HLA-G-Luciferase细胞，用胰酶(购自Gibco)消化收集后200g离心5min，弃上清，用检测缓冲液(RPMI 1640培养基)清洗一次，将细胞密度调整为4×105/mL，96孔板内每孔接种25μL、1×104个细胞。
(3)收集原代NK细胞，200g离心5min，弃上清，用检测缓冲液清洗一次，将细胞密度调整为3×106/mL，96孔板内每孔接种25μL、7.5×104个细胞，使效靶比为7.5:1。
(3)取阳性对照BND-22以及嵌合抗体稀释至200μg/mL，随后进行5倍梯度稀释至抗体浓度依次为200μg/mL、40μg/mL、8μg/mL，96孔板内每孔加入50μL，使每孔抗体终浓度依次为100μg/mL、20μg/mL、4μg/mL。置于37℃、5％CO2条件下共培养5h。
(4)孵育结束后，将96孔板于200g离心5min，吸弃150μL上清。
(5)每孔加入50μL Bio-Lite染料(购自诺唯赞)，使用BMG CLARIOstar plus多功能酶标仪读取Luciferase荧光数值，并计算各孔NK细胞对靶细胞的杀伤率。
图7(嵌合抗体促进原代NK细胞杀伤实验的检测结果图)示出了原代NK细胞杀伤实验的检测结果，可以看出，嵌合抗体和阳性对照相比IgG4同型对照均可增强原代NK细胞对A375靶细胞的杀伤，嵌合抗体6-B8-C3-G6-chi、40-H9-G4-D8-chi增强杀伤的活性优于BND-22。
实施例11：检测嵌合抗体促进巨噬细胞杀伤的实验
按照实施例6所述步骤检测嵌合抗体6-B8-C3-G6-chi、40-H9-G4-D8-chi以及阳性对照BND-22促进原代巨噬细胞对肿瘤细胞杀伤的活性。其中，步骤(1)采用CD14+磁珠(购自美天旎)分选单核细胞，步骤(4)每孔抗体终浓度依次为100μg/mL、20μg/mL、4μg/mL。图8(嵌合抗体促进巨噬细胞杀伤实验的检测结果图)示出了巨噬细胞杀伤实验的检测结果，可以看出，嵌合抗体和阳性对照均可剂量依赖性增强原代巨噬细胞对A375靶细胞的杀伤，嵌合抗体6-B8-C3-G6-chi、40-H9-G4-D8-chi增强杀伤的活性优于阳性对照BND-22。
实施例12：抗体人源化
对嵌合抗体6-B8-C3-G6-chi、40-H9-G4-D8-chi进行抗体人源化改造，基于其鼠源抗体重链和轻链的氨基酸序列进行人源化设计，保留原始抗体的CDR区序列不变，根据germline alignment的结果以及抗体结构模拟的结果，重链和轻链分别选择不同的人源抗体模板，并在人源化之后对框架区进行回复突变，设计候选的人源化抗体序列，同时以氨基酸替换的方式对CDR区存在的翻译后修饰位点进行消除，例如将40-H9-G4-D8-chi轻链CDR3的潜在脱酰胺位点QH替换为SH，重链CDR3的潜在异构化位点DS替换为DA、DV、TS或GT，将6-B8-C3-G6-chi轻链CDR3的潜在脱酰胺位点QH替换为SH或AH。将设计的人源化抗体的重链和轻链分别进行基因合成，重链亚克隆至pcDNA3.4-hIgG4表达载体，轻链亚克隆至pcDNA3.4-hIgKc表达载体。构建的载体经测序验证无误后，用CHO细胞进行瞬转表达。代表性人源化抗体的CDR序列见表4，可变区和恒定区序列见表5。
表4人源化抗体的CDR序列

表5人源化抗体的可变区和恒定区序列


实施例13：人源化抗体的报告基因法活性检测
按照实施例2所述步骤采用报告基因法对嵌合抗体和人源化抗体进行活性检测，显示出嵌合抗体6-B8-C3-G6-chi、40-H9-G4-D8-chi经过人源化和突变改造仍然保持了较高的活性，结果如表6所示。
表6人源化抗体报告基因法的活性检测结果

实施例14：检测人源化抗体促进巨噬细胞杀伤的实验
按照实施例11所述步骤检测人源化抗体以及阳性对照BND-22促进原代巨噬细胞对肿瘤细胞杀伤的活性。图9A和图9B(人源化抗体促进巨噬细胞杀伤实验的检测结果图)示出了巨噬细胞杀伤实验的检测结果，可以看出，人源化抗体和阳性对照均可剂量依赖性增强原代巨噬细胞对A375靶细胞的杀伤，就高浓度下促进杀伤的作用来看，人源化抗体40-H9-F1-M20、6-B8-F2、40-H9-F1-M17、40-H9-F1、40-H9-F4-M19增强杀伤的活性优于阳性对照BND-22或与其相当。
实施例15：人源化抗体在人源化小鼠中的体内药效
对人源化抗体40-H9-F4-M19以及阳性对照抗体BND-22进行人源化小鼠体内药效研究。使用转入了人LILRB1/LILRB4基因的B-Tg(hLILRB1/hLILRB4)人源化C57BL/6小鼠(购自百奥赛图)。取雌性B-Tg(hLILRB1/hLILRB4)小鼠，按5E5/只的数量皮下接种过表达人HLA-G的小鼠结肠癌MC38细胞，建立皮下荷瘤模型。当平均肿瘤体积达到约60mm3时，根据小鼠肿瘤体积和体重将小鼠随机分配到3个实验组中，每组6只，然后按照表7所示方案开始给药。研究过程中每天观察小鼠存活情况及一般状态，每周2次测量肿瘤体积和体重，并计算肿瘤生长抑制率(TGI)。
至研究终点的肿瘤生长曲线图和体重变化趋势图分别如图10A、图10B所示，研究结果显示，人源化抗体40-H9-F4-M19在该小鼠模型中表现出抑瘤作用，并且其抑瘤作用高于等剂量BND-22，实验过程中各组动物体重均无明显下降。
表7人源化抗体在人源化小鼠中的体内药效研究方案
将本申请的序列列于下表6中。
表6本申请的序列




注：X1为I、N、F、V、S、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D；
X2为I、N、F、V、S、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D；
X3为I、N、F、V、S、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D；
X4为I、N、F、V、S、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D；
X5为I、N、F、V、S、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D；
X6为I、N、F、V、R、A、T、E、G、H、K、L、P、W、Y、C、M、Q或D。
为了描述和公开的目的，以引用的方式将所有的专利、专利申请和其它出版物在此明确地并入本文。这些出版物仅因为它们的公开早于本申请的申请日而提供。所有关于这些文件的日期的声明或这些文件的内容的表述是基于申请者可得的信息，并且不构成任何关于这些文件的日期或这些文件的内容的正确性的承认。而且，在任何国家，在本中对这些出版物的任何引用并不构成关于该出版物成为本领域的公知常识的一部分的认可。
