Anti-lilrb1 nanobodies or antigen binding fragments thereof, methods of making and uses

Abstract

The present application relates to the field of immunology, in particular to anti-LILRB1 antibodies or antigen-binding fragments thereof, preparation methods and uses, especially anti-LILRB1 nanobodies or antigen-binding fragments thereof, preparation methods and uses. The anti-LILRB1 antibodies or antigen-binding fragments thereof can inhibit the immune escape mechanism of tumor cells, so that immune cells can exhibit their anti-cancer effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology, in particular to anti-LILRB1 antibodies or antigen-binding fragments thereof, preparation methods and uses, in particular anti-LILRB1 nanobodies or antigen-binding fragments thereof, preparation methods and uses. BACKGROUND

[0002] Leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1; also known as ILT2, CD85j or LIR-1) 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 ligands and an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) that can recruit downstream phosphatases such as SHP1, SHP2, SHIP, etc., and inhibit the activation of immune cells through a series of signaling pathways.

[0003] 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 propensity to bind HLA-G. At the same time, a variety of cancer cells overexpress HLA-G to evade immunity. Currently, the interaction of LILRB1 with 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 inhibited immune cell activity, thereby exerting an anti-tumor effect.

[0004] Therefore, there is a need to develop an agent that binds to LILRB1 and blocks the action of LILRB1 and HLA-G. SUMMARY

[0005] To solve the above problems existing in the prior art, the present application provides an anti-LILRB1 antibody or antigen-binding fragment thereof, a preparation method and a use, which can inhibit the immune escape mechanism of tumor cells so that immune cells can exhibit their anti-cancer effect.

[0006] The first aspect of the present application provides an anti-LILRB1 antibody or antigen-binding fragment thereof, which contains a VHH chain comprising the complementarity determining regions CDR1, CDR2 and CDR3 as shown in the following amino acid sequences:

[0007] The CDR1 amino acid sequence is one of the sequences shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 29, 32, 33, 34, 36, 39, 43, 46, 49, 51, 54, 56, 58, 61, 64, 67, 70;

[0008] the CDR2 amino acid sequence is one of the sequences set forth in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 24, 26, 27, 30, 37, 40, 44, 47, 52, 55, 57, 59, 62, 65, 68;

[0009] the CDR3 amino acid sequence is one of the sequences set forth in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 25, 28, 31, 35, 38, 41, 42, 45, 48, 50, 53, 60, 63, 66, 69.

[0010] In some embodiments of the application, the VHH chain comprises:

[0011] (1) CDR1 set forth in SEQ ID NO: 1, CDR2 set forth in SEQ ID NO: 2, CDR3 set forth in SEQ ID NO: 3; or

[0012] (2) CDR1 set forth in SEQ ID NO: 4, CDR2 set forth in SEQ ID NO: 5, CDR3 set forth in SEQ ID NO: 6; or

[0013] (3) CDR1 set forth in SEQ ID NO: 7, CDR2 set forth in SEQ ID NO: 8, CDR3 set forth in SEQ ID NO: 9; or

[0014] (4) CDR1 set forth in SEQ ID NO: 10, CDR2 set forth in SEQ ID NO: 11, CDR3 set forth in SEQ ID NO: 12; or

[0015] (5) CDR1 set forth in SEQ ID NO: 13, CDR2 set forth in SEQ ID NO: 14, CDR3 set forth in SEQ ID NO: 15; or

[0016] (6) CDR1 set forth in SEQ ID NO: 16, CDR2 set forth in SEQ ID NO: 17, CDR3 set forth in SEQ ID NO: 15; or

[0017] (7) CDR1 set forth in SEQ ID NO: 10, CDR2 set forth in SEQ ID NO: 11, CDR3 set forth in SEQ ID NO: 18; or

[0018] (8) CDR1 set forth in SEQ ID NO: 19, CDR2 set forth in SEQ ID NO: 20, CDR3 set forth in SEQ ID NO: 21; or

[0019] (9) a CDR1 as set forth in SEQ ID NO: 22, a CDR2 as set forth in SEQ ID NO: 23, a CDR3 as set forth in SEQ ID NO: 3; or

[0020] (10) a CDR1 as set forth in SEQ ID NO: 10, a CDR2 as set forth in SEQ ID NO: 24, a CDR3 as set forth in SEQ ID NO: 25;

[0021] (11) a CDR1 as set forth in SEQ ID NO: 13, a CDR2 as set forth in SEQ ID NO: 26, a CDR3 as set forth in SEQ ID NO: 25; or

[0022] (12) a CDR1 as set forth in SEQ ID NO: 10, a CDR2 as set forth in SEQ ID NO: 11, a CDR3 as set forth in SEQ ID NO: 25; or

[0023] (13) a CDR1 as set forth in SEQ ID NO: 10, a CDR2 as set forth in SEQ ID NO: 27, a CDR3 as set forth in SEQ ID NO: 28; or

[0024] (14) a CDR1 as set forth in SEQ ID NO: 29, a CDR2 as set forth in SEQ ID NO: 30, a CDR3 as set forth in SEQ ID NO: 31; or

[0025] (15) a CDR1 as set forth in SEQ ID NO: 32, a CDR2 as set forth in SEQ ID NO: 11, a CDR3 as set forth in SEQ ID NO: 18; or

[0026] (16) a CDR1 as set forth in SEQ ID NO: 33, a CDR2 as set forth in SEQ ID NO: 11, a CDR3 as set forth in SEQ ID NO: 18; or

[0027] (17) a CDR1 as set forth in SEQ ID NO: 34, a CDR2 as set forth in SEQ ID NO: 23, a CDR3 as set forth in SEQ ID NO: 35; or

[0028] (18) a CDR1 as set forth in SEQ ID NO: 36, a CDR2 as set forth in SEQ ID NO: 37, a CDR3 as set forth in SEQ ID NO: 38; or

[0029] (19) a CDR1 as set forth in SEQ ID NO: 39, a CDR2 as set forth in SEQ ID NO: 40, a CDR3 as set forth in SEQ ID NO: 41; or

[0030] (20) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:42; or

[0031] (21) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:44, and CDR3 shown in SEQ ID NO:45; or

[0032] (22) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:48; or

[0033] (23) CDR1 shown in SEQ ID NO:49, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:50; or

[0034] (24) CDR1 shown in SEQ ID NO:51, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:53; or

[0035] (25) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:53; or

[0036] (26) CDR1 shown in SEQ ID NO:56, CDR2 shown in SEQ ID NO:57, and CDR3 shown in SEQ ID NO:53; or

[0037] (27) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:60; or

[0038] (28) CDR1 shown in SEQ ID NO:61, CDR2 shown in SEQ ID NO:62, and CDR3 shown in SEQ ID NO:63; or

[0039] (29) CDR1 shown in SEQ ID NO:64, CDR2 shown in SEQ ID NO:65, and CDR3 shown in SEQ ID NO:66; or

[0040] (30) CDR1 shown in SEQ ID NO:67, CDR2 shown in SEQ ID NO:68, and CDR3 shown in SEQ ID NO:69; or

[0041] (31) CDR1 shown in SEQ ID NO:70, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:60.

[0042] In a preferred embodiment, the VHH chain comprises:

[0043] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3;

[0044] (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6;

[0045] (3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9;

[0046] (4) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12;

[0047] (5) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:44, and CDR3 shown in SEQ ID NO:45;

[0048] (6) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:48;

[0049] (7) CDR1 shown in SEQ ID NO:51, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:53;

[0050] (8) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:53;

[0051] (9) CDR1 shown in SEQ ID NO:56, CDR2 shown in SEQ ID NO:57, and CDR3 shown in SEQ ID NO:53;

[0052] (10) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:60; or

[0053] (11) CDR1 shown in SEQ ID NO:61, CDR2 shown in SEQ ID NO:62, and CDR3 shown in SEQ ID NO:63.

[0054] In a preferred embodiment, the VHH chain comprises:

[0055] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3;

[0056] (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6;

[0057] (3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9;

[0058] (4) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12;

[0059] (5) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:44, and CDR3 shown in SEQ ID NO:45;

[0060] (6) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:48;

[0061] (7) CDR1 shown in SEQ ID NO:51, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:53;

[0062] (8) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:53; or

[0063] (9) CDR1 shown in SEQ ID NO:56, CDR2 shown in SEQ ID NO:57, and CDR3 shown in SEQ ID NO:53.

[0064] In a further preferred embodiment, the VHH chain comprises:

[0065] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; or

[0066] (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6.

[0067] In some embodiments of this application, the VHH chain has an amino acid sequence shown in SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 or 82, 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: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 or 82.

[0068] In some embodiments, the amino acid sequences represented by SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 or 82 and the amino acid sequences having at least 80% identity with them, are predominantly or entirely located in the FR region of the VHH chain.

[0069] In a preferred embodiment, the VHH chain has the amino acid sequence shown in SEQ ID NO: 71, 77, 81 or 82.

[0070] In some embodiments of this application, the antibody is a nanobody or a heavy chain antibody (HcAb).

[0071] In some embodiments of this application, the antibody is a nanobody.

[0072] In some embodiments of this application, the anti-LILRB1 antibody is a heavy chain antibody (HcAb).

[0073] In some embodiments of this application, the LILRB1 antibody or antigen-binding fragment further comprises an antibody constant region sequence lacking a CH1 fragment; more preferably, the antibody or antigen-binding fragment further comprises an antibody constant region sequence having CH2 and CH3 fragments, or the antibody or antigen-binding fragment further comprises an antibody Fc region. Preferably, the antibody constant region or antibody Fc region is linked to the antibody or antigen-binding fragment by or without a linker peptide. In some embodiments, the antibody or antigen-binding fragment comprises CH2 and CH3 fragments or an Fc region of IgG (e.g., IgG1, IgG2, IgG3, or IgG4, preferably IgG4, more preferably human IgG4), preferably CH2 and CH3 fragments or an Fc region of IgG4, the amino acid sequence of which is shown in SEQ ID NO: 87.

[0074] In some embodiments of this application, the LILRB1 is mammalian LILRB1, and the anti-LILRB1 antibody is preferably a camel-derived nanobody or a humanized nanobody.

[0075] In some specific embodiments of this application, the anti-LILRB1 antibody is a camel-derived nanobody.

[0076] In some embodiments of this application, the antibody is an IgG antibody, which may be an IgG1, IgG2, IgG3, or IgG4 antibody, preferably an IgG4 antibody. In some specific embodiments of this application, the antibody is an IgG4 antibody.

[0077] In some embodiments of this application, the antibody is selected from monoclonal antibodies, polyclonal antibodies, monovalent antibodies, multivalent antibodies, monospecific antibodies, and multispecific antibodies.

[0078] In some embodiments of this application, the antibody is a multivalent antibody, which comprises at least two of the VHH chains. Preferably, the multivalent antibody is a bivalent, trivalent, or tetravalent antibody. More preferably, the multivalent antibody is a bivalent antibody.

[0079] In some embodiments of this application, the antibody is a multispecific antibody that specifically binds to LILRB1 and additionally specifically binds to one or more other targets. Preferably, the multispecific antibody, such as a bispecific antibody, further comprises at least one second antibody having a second binding specificity against a second target. Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody. More preferably, the multispecific antibody is a bispecific antibody.

[0080] In some embodiments of this application, the antibody or its antigen-binding fragment comprises a framework region of a camel-derived heavy chain antibody or a heavy chain framework region derived from human immunoglobulin.

[0081] A second aspect of this application provides a nucleic acid molecule that encodes the anti-LILRB1 antibody or its antigen-binding fragment as described in the first aspect.

[0082] A third aspect of this application provides a recombinant vector comprising the nucleic acid molecules described in the second aspect.

[0083] The fourth aspect of this application provides a recombinant cell comprising the nucleic acid molecule described in the second aspect or the recombinant vector described in the third aspect.

[0084] The fifth aspect of this application provides a method for preparing an anti-LILRB1 antibody or an antigen-binding fragment thereof, comprising culturing the recombinant cells described in the fourth aspect.

[0085] The sixth aspect of this application provides the use of the anti-LILRB1 antibody or its antigen-binding fragment 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 the preparation of a medicament.

[0086] Preferably, the drug is a drug for the prevention and / or treatment of cancer.

[0087] In some embodiments, this application relates to an anti-LILRB1 antibody or its antigen-binding fragment as described in the first aspect of this application, a nucleic acid molecule as described in the second aspect, a recombinant vector as described in the third aspect, or a recombinant cell as described in the fourth aspect, for the prevention and / or treatment of LILRB1-related diseases. Alternatively, this application relates to a method for the prevention and / or treatment of LILRB1-related diseases, comprising administering to a subject in need an anti-LILRB1 antibody or its antigen-binding fragment as described in the first aspect of this application, a nucleic acid molecule as described in the second aspect, a recombinant vector as described in the third aspect, or a recombinant cell as described in the fourth aspect.

[0088] The seventh aspect of this application provides the use of the anti-LILRB1 antibody or its antigen-binding fragment described in the first aspect in the preparation of reagents for diagnosing LILRB1-related diseases. In some embodiments, this application relates to the anti-LILRB1 antibody or its antigen-binding fragment described in the first aspect of this 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 diagnosing LILRB1-related diseases.

[0089] Preferably, the LILRB1-related disease is cancer. In some embodiments of this application, the cancer is a cancer expressing MHC class I molecules, preferably a cancer expressing HLA-G.

[0090] In some embodiments, this 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 its antigen-binding fragment described herein.

[0091] In some embodiments, this application provides a kit comprising the anti-LILRB1 antibody described in this application or its antigen-binding fragment, or a chimeric antigen receptor.

[0092] The beneficial effects of this application are:

[0093] The anti-LILRB1 antibody or its antigen-binding fragment provided in this application can inhibit the immune escape mechanism of tumor cells, thereby enabling immune cells to exhibit their anti-cancer effects. It has the activity of promoting the killing of tumor cells by NK92 cells, primary NK cells, and macrophages, and can bind to LILRB1, blocking the binding of LILRB1 to HLA-G.

[0094] The anti-LILRB1 antibody or its antigen-binding fragment contained in this application contains a VHH chain and can be a nanobody or heavy chain antibody, exhibiting good biological activity. Attached Figure Description

[0095] Figure 1 This is a graph showing the reporter gene activity results of the positive control BND-22 in this application;

[0096] Figure 2 The image shows the activity detection results of the nanobody reporter gene method (representative image);

[0097] in, Figure 2 Figure A shows the activity detection results of the BND-22 nanobody using the reporter gene assay. Figure 2 B shows the activity detection results of the 3-A1 nanobody reporter gene assay. Figure 2 Figure C shows the activity detection results of the 3-C8 nanobody reporter gene assay. Figure 2 D is the activity detection result of the nanobody 3-D8 reporter gene method;

[0098] Figure 3 The image shows the results of the binding detection of nanobodies to the stable LILRB1 cell line of cynomolgus monkeys;

[0099] Figure 4 The image shows the results of the binding detection of nanobodies to the stable LILRB1 cell line of cynomolgus monkeys;

[0100] Figure 5 The image shows the detection results of the NK92 nanobody cell killing experiment (representative image);

[0101] in, Figure 5 Figure A shows the detection results of the cell killing assay using the BND-22NK92 ​​nanobody. Figure 5 B shows the detection results of the 3-A1 nanobody NK92 cell killing assay. Figure 5 Figure C shows the detection results of the 3-C8 nanobody NK92 cell killing assay. Figure 5 D is the detection result of the 3-D8 nanobody NK92 cell killing experiment;

[0102] Figure 6 The image shows the detection results of the nanobody primary NK cell killing experiment.