本领域技术人员将认识到，本申请的范围并不限于上文描述的各种具体实施方式和实施例，而是能够在不脱离本申请的精神的情况下，进行各种修改、替换、或重新组合，这都落入了本申请的保护范围内。

Claims (15)

  1. 一种抗LILRB1抗体或其抗原结合片段，其包含：
    (a)包含下述互补决定区的重链可变区:
    HCDR1，其包含SEQ ID NO:1或7所示的氨基酸序列，或与SEQ ID NO：1或7所示的氨基酸序列具有至少80％同一性的氨基酸序列，或SEQ ID NO：1或7所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：1或7所示的氨基酸序列功能相同或相似；
    HCDR2，其包含SEQ ID NO:2或8所示的氨基酸序列，或与SEQ ID NO：2或8所示的氨基酸序列具有至少80％同一性的氨基酸序列，或SEQ ID NO：2或8所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：2或8所示的氨基酸序列功能相同或相似；
    HCDR3，其包含SEQ ID NO:119、3或9所示的氨基酸序列，或与SEQ ID NO：119、3或9所示的氨基酸序列具有至少80％同一性的氨基酸序列，或SEQ ID NO：119、3或9所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：119、3或9所示的氨基酸序列功能相同或相似；和/或
    (b)包含下述互补决定区的轻链可变区:
    LCDR1，其包含SEQ ID NO:4或10所示的氨基酸序列，或与SEQ ID NO：4或10所示的氨基酸序列具有至少80％同一性的氨基酸序列，或SEQ ID NO：4或10所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：4或10所示的氨基酸序列功能相同或相似；
    LCDR2，其包含SEQ ID NO:5所示的氨基酸序列，或与SEQ ID NO：5所示的氨基酸序列具有至少80％同一性的氨基酸序列，或SEQ ID NO：5所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：5所示的氨基酸序列功能相同或相似；
    LCDR3，其包含SEQ ID NO:6或11所示的氨基酸序列，或与SEQ ID NO：6或11所示的氨基酸序列具有至少80％同一性的氨基酸序列，或SEQ ID NO：6或11所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NO：6或11所示的氨基酸序列功能相同或相似。
  2. 根据权利要求1所述的抗LILRB1抗体或其抗原结合片段，其中，所述抗LILRB1抗体或其抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3：
    (1)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、2和119所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和6所示的氨基酸序列；
    (2)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、2和3所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和6所示的氨基酸序列；或
    (3)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和11所示的氨基酸序列。
  3. 根据权利要求1或2所述的抗LILRB1抗体或其抗原结合片段，其中，所述抗LILRB1抗体或其抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3：
    (1)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和93所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
    (2)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和13所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
    (3)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和15所示的氨基酸序列；
    (4)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和13所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和58所示的氨基酸序列；
    (5)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和92所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
    (6)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和42所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
    (7)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和94所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
    (8)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和77所示的氨基酸序列；或者
    (9)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和79所示的氨基酸序列；
    优选地，所述抗LILRB1抗体或其抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2 和LCDR3：
    (1)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和93所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