[0103] Figure 7 The image shows the detection results of the nanobody macrophage killing experiment;

[0104] Figure 8 The image shows the detection results of the humanized nanobody primary NK cell killing experiment. Detailed Implementation

[0105] To make this application easier to understand, the following detailed description will be provided with reference to embodiments. These embodiments are for illustrative purposes only and do not limit the scope of application of this application.

[0106] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Inc., New York, USA (2012); Abbas et al., Cellular and Molecular Immunology, Elsevier Science Health Science div (2009); He Wei et al., Medical Immunology (2nd ed.), People's Medical Publishing House, 2010.

[0107] Unless otherwise stated, the terms “comprise,” “comprises,” and “comprising,” or their equivalents (contain, contain, containing, include, include, including) used herein are open-ended expressions, meaning that they may cover other unspecified elements, components, and steps in addition to those listed. Unless otherwise stated, the terms “optional” or “optionally” used herein indicate the presence or absence, occurrence or non-occurrence of the object or event they modify. Unless otherwise stated, the term “subject” used herein covers any vertebrate, such as mammals and non-mammals, including humans, non-human primates, sheep, dogs, cats, horses, cattle, chickens, pigs, rats, etc. Unless otherwise stated, all figures used herein to indicate the amount, measurement, or reaction conditions of an ingredient should be understood to be modified by the term “about” in all cases. When used with percentages, the term “about” may mean, for example, ±1%, preferably ±0.5%, more preferably ±0.1%. In this document, unless the context clearly indicates otherwise, singular terms cover plural references, and vice versa. Unless the context clearly indicates otherwise, the word "or" in this article is intended to include "and".

[0108] Unless otherwise stated, the term “treatment” as used herein means that, after administration, it can inhibit, suppress, reduce, improve, slow down, alleviate or eliminate a disease or its associated symptoms, delay, slow down, stop or terminate the progression of a disease or its associated symptoms, or prevent, control or reduce the recurrence of a disease or its associated symptoms.

[0109] The term "antibody" refers to a polypeptide encoded by immunoglobulin genes that can bind to antigens.

[0110] "Conventional" antibodies are tetrameric (as is the case with human immunoglobulin G (IgG)), consisting of two identical polypeptide chains called light chains and two identical polypeptide chains called heavy chains. Variable domains are located at the N-terminus of each chain ("VH" for the heavy chain and "VL" for the light chain), and the two variable domains, VH and VL, recognize the antigen. Each variable domain typically contains four "framework regions" (called FR1, FR2, FR3, and FR4) and three regions directly responsible for antigen binding, called "CDRs" (called CDR1, CDR2, and CDR3). Typically, each variable domain has the following form: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Binding to the antigen is usually accomplished through pairs of variable domains (i.e., the light chain variable domain VL and the heavy chain variable domain VH) (as is the case with human IgG), meaning that six CDRs are involved in the formation of one antigen-binding site.

[0111] The term "heavy chain antibody" or "HcAb" refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in 4-chain antibodies. Cameloideas (e.g., camels, llamas, or alpacas) are known to produce HcAbs. Heavy chain antibodies lack the light chain and the heavy chain constant region 1 (CH1), containing only two heavy chains consisting of a variable region (VHH) and other constant regions. The variable region is linked to the constant region via a hinge-like structure. Each heavy chain of cameloid heavy chain antibodies contains one variable region (VHH) and two constant regions (CH2 and CH3).

[0112] The antigen-binding fragment of a heavy chain antibody includes a separated CDR region, VHH, and a single-chain heavy chain antibody. By fusing with the constant region of human IgG Fc, a heavy chain antibody can possess the CH2 and CH3 regions of human IgG Fc. The terms "heavy chain variable region domain of heavy chain antibody," "VHH," and "nanobody" are used interchangeably.

[0113] Unless otherwise stated, the scope of CDRs for antibodies described in this application has been defined according to the ImMunoGenTics (IMGT) numbering rules. However, those skilled in the art will understand that CDR sequences numbered according to any one or more of the AbM numbering rules, Kabat numbering rules, Chothia numbering rules, and Contact numbering rules (based on the analysis of available complex crystal structures) also fall within the scope of protection of this application.

[0114] In some embodiments of this application, an anti-LILRB1 antibody or its antigen-binding fragment is provided, which contains a VHH chain, said VHH chain comprising complementarity-determining regions CDR1, CDR2, and CDR3 with the following amino acid sequences:

[0115] The amino acid sequence of CDR1 is one of the sequences shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 29, 32, 33, 34, 36, 39, 43, 46, 49, 51, 54, 56, 58, 61, 64, 67, 70;

[0116] The CDR2 amino acid sequence is one of the sequences shown in SEQ ID NO:2, 5, 8, 11, 14, 17, 20, 23, 24, 26, 27, 30, 37, 40, 44, 47, 52, 55, 57, 59, 62, 65, 68;

[0117] The amino acid sequence of CDR3 is one of the sequences shown in SEQ ID NO:3, 6, 9, 12, 15, 18, 21, 25, 28, 31, 35, 38, 41, 42, 45, 48, 50, 53, 60, 63, 66, 69;

[0118] In some embodiments of this application, the VHH chain comprises:

[0119] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; or

[0120] (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; or

[0121] (3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9; or

[0122] (4) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12; or

[0123] (5) CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:15; or

[0124] (6) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, and CDR3 shown in SEQ ID NO:15; or

[0125] (7) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:18; or

[0126] (8) CDR1 shown in SEQ ID NO:19, CDR2 shown in SEQ ID NO:20, and CDR3 shown in SEQ ID NO:21; or

[0127] (9) CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:3; or

[0128] (10) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:24, and CDR3 shown in SEQ ID NO:25;

[0129] (11) CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:26, and CDR3 shown in SEQ ID NO:25; or

[0130] (12) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:25; or

[0131] (13) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:28; or

[0132] (14) CDR1 shown in SEQ ID NO:29, CDR2 shown in SEQ ID NO:30, and CDR3 shown in SEQ ID NO:31; or

[0133] (15) CDR1 shown in SEQ ID NO:32, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:18; or

[0134] (16) CDR1 shown in SEQ ID NO:33, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:18; or

[0135] (17) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:35; or

[0136] (18) CDR1 shown in SEQ ID NO:36, CDR2 shown in SEQ ID NO:37, and CDR3 shown in SEQ ID NO:38; or

[0137] (19) CDR1 shown in SEQ ID NO:39, CDR2 shown in SEQ ID NO:40, and CDR3 shown in SEQ ID NO:41; or

[0138] (20) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:42; or

[0139] (21) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:44, and CDR3 shown in SEQ ID NO:45; or

[0140] (22) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:48; or

[0141] (23) CDR1 shown in SEQ ID NO:49, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:50; or

[0142] (24) CDR1 shown in SEQ ID NO:51, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:53; or

[0143] (25) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:53; or

[0144] (26) CDR1 shown in SEQ ID NO:56, CDR2 shown in SEQ ID NO:57, and CDR3 shown in SEQ ID NO:53; or

[0145] (27) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:60; or

[0146] (28) CDR1 shown in SEQ ID NO:61, CDR2 shown in SEQ ID NO:62, and CDR3 shown in SEQ ID NO:63; or

[0147] (29) CDR1 shown in SEQ ID NO:64, CDR2 shown in SEQ ID NO:65, and CDR3 shown in SEQ ID NO:66; or

[0148] (30) CDR1 shown in SEQ ID NO:67, CDR2 shown in SEQ ID NO:68, and CDR3 shown in SEQ ID NO:69; or

[0149] (31) CDR1 shown in SEQ ID NO:70, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:60.