    (2)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和94所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；
    (3)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：7、8和9所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：10、5和77所示的氨基酸序列；
    (4)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和92所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列；或者
    (5)所述HCDR1、HCDR2和HCDR3分别具有SEQ ID NO：1、12和13所示的氨基酸序列，所述LCDR1、LCDR2和LCDR3分别具有SEQ ID NO：4、5和14所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的抗LILRB1抗体或其抗原结合片段，其中，所述抗LILRB1抗体或其抗原结合片段包含重链可变区和轻链可变区：
    所述重链可变区包括SEQ ID NOs：95、102-106、110、112所示的氨基酸序列或与SEQ ID NOs：95、102-106、110、112所示的氨基酸序列具有至少80％同一性的氨基酸序列，或SEQ ID NOs：95、102-106、110、112所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NOs：95、102-106、110、112所示的氨基酸序列功能相同或相似；
    所述轻链可变区包括SEQ ID NOs：96-101、107-109、111所示的氨基酸序列或与SEQ ID NOs：96-101、107-109、111所示的氨基酸序列具有至少80％同一性的氨基酸序列，或SEQ ID NOs：96-101、107-109、111所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列且与SEQ ID NOs：96-101、107-109、111所示的氨基酸序列功能相同或相似。
  5. 根据权利要求4所述的抗LILRB1抗体或其抗原结合片段，其中，所述抗LILRB1抗体或其抗原结合片段包含重链可变区和轻链可变区：
    (1)所述重链可变区包含SEQ ID NO：104所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：98所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (2)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：97所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (3)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：98所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (4)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：99所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (5)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：100所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (6)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：101所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (7)所述重链可变区包含SEQ ID NO：102所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：96所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (8)所述重链可变区包含SEQ ID NO：103所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：96所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (9)所述重链可变区包含SEQ ID NO：104所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：96所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (10)所述重链可变区包含SEQ ID NO：105所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：96所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (11)所述重链可变区包含SEQ ID NO：102所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：98所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (12)所述重链可变区包含SEQ ID NO：103所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：98所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (13)所述重链可变区包含SEQ ID NO：95所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：96所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (14)所述重链可变区包含SEQ ID