[0150] In a preferred embodiment, the VHH chain comprises:

[0151] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3;

[0152] (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6;

[0153] (3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9;

[0154] (4) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12;

[0155] (5) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:44, and CDR3 shown in SEQ ID NO:45;

[0156] (6) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:48;

[0157] (7) CDR1 shown in SEQ ID NO:51, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:53;

[0158] (8) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:53;

[0159] (9) CDR1 shown in SEQ ID NO:56, CDR2 shown in SEQ ID NO:57, and CDR3 shown in SEQ ID NO:53;

[0160] (10) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:60; or

[0161] (11) CDR1 shown in SEQ ID NO:61, CDR2 shown in SEQ ID NO:62, and CDR3 shown in SEQ ID NO:63.

[0162] In a preferred embodiment, the VHH chain comprises:

[0163] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3;

[0164] (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6;

[0165] (3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9;

[0166] (4) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12;

[0167] (5) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:44, and CDR3 shown in SEQ ID NO:45;

[0168] (6) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:48;

[0169] (7) CDR1 shown in SEQ ID NO:51, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:53;

[0170] (8) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:53; or

[0171] (9) CDR1 shown in SEQ ID NO:56, CDR2 shown in SEQ ID NO:57, and CDR3 shown in SEQ ID NO:53.

[0172] This application also provides conserved modified forms of anti-LILRB1 antibodies or antigen-binding fragments thereof. Those skilled in the art will recognize that a conserved amino acid substitution is the replacement of an amino acid with another amino acid having similar structural or chemical properties (e.g., like a similar side chain). Exemplary conserved substitutions are described in the art, for example, in Watson et al., Molecular Biology of the Gene, The Bengamin / Cummings Publication Company, 4th edition (1987).

[0173] In a further preferred embodiment, the VHH chain comprises:

[0174] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; or

[0175] (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6.

[0176] In some embodiments of this application, the VHH chain has an amino acid sequence shown in SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 or 82, 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: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 or 82. In some embodiments, all the amino acid sequences represented by SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 or 82 and the amino acid sequences having at least 80% identity with them are located in the FR region of the VHH chain.

[0177] In a preferred embodiment, the VHH chain has the amino acid sequence shown in SEQ ID NO: 71, 77, 81 or 82.

[0178] In some embodiments of this application, the antibody is a nanobody or a heavy chain antibody (HcAb).

[0179] In some embodiments of this application, the antibody is a nanobody.

[0180] In some embodiments of this application, the anti-LILRB1 antibody is a heavy chain antibody (HcAb).

[0181] In some embodiments of this application, the LILRB1 antibody or antigen-binding fragment further comprises an antibody constant region sequence lacking the CH1 fragment; more preferably, the antibody or antigen-binding fragment further comprises an antibody constant region sequence having CH2 and CH3 fragments, or the antibody or antigen-binding fragment further comprises an antibody Fc region. Preferably, the antibody constant region or antibody Fc region is connected, or not connected, via a linker peptide (e.g., (G4S)). n (where n is an integer selected from 1 to 5) connects the antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment comprises the CH2 and CH3 fragments or Fc region of IgG (e.g., human IgG1, IgG2, IgG3, or IgG4), preferably the CH2 and CH3 fragments or Fc region of IgG4, whose amino acid sequence is shown in SEQ ID NO: 87.

[0182] In some embodiments of this application, the LILRB1 is mammalian LILRB1, and the anti-LILRB1 antibody is preferably a camel-derived nanobody or a humanized nanobody.

[0183] In some specific embodiments of this application, the antibody is a camel-derived nanobody.

[0184] In some embodiments of this application, the antibody is an IgG antibody, which may be an IgG1, IgG2, IgG3 or IgG4 antibody, preferably an IgG4 antibody.

[0185] In some embodiments of this application, the antibody is selected from monoclonal antibodies, polyclonal antibodies, monovalent antibodies, multivalent antibodies, monospecific antibodies, and multispecific antibodies.

[0186] In some embodiments of this application, the antibody is a multivalent antibody, which comprises at least two of the VHH chains. Preferably, the multivalent antibody is a bivalent, trivalent, or tetravalent antibody. More preferably, the multivalent antibody is a bivalent antibody.

[0187] In some embodiments of this application, the antibody is a multispecific antibody that specifically binds to LILRB1 and additionally specifically binds to one or more other targets. Preferably, the multispecific antibody, such as a bispecific antibody, further comprises at least one second antibody having a second binding specificity against a second target. Preferably, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody. More preferably, the multispecific antibody is a bispecific antibody.

[0188] As a non-limiting example, the anti-LILRB1 antibody or its antigen-binding fragment described in this application may exhibit the following properties:

[0189] (1) Combining with LILRB1; and / or

[0190] (2) Blocking the binding of LILRB1 to MHC class I molecules (preferably HLA-G); and / or

[0191] (3) Enhance the killing effect of immune cells on tumor cells.

[0192] In some embodiments, binding to LILRB1 includes binding to LILRB1 in mammals, including humans, non-human primates (such as cynomolgus monkeys), and camels. In some embodiments, binding to LILRB1 includes binding to cells expressing LILRB1. The anti-LILRB1 antibody or its antigen-binding fragment is capable of cross-binding with human or cynomolgus monkey LILRB1, or cross-binding with cells expressing human or cynomolgus monkey LILRB1.

[0193] In some embodiments, enhancing the killing effect of immune cells on tumor cells includes enhancing the killing effect of NK cells and / or macrophages on tumor cells. In some embodiments, the anti-LILRB1 antibody or its antigen-binding fragment blocks the IC50 of HLA-G binding to LILRB1. 50 <2.6μg / mL, <1.1μg / mL, <0.2μg / mL or <0.05μg / mL.

[0194] In some embodiments of this application, a nucleic acid molecule is provided that encodes the anti-LILRB1 antibody or an antigen-binding fragment thereof.

[0195] In some embodiments of this application, a recombinant vector is provided that contains the aforementioned nucleic acid molecule.

[0196] The term "vector" refers to a tool used to express a target gene in a host cell, such as plasmid vectors, granular vectors, and viral vectors such as phage vectors, lentiviral vectors, adenovirus vectors, retroviral vectors, and adeno-associated virus vectors. Recombinant vectors can be constructed from or by manipulating plasmids (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, M13, etc.), or viral vectors (e.g., SV40, etc.), which are commonly used in the art.

[0197] In recombinant vectors, nucleic acid molecules can be operatively linked to promoters. The term "operatively linked" refers to a functional connection between a target nucleotide sequence and an expression regulatory sequence (e.g., a promoter sequence). When "operatively linked," the regulatory element can control the transcription and / or translation of the target polynucleotide.

[0198] Recombinant vectors can typically be constructed as cloning vectors or expression vectors. For recombinant expression vectors, vectors commonly available in the relevant art for expressing foreign proteins in plant, animal, or microbial cells can be used. Various methods well-known in the art can be used to construct recombinant vectors.

[0199] For use in a host, such as a prokaryotic or eukaryotic cell, a recombinant vector can be constructed accordingly. For example, when constructing a vector as an expression vector for a prokaryotic host, the vector typically includes a strong promoter for transcription (e.g., pLλ 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. On the other hand, expression vectors for eukaryotic hosts include, but are not limited to, origins of replication operable in eukaryotic cells, such as the f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adenovirus origin of replication, AAV origin of replication, and BBV origin of replication. Additionally, expression vectors typically include promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter, etc.), and a polyadenylated sequence as a transcription termination sequence. Recombinant cells can be prepared by introducing the recombinant vector into a suitable host cell. Any host cell known in the art can be used in this disclosure, provided that it allows the recombinant vector to be continuously cloned and expressed in a stable manner. Examples of prokaryotic host cells that can be used in this disclosure include Escherichia coli species such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, and E. coli W3110; Bacillus species such as Bacillus subtilis and Bacillus thuringiensis; and Enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. Eukaryotic host cells suitable for transformation can be selected from, but are 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 fields. For example, when the host cell is a prokaryotic cell, such transfection can be performed using CaCl2 or electroporation. For eukaryotic host cells, gene introduction can be achieved using, but is not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, or particle bombardment.

[0200] To select host cells for transformation, phenotypes associated with selection markers can be utilized according to methods well-known in the art. For example, when the selection marker is a gene conferring resistance to certain antibiotics, host cells can be grown in a culture medium in the presence of antibiotics to select target transformants.

[0201] In some embodiments of this application, a recombinant cell is provided that comprises the nucleic acid molecules or the recombinant vector described herein.

[0202] In some embodiments, the recombinant cells may be recombinant CHO cells (such as recombinant CHO-S cells, recombinant CHO-K1 cells), recombinant 293 cells, or recombinant 293T cells.

[0203] In some embodiments of this application, a method for preparing an anti-LILRB1 antibody or an antigen-binding fragment thereof is provided, the method comprising culturing the recombinant cells. The method may further include isolating and / or purifying the antibody or fragment thereof from the cell culture after an expression or culture step, for example, using a Protein A affinity column.

[0204] In some embodiments of this application, the use of the anti-LILRB1 antibody or its antigen-binding fragment, the nucleic acid molecule, the recombinant vector, or the recombinant cell in the preparation of a drug is provided.

[0205] In some embodiments of this application, the drug is a drug for the prevention and / or treatment of cancer.

[0206] In some embodiments of this application, the use of the described anti-LILRB1 antibody or its antigen-binding fragment thereof in the preparation of reagents for diagnosing LILRB1-related diseases is provided.

[0207] Preferably, the LILRB1-related disease is cancer.

[0208] The cancer cells that can be killed according to this disclosure include, but are not limited to, cancer cells from the following: bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus.

[0209] In addition, cancer can specifically have the following histological types, although not limited to these: tumors, malignant tumors; carcinomas; undifferentiated carcinomas; giant cell and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; lymphoepithelial carcinomas; basal cell carcinomas; epidermal carcinomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; gastrinomas, malignant tumors; cholangiocarcinomas; hepatocellular carcinomas; mixed hepatocellular carcinomas and cholangiocarcinomas; trabecular adenocarcinomas; adenoid cystic carcinomas; adenomas among adenomatous polyps; adenocarcinomas, familial adenomatous polyposis; solid carcinomas; carcinoid tumors, malignant tumors; branched alveolar adenocarcinomas; papillary adenocarcinomas; chromophobe adenocarcinomas; eosinophilic cell carcinomas; eosinophilic adenocarcinomas; basophilic cell carcinomas; clear cell adenocarcinomas; granular cell carcinomas; follicular adenocarcinomas; papillary and follicular adenocarcinomas; non-encapsulated Sclerosing carcinoma; Adrenocortical carcinoma; Endometrioid carcinoma; Skin appendage carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Cerumen gland carcinoma; Mucoepidermoid carcinoma; Cystic adenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous gland carcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma, malignant tumor; Ovarian stromal tumor, malignant tumor; Theca cell tumor, malignant tumor; Granulosa cell tumor, malignant tumor; Androgenetic adenocarcinoma, malignant tumor; Sertoli cell carcinoma; Radich cell tumor, malignant tumor; Lipiocytoma, malignant tumor; Paraganglioma, malignant tumor; Extramammary paraganglioma, malignant tumor; Pheochromocytoma; Glomangiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficially expanded melanoma; Malignant melanoma in giant nevus; Epithelioid cell melanoma; Blue nevus, malignant tumor; Sarcoma; Fibrosarcoma; Fibrohistocytoma, malignant tumor; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor, malignant tumor; Müllerian duct mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Stromal tumor, malignant tumor; Brenner's tumor, malignant tumor; Phyllodes tumor, malignant tumor; Synovial sarcoma; Mesothelioma, malignant tumor; Dysgerminoma; Embryonic carcinoma; Teratoma, malignant tumor; Thyroid gland Tumor-like ovarian tumor, malignant tumor; choriocarcinoma; mesonephric tumor, malignant tumor; angiosarcoma; hemangioendothelioma, malignant tumor; Kaposi's sarcoma; hemangiopericytoma, malignant tumor; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant tumor; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant tumor; ameloblastic odontosarcoma; ameloblastoma, malignant tumor; ameloblastic fibrosarcoma; pineal tumor, malignant tumor; chordoma; glioma, malignant tumor; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor;Cerebellar sarcoma; Ganglioblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant tumor; Neurofibrosarcoma; Schwannoma, malignant tumor; Granulosarcoma, malignant tumor; Malignant lymphoma; Hodgkin's disease; Paragranuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other designated non-Hodgkin's lymphoma; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoplasmic small bowel disease; Leukemia; Lymphocytic leukemia; Plasma cell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myeloid leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; Megakaryocytic leukemia; Myeloid sarcoma and hairy cell leukemia. In some respects, tumors can include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing sarcoma, glioblastoma, neuroblastoma, or leukemia.

[0210] In some embodiments of this application, 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; more preferably, by overexpression of HLA-G.

[0211] In a specific embodiment, the cancer is a melanoma expressing HLA-G.

[0212] The dosage and frequency of administration of the anti-LILRB1 antibody or its antigen-binding fragment described in this application may be determined by a clinician based on factors such as the subject's age, weight, sex, general condition, and disease severity; they may be administered via any conventional route known in the art, such as parenteral, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal administration.

[0213] Example

[0214] The present application will be further described below with reference to the embodiments. However, those skilled in the art will understand that the scope of protection of the present application is not limited to these embodiments.

[0215] Unless otherwise specified, the reagents, materials or instruments used in the following examples are commercially available.

[0216] Example 1: Construction of stable human LILRB1 cell lines and stable cynomolgus monkey LILRB1 cell lines

[0217] (1) Construction of overexpression lentiviral vector

[0218] The LILRB1 nucleic acid sequence can be inserted into an appropriate expression vector to achieve heterologous expression of the aforementioned nucleic acid sequence. For LILRB1, LILRB1 derived from mammals, such as human LILRB1, monkey LILRB1, rabbit LILRB1, etc., can be selected, and LILRB1 and its nucleic acid sequence can be synthesized based on known sequences published in commonly used nucleic acid and protein sequence databases such as NCBI, ENSMBL, or UniProt.

[0219] Here, human LILRB1 and cynomolgus monkey LILRB1 are used as examples. The recombinant human LILRB1 and cynomolgus monkey LILRB1 proteins used for mouse immunization and activity assays were purchased from ACRO Biosystems.