NO：105所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：98所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (15)所述重链可变区包含SEQ ID NO：106所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：107所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (16)所述重链可变区包含SEQ ID NO：106所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：108所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (17)所述重链可变区包含SEQ ID NO：106所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：109所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (18)所述重链可变区包含SEQ ID NO：110所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：111所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (19)所述重链可变区包含SEQ ID NO：110所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：109所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    (20)所述重链可变区包含SEQ ID NO：112所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：107所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；或者
    (21)所述重链可变区包含SEQ ID NO：112所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列，所述轻链可变区包含SEQ ID NO：108所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列；
    优选地，所述抗LILRB1抗体或其抗原结合片段包含重链可变区和轻链可变区：
    (1)SEQ ID NO：104所示的重链可变区，SEQ ID NO：98所示的轻链可变区；
    (2)SEQ ID NO：105所示的重链可变区，SEQ ID NO：96所示的轻链可变区；
    (3)SEQ ID NO：106所示的重链可变区，SEQ ID NO：107所示的轻链可变区；
    (4)SEQ ID NO：102所示的重链可变区，SEQ ID NO：96所示的轻链可变区；或者
    (5)SEQ ID NO：95所示的重链可变区，SEQ ID NO：96所示的轻链可变区。
  6. 根据权利要求1-5中任一项所述的抗LILRB1抗体或其抗原结合片段，其中，所述抗LILRB1抗体或其抗原结合片段还包含重链恒定区和轻链恒定区，所述重链恒定区的氨基酸序列如SEQ ID NO：113所示或与其具有至少80％同一性；所述轻链恒定区的氨基酸序列如SEQ ID NO：114所示或与其具有至少80％同一性。
  7. 根据权利要求1-6中任一项所述的抗LILRB1抗体或其抗原结合片段，其中，所述抗LILRB1抗体或其抗原结合片段包含重链和轻链，其中，
    (1)所述重链包含具有SEQ ID NO：104所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的重链可变区和具有SEQ ID NO：113所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的重链恒定区，
    所述轻链包含具有SEQ ID NO：98所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的轻链可变区和具有SEQ ID NO：114所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的轻链恒定区；
    (2)所述重链包含具有SEQ ID NO：105所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的重链可变区和具有SEQ ID NO：113所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的重链恒定区，
    所述轻链包含具有SEQ ID NO：96所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的轻链可变区和具有SEQ ID NO：114所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的轻链恒定区；
    (3)所述重链包含具有SEQ ID NO：106所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的重链可变区和具有SEQ ID NO：113所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的重链恒定区，
    所述轻链包含具有SEQ ID NO：107所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的轻链可变区和具有SEQ ID NO：114所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的轻链恒定区；
    (4)所述重链包含具有SEQ ID NO：102所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的重链可变区和具有SEQ ID NO：113所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的重链恒定区，
    所述轻链包含具有SEQ ID NO：96所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的轻链可变区和具有SEQ ID NO：114所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的轻链恒定区；或者
    (5)所述重链包含具有SEQ ID NO：95所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的重链可变区和具有SEQ ID NO：113所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的重链恒定区，
    所述轻链包含具有SEQ ID NO：96所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的轻 链可变区和具有SEQ ID NO：114所示的氨基酸序列或与其具有至少80％同一性的氨基酸序列的轻链恒定区；
    优选地，所述抗LILRB1抗体或其抗原结合片段包含重链和轻链，其中，
    (1)所述重链包含SEQ ID NO：104所示的重链可变区和SEQ ID NO：113所示的重链恒定区，所述轻链包含SEQ ID NO：98所示的轻链可变区和SEQ ID NO：114所示的轻链恒定区；
    (2)SEQ ID NO：105所示的重链可变区和SEQ ID NO：113所示的重链恒定区，所述轻链包含SEQ ID NO：96所示的轻链可变区和SEQ ID NO：114所示的轻链恒定区；
    (3)SEQ ID NO：106所示的重链可变区和SEQ ID NO：113所示的重链恒定区，所述轻链包含SEQ ID NO：107所示的轻链可变区和SEQ ID NO：114所示的轻链恒定区；
    (4)SEQ ID NO：102所示的重链可变区和SEQ ID NO：113所示的重链恒定区，所述轻链包含SEQ ID NO：96所示的轻链可变区和SEQ ID NO：114所示的轻链恒定区；或者
    (5)SEQ ID NO：95所示的重链可变区和SEQ ID NO：113所示的重链恒定区，所述轻链包含SEQ ID NO：96所示的轻链可变区和SEQ ID NO：114所示的轻链恒定区。
  8. 根据权利要求1或2所述的抗LILRB1抗体或其抗原结合片段，其中，
    所述HCDR2具有SEQ ID NO：12所示的氨基酸序列；和/或
    所述HCDR3具有SEQ ID NO：13所示的氨基酸序列；和/或
    所述LCDR3具有SEQ ID NO：14所示的氨基酸序列；和/或
    所述LCDR3具有SEQ ID NO：15所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的抗LILRB1抗体或其抗原结合片段，其中，所述LILRB1为哺乳动物LILRB1，所述抗体优选为鼠源抗体、猴源抗体、兔源抗体、嵌合抗体、人源化抗体、人源抗体；和/或
    所述抗体为IgG抗体，优选为IgG1、IgG2、IgG3或IgG4抗体，更优选为IgG4抗体；和/或
    所述抗LILRB1抗体或其抗原结合片段选自单克隆抗体、多克隆抗体、Fab片段、Fab'片段、F(ab')2片段、Fd片段、Fv片段、dAb片段、分离的CDR区、scFv和纳米抗体。
  10. 一种核酸分子，所述核酸分子编码权利要求1-9中任一项所述的抗LILRB1抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
  11. 一种重组载体，所述重组载体包含权利要求10所述的核酸分子。
  12. 一种重组细胞，所述重组细胞包含权利要求10所述的核酸分子或权利要求11所述的包含所述核酸分子的重组载体。
  13. 一种试剂盒，包含权利要求1-9中任一项所述的抗LILRB1抗体或其抗原结合片段。
  14. 用于预防和/或治疗LILRB1相关疾病的权利要求1-9中任一项所述的抗LILRB1抗体或其抗原结合片段、权利要求10所述的核酸分子、权利要求11所述的重组载体或权利要求12所述的重组细胞；
    优选地，所述疾病为癌症；
    更优选地，所述癌症为表达MHC I类分子的癌症，优选为表达HLA-G的癌症。
  15. 用于诊断LILRB1相关疾病的权利要求1-9中任一项所述的抗LILRB1抗体或其抗原结合片段、或权利要求10所述的核酸分子；
    优选地，所述疾病为癌症；
    更优选地，所述癌症为表达MHC I类分子的癌症，优选为表达HLA-G的癌症。
PCT/CN2024/070594 2023-04-04 2024-01-04 抗lilrb1抗体或其抗原结合片段、制备方法和用途 WO2024207855A1 (zh)

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WO2021028921A1 (en) * 2019-08-12 2021-02-18 Biond Biologics Ltd. Antibodies against ilt2 and use thereof
CN113330033A (zh) * 2018-12-26 2021-08-31 先天制药公司 白细胞免疫球蛋白样受体2中和性抗体
WO2022026360A2 (en) * 2020-07-28 2022-02-03 The Board Of Regents Of The University Of Texas System Monoclonal antibodies against lilrb1 for diagnostic and therapeutic use
US20220048991A1 (en) * 2020-08-12 2022-02-17 Biond Biologics Ltd. Antibodies Against ILT2 and Use Thereof
CN114437214A (zh) * 2020-11-03 2022-05-06 南京北恒生物科技有限公司 靶向lir1的抗体及其用途
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CN113330033A (zh) * 2018-12-26 2021-08-31 先天制药公司 白细胞免疫球蛋白样受体2中和性抗体
WO2021028921A1 (en) * 2019-08-12 2021-02-18 Biond Biologics Ltd. Antibodies against ilt2 and use thereof
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