[0220] The coding region sequence information of human LILRB1 and cynomolgus monkey LILRB1 was retrieved from the UniProt database, as shown below:

[0221] Human LILRB1 full-length sequence, SEQ NO ID:83:

[0222] MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH

[0223] Full-length sequence of cynomolgus monkey LILRB1, SEQ NO ID: 84:

[0224] MHRGLLHPQSRAVGGDAMTPILTVLICLGLSLGPRTHVQAGILPKPMLWAEPDRVITQGSPVTLRCQGNLEALGYHLYRERKSSASWITSIRPELVRKGQFPIPSITWEDAGRYRCQYYSHSWWSEHSDPLELVVTGAYSKPTLSALPSPVVASGGNVTLQCDSRVA FDGFILCKEGEDEHSQCLNSQPRTRGSSRAVFSVGPVSPSRRWSYRCYGYDSSFPYVWSLPSDLLELLVSGVSKKPSLSVQPGPVVAPGDKVTLQCGSDAGYDRFVLYKEGEREFLQRPGRQPQAGLSQANFTLGPVSHSHGGQYRCCGAHNLSSEWSAPSDPLDIL ISGQTHARPSLSVQPGPTVASGENVTLLCQSQGWMDTFFLTKEGAADAPLHLKSKRRSHIYQAEFPMGPVTSAHAGTYRCYGSFSSNPYLLTHPSEPLELVVSGPSGSPSPPLTGPTPTPGPEDQPLTPTGSAPQSGLGRHLGVVTGILVAFVLLLFLLLLLFLVL RHQRQGKHWTSAQRKADFQHPAGAVEPEPRDRGFQRRSSPAADTQEENLYAAVKDTQPEDRVELDSRSPHDEDPQAVTYAQVKHSGPRREMTSPPSPLSEEFLDTKDTQAEEDRQRDTEAAASEDPQDVTYAQLQSLTLRREATEPPPTQEREPPAEPSVYATLAIH

[0225] Based on the above information, the full genes encoding the 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-cynoILT2 overexpression vector (constructed by GenScript). Plasmids were extracted using a plasmid extraction kit (purchased from Novizumi) for later use.

[0226] (2) Lentiviral packaging

[0227] S1. Prepare a 15cm cell culture dish with 5×10⁻⁶ cells / mL. 6 293T cells (purchased from Genio Biotech) were seeded per dish, and complete culture medium (DMEM high-glucose medium supplemented with 10% FBS) was added. The cells were then incubated overnight at 37°C in a 5% CO2 incubator.

[0228] S2. Take out the transfection reagent LVTransm (purchased from Aikon), expression vectors (Lenti-CMV-humanILT2 overexpression vector and Lenti-CMV-cyno ILT2 overexpression vector) and lenti-packaging mix from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down.

[0229] S3. Prepare the transfection complex according to the following procedure:

[0230] S3-1. Warm 1×PBS buffer to room temperature, then add 2 mL of PBS buffer to one well of a 6-well plate, and then add [the following to be added to the remaining wells]...

[0231] (A) 10 μg Lenti-CMV-human ILT2 overexpression vector, 30 μL Lenti-packaging Mix; and

[0232] (B) 10 μg Lenti-CMV-cyno ILT2 overexpression vector, 30 μL Lenti-packaging Mix;

[0233] S3-2. After thoroughly mixing by pipetting up and down with a pipette, add 50 μL of LVTransm, immediately mix by pipetting up and down with a pipette, and let stand at room temperature for 10-15 minutes.

[0234] S3-3. Add the above transfection complex dropwise to the 15cm cell culture dish from S1, gently shake the dish to mix thoroughly. Incubate the dish at 37℃ in a 5% CO2 incubator for 6-8 hours, then remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium. Return the dish to the incubator to continue culturing.

[0235] S4. After continuous incubation for 24 hours, collect the supernatant containing the virus from the culture dish into a 50mL centrifuge tube; add about 25mL of fresh complete culture medium back into the culture dish and continue incubation for 24 hours.

[0236] S5. Collect the supernatant of the culture medium containing the virus from the culture dish into a 50mL centrifuge tube; add about 25mL of fresh complete culture medium back into the culture dish and continue to incubate for 24 hours;

[0237] S6. Collect the virus-containing culture medium supernatant from the culture dish and mix it with the two culture medium supernatants collected previously;

[0238] S7. After filtering the culture medium supernatant through a 0.45 μm PES filter membrane, transfer the filtrate to a sterile centrifuge tube and centrifuge at 50,000 × g and 4 °C for 2 hours. After centrifugation, carefully aspirate the liquid from the centrifuge tube in a biosafety cabinet, add 1 mL of PBS buffer to resuspend the precipitate, and store the obtained Lenti-CMV-human ILT2 overexpressing lentivirus and Lenti-CMV-cyno ILT2 overexpressing lentivirus at -80 °C.

[0239] (3) Screening for stable CHO-S-human LILRB1 cell lines and CHO-S-cyno LILRB1 cell lines

[0240] Recombinant CHO cells capable of stably expressing LILRB1 on their cell surface were prepared according to the following procedure:

[0241] S1. CHO-S cells were resuscitated from liquid nitrogen and cultured in serum-free CHO Grow CD1 medium at 37°C in a 5% CO2 incubator. The cells were passaged five times consecutively to induce logarithmic growth.

[0242] S2. Take a new 6-well plate and prepare it according to 5×10 6 At a density of cells / well, the above-mentioned logarithmic growth phase CHO-S cells were seeded into 6-well plates, and 3 mL of serum-free CHO Grow CD1 medium and 10 mL of the prepared Lenti-CMV-humanILT2 overexpressing lentivirus and Lenti-CMV-cyno ILT2 were added and gently mixed with a pipette.

[0243] S3. Place the 6-well plate in a centrifuge and centrifuge at 800×g at room temperature for 1 hour;

[0244] S4. After centrifugation, remove the 6-well plate and place it in a 37°C, 5% CO2 incubator for 24 hours.

[0245] S5. After culturing for 24 hours, replace the culture medium in the 6-well plate with fresh CHO Grow CD1 serum-free medium and continue culturing for another 24 hours.

[0246] S6. Replace the culture medium in the 6-well plate with serum-free CHO Grow CD1 medium containing 10 μg / mL Puromycin, and screen for recombinant cell lines to obtain stable CHO-S-human LILRB1 and CHO-S-cynoLILRB1 cell lines.

[0247] Example 2: Establishment of a reporter gene method for evaluating functional activity

[0248] Using Biond Biologics' LILRB1 antibody BND-22 as a positive control antibody, the amino acid sequence of BND-22 is described in WO2021028921A1, as shown below:

[0249] BND-22 heavy chain sequence, SEQ NO ID:85 of this application:

[0250] DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEWMGYISYDGSNNYNPSLKNRITISRDTDKNQFSLKLNSVTAADTATYYCAHGYSYYYAMDAWGQ GTSSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0251] BND-22 light chain sequence, SEQ NO ID:86 of this application:

[0252] DIQMTQSPSSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLISYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0253] The light and heavy chain genes of BND-22 were synthesized and subcloned into the pcDNA3.4 expression vector, respectively. CHO-K1 cells were transfected with BIOHUB78PEI transfection reagent (purchased from Shanghai Qifa). After culturing at 37℃ and 5% CO2, the cell culture was collected, centrifuged, and purified using a Protein A affinity column.

[0254] The coding region nucleic acid sequence information of HLA-G and β2M was obtained from the UniProt database. After codon optimization, the genes were synthesized and subcloned into Lenti-CMV-puro lentiviral expression vectors, respectively. Lentiviral viruses were prepared (see, for example, Neha Tandon et al., Bio Protoc. 2018 Nov 5; 8(21):e3073), and A375 melanoma cells were infected. Recombinant cell lines that stably overexpressed HLA-G were screened using 2 μg / mL puromycin antibiotic. These cells were then further infected with the CD3 antibody membrane expression vector Lenti-EF1a-Muromonab for stable cell line selection, and A375-HLA-G-Muromonab cells were obtained as target cells for the reporter gene method. Jurkat-LILRB1 Reporter cells (purchased from Jiman Biotechnology) were used as effector cells to establish a LILRB1 antibody reporter gene method with A375-HLA-G-Muromonab cells. The activation effect of the positive control BND-22 on reporter gene cell lines was verified by following these steps.

[0255] (1) Jurkat-LILRB1 reporter cells in the logarithmic growth phase and A375-HLA-G-Muromonab cells were washed twice with 1×PBS and mixed at an effector-to-target ratio of 5:1. The density was adjusted to 1.2×10⁶ cells / year with RPMI 1640 medium. 6 / mL, 100μL per well, 1.2×10 5 Each cell.

[0256] (2) The positive control antibody BND-22 was diluted to 800 μg / mL, followed by a 3-fold serial dilution. 100 μL was added to each well of a 96-well plate, resulting in final antibody concentrations of 400 μg / mL, 133.33 μg / mL, 44.44 μg / mL, 14.81 μg / mL, 4.94 μg / mL, 1.65 μg / mL, 0.55 μg / mL, 0.18 μg / mL, and 0.06 μg / mL, respectively. The plate was incubated overnight at 37°C and 5% CO2.

[0257] (3) Add 20 μL of One-Glo luminescent substrate (purchased from Promega) to each well, incubate at room temperature in the dark for 5 min, and read the Luciferase fluorescence values ​​using a Tecan M1000 multi-functional microplate reader. Data are as follows: Figure 1 (The reporter gene activity results of the positive control BND-22 are shown in the figure.) This indicates that the positive control BND-22 can block HLA-G binding to LILRB1 and thereby activate downstream signaling pathways in a dose-dependent manner. This functional experimental system can be used to evaluate the activity of candidate antibodies.

[0258] Example 3: Construction and Screening of an Alpaca Immune Bank

[0259] Two alpacas were immunized with recombinant human LILRB1 protein and recombinant cynomolgus monkey LILRB1 protein (both purchased from ACRO Biosystems). The first three immunizations were performed with 500 μg, 250 μg, and 250 μg of human LILRB1 protein, respectively. The fourth immunization was performed with 250 μg of cynomolgus monkey LILRB1 protein. The immunization interval was 21 days. Ten days after the last immunization, peripheral blood was collected to determine the immunogenicity. Peripheral blood was collected from alpacas with higher immunogenicity. PBMCs were isolated, and RNA was extracted according to the instructions of the RNA extraction kit (purchased from TakaRa). PrimeScript was used to analyze the RNA. TM II. The 1st Strand cDNA Synthesis Kit (purchased from TaKaRa) was used to reverse transcribe the DNA into cDNA. The VHH gene fragment was amplified from the cDNA sample by PCR using universal degenerate primers (synthesized by Nanjing GenScript) and DNA polymerase (purchased from XinHai Biotechnology). The PCR amplification products were subjected to 1% agarose gel electrophoresis, and the PCR amplification products were recovered using a gel recovery kit (purchased from Qiagen) according to the manufacturer's instructions. The VHH PCR amplification products and the yeast display vector pDisplay, which was linearized by SfiI digestion (purchased from NEB), were co-electrolyzed into EYB100 yeast competent cells. The cells were amplified and cultured in SDCAA medium at 37°C and 5% CO2. After centrifugation and washing with 1×PBS, a yeast nanobody display library was constructed through yeast homologous recombination. Yeast strains were enriched by sorting using biotin-labeled human LILRB1 and streptavidin magnetic beads, and yeast clones that bound to biotin-labeled human LILRB1 and biotin-labeled cynomolgus monkey LILRB1 were screened by FACS. A total of 31 positive clones that cross-binded to human and cynomolgus monkey LILRB1 were obtained. Their VHHs were sequenced, and the nanobody CDR sequences are shown in Table 1.

[0260] Table 1. CDR sequences of nanobodies

[0261]

[0262]

[0263] Example 4: Detection of nanobody activity using reporter gene assay

[0264] Thirty-one nanobody sequences obtained from the alpaca immune library were expressed. The VHH chain of the nanobody was synthesized and subcloned into the pcDNA3.4-hIgG4 expression vector. After the constructed vector was verified by sequencing, it was transiently transfected into CHO-K1 cells. 12 μg of BIOHUB 78 PEI transfection reagent (purchased from Shanghai Qifa) was added to 1.5 mL of OPM-Trans CHO transfection medium (purchased from OPMA), mixed thoroughly, and then added to 1.5 mL of OPM-Trans CHO transfection medium containing 30 μg of the expression vector. After thorough mixing, the mixture was incubated at room temperature for 20 minutes. The complex was then added to a medium with a density of 4 × 10⁻⁶ cells / mL. 6 In 30 mL of CHO-K1 cells, the sample was gently shaken to mix thoroughly. The cells were then cultured at 37°C, 5% CO2, and 100 rpm. On days 1, 3, and 5 post-transfection, 2.5% OPM-CHO ProFeed medium (purchased from OPMA) was added. On day 8, the supernatant was collected by centrifugation and purified using protein A affinity chromatography. The purified nanobody and the positive control BND-22 were then analyzed using the reporter gene assay according to the steps described in Example 2, and the IC50 was calculated. 50 value.

[0265] Table 2 shows the activity detection results of the reporter gene assay. It can be seen that both nanobodies and positive controls can dose-dependently activate the fluorescence signal of the reporter gene cell line. Except for 3-C8 (whose activity is substantially equivalent to the positive control BND-22), the nanobodies in Table 2 exhibit superior activity compared to the positive control BND-22. Representative results are shown in... Figure 2 The results of the activity detection using the nanobody reporter gene assay (representative figure) are shown in the figure; among them, Figure 2 Figure A shows the activity detection results of the BND-22 nanobody using the reporter gene assay. Figure 2 B shows the activity detection results of the 3-A1 nanobody reporter gene assay. Figure 2 Figure C shows the activity detection results of the 3-C8 nanobody reporter gene assay. Figure 2 Figure D shows the activity detection results of the nanobody 3-D8 reporter gene method.

[0266] Table 2. Activity detection results of nanobody reporter gene assay

[0267] No. Antibody No. IC 50 (μg / mL) No. Antibody No. IC 50 (μg / mL) BND-22 11.09 16 3-F9 0.2935 1 3-A1 0.6939 17 3-B2 2.145 2 3-C8 14 18 3-F5 1.107 3 3-D8 1.279 19 3-A6 0.4467 4 3-A3 0.7891 20 3-E10 0.4569 5 3-A2 0.4504 21 3-E1 0.9787 6 3-H7 0.8149 22 3-C12 2.23 7 3-D4 0.696 23 3-G8 0.6191 8 3-A4 1.16 24 3-G4 1.576 9 3-H10 0.6697 25 3-H5 2.677 10 3-C9 0.3979 26 3-A5 1.654 11 3-F3 0.6911 27 2-D11 0.2992 12 3-B1 0.3643 28 2-E2 0.2644 13 3-B9 0.4492 29 2-E8 0.307 14 3-H6 0.6618 30 2-E10 0.2893 15 3-A8 1.405 31 2-F4 0.4489

[0268] Example 5: Experiment to detect the binding of nanobodies to the stable LILRB1 cell line of cynomolgus monkeys

[0269] The binding of the preferred nanobodies (3-E1, 3-C12, 3-G4, 3-C8, 3-H5, 3-A5, 2-D11, 2-E2, 3-A3, 3-A1, 3-D8) and the positive control BND-22 to the stable transgenic cynomolgus monkey LILRB1 cell line obtained in Example 1 was detected according to the following steps.

[0270] (1) Take CHO-S-cyno LILRB1 cells in the logarithmic growth phase, centrifuge at 200g for 5 min, discard the supernatant, wash once with detection buffer (RPMI 1640 medium containing 1% FBS), and adjust the cell density to 4×10⁶ cells / year. 6 / mL, inoculate 50μL of 2×10⁶ mg / well in each well of a 96-well U-plate. 5 Each cell.

[0271] (2) The positive control BND-22 and the nanobody were diluted to 60 μg / mL, and then serially diluted 3-fold. 50 μL was added to each well of a 96-well U-plate to make the final antibody concentrations in each well 30 μg / mL, 10 μg / mL, 3.33 μg / mL, 1.11 μg / mL, 0.37 μg / mL, 0.123 μg / mL, 0.041 μg / mL, 0.014 μg / mL, 0.0046 μg / mL, and 0.0015 μg / mL, respectively. The plates were incubated at room temperature for 1.5 h.

[0272] (3) After incubation, add 200 μL of detection buffer to each well to resuspend the cells. Centrifuge the 96-well U plate at 200g for 5 min, discard the supernatant, and repeat the washing once.

[0273] (4) Add 1:1000 diluted GAH-PE (purchased from Biolegend) to resuspend the cells, 100 μL per well, and react at room temperature in the dark for 0.5 h.

[0274] (5) After the reaction, add 200 μL of detection buffer to each well to resuspend the cells. Centrifuge the 96-well U plate at 200g for 5 min, discard the supernatant, and repeat the washing once.

[0275] (6) Add 100 μL of detection buffer to each well to resuspend the cells and read the values ​​using a Beckman Coulter CytoFlex flow cytometer.

[0276] Figure 3 (Image showing the binding detection results of nanobodies to the stable LILRB1 cell line of cynomolgus monkeys) and Figure 4(Figure showing the binding detection results of nanobodies to the stable LILRB1 cell line of cynomolgus monkeys) The figure shows the FACS detection results of the binding of nanobodies to the stable LILRB1 cell line of cynomolgus monkeys. It can be seen that, except for 2-D11 and 2-E2 which have weak binding, the other nanobodies and the positive control BND-22 have obvious binding to the stable LILRB1 cell line of cynomolgus monkeys.

[0277] Example 6: Experiment to detect the effect of nanobodies on NK92 cell killing

[0278] The following steps were followed to detect the activity of the selected nanobodies (antibody numbers are shown in Table 3) and the positive control BND-22 in promoting the killing of tumor cells by NK92 cells.

[0279] (1) Take logarithmically proliferating NK92 cells, wash twice with PBS, and adjust the density to 3×10⁻⁶ cells with RPMI 1640 medium. 6 / mL, 50μL of 1.5×10⁶ mcg solution was inoculated into each well of a 96-well plate. 5 Each cell.

[0280] (2) The positive control BND-22 and the nanobody were diluted to 40 μg / mL, and then serially diluted 5-fold to antibody concentrations of 40 μg / mL, 8 μg / mL, 1.6 μg / mL, 0.32 μg / mL, 0.064 μg / mL, 0.0128 μg / mL, 0.00256 μg / mL, and 0.000512 μg / mL, respectively. 100 μL was added to each well of a 96-well plate. The plate was incubated at 37°C and 5% CO2 for 2 h.

[0281] (3) The constructed stable HLA-G and luciferase-transfected A375 cells (A375-HLA-G-Luciferase) were used as target cells. Log-proliferating A375-HLA-G-Luciferase cells were washed twice with 1×PBS and then adjusted to a density of 4×10⁶ cells / year using RPMI 1640 medium. 5 / mL, inoculate 50μL of 2×10⁶ mg / well in each well of a 96-well plate. 4 Cells were used to achieve an effector-to-target ratio of 7.5:1, with final antibody concentrations per well of 20 μg / mL, 4 μg / mL, 0.8 μg / mL, 0.16 μg / mL, 0.032 μg / mL, 0.0064 μg / mL, 0.00128 μg / mL, and 0.000256 μg / mL, respectively. The cells were co-cultured at 37°C and 5% CO2 for 5 hours.

[0282] (4) Add 20 μL of One-Glo luminescent substrate to each well, place at room temperature in the dark for 5 min, read the Luciferase fluorescence value using a Tecan M1000 multi-functional microplate reader, and calculate the killing rate of NK92 cells against target cells in each well.

[0283] Table 3 shows the results of the NK92 cell killing assay. It can be seen that both the nanobody and the positive control can enhance the killing effect of NK92 cells on A375 target cells. The nanobody's activity in enhancing killing is superior to or comparable to that of the positive control BND-22. Representative results are shown in... Figure 5 ( Figure 5 The results of the NK92 nanobody cell killing assay are shown in the figure (representative figure); among them, Figure 5 Figure A shows the detection results of the cell killing assay using the BND-22NK92 ​​nanobody. Figure 5 B shows the detection results of the 3-A1 nanobody NK92 cell killing assay. Figure 5 Figure C shows the detection results of the 3-C8 nanobody NK92 cell killing assay. Figure 5 Figure D shows the detection results of the 3-D8 NK92 cell killing assay using nanobody.

[0284] Table 3. Results of the NK92 nanobody cell killing assay

[0285] No. Antibody No. EC 50 (μg / mL) No. Antibody No. EC 50 (μg / mL) BND-22 0.2077 16 3-F9 0.0008358 1 3-A1 0.001312 17 3-B2 0.01878 2 3-C8 0.4964 18 3-F5 0.002315 3 3-D8 0.0004175 19 3-A6 0.000593 4 3-A3 1.04E-07 20 3-E10 0.007988 5 3-A2 0.0001772 21 3-E1 0.0004056 6 3-H7 0.0002865 22 3-C12 0.2286 7 3-D4 0.0009565 23 3-G8 0.01953 8 3-A4 0.01271 24 3-G4 0.01982 9 3-H10 0.002743 25 3-H5 0.04233 10 3-C9 0.00108 26 3-A5 0.02663 11 3-F3 0.001158 27 2-D11 0.005934 12 3-B1 0.0004582 28 2-E2 0.001653 13 3-B9 0.00171 29 2-E8 0.002058 14 3-H6 0.001611 30 2-E10 0.002077 15 3-A8 0.0005918 31 2-F4 0.0001903

[0286] Example 7: Experiment to detect the effect of nanobodies on primary NK cell killing

[0287] The following steps were performed to detect the activity of selected nanobodies (2-D11, 2-E2, 3-A3, 3-A1, 3-D8, 3-E1, 3-C8, 3-C12, 3-G4, 3-H5, 3-A5) and the positive control BND-22 in promoting the killing of tumor cells by primary NK cells.

[0288] (1) Collect A375-HLA-G-Luciferase cells in the logarithmic growth phase, digest them with trypsin (purchased from Gibco), centrifuge at 200g for 5 min, discard the supernatant, wash once with detection buffer (RPMI 1640 medium), and adjust the cell density to 4×10⁶ cells / year. 5 / mL, inoculate 25μL of 1×10⁶ mg / well in each well of a 96-well plate. 4 Each cell.

[0289] (3) Collect primary NK cells, centrifuge at 200g for 5 min, discard the supernatant, wash once with detection buffer, and adjust the cell density to 3×10⁶ cells / year. 6 / mL, 25μL of 7.5×10⁶ mcg solution was inoculated into each well of a 96-well plate.4 The target ratio was 7.5:1, which was achieved by using 10 cells.

[0290] (3) Take the positive control BND-22 and the nanobody and dilute them to 40 μg / mL. Add 50 μL to each well of a 96-well plate to make the final antibody concentration 20 μg / mL. Incubate at 37℃ and 5% CO2 for 5 h.

[0291] (4) After incubation, centrifuge the 96-well plate at 200g for 5 min and discard 150μL of supernatant.

[0292] (5) Add 50 μL of Bio-Lite dye (purchased from Novizan) to each well, read the Luciferase fluorescence value using a BMG CLARIOstar plus multi-functional microplate reader, and calculate the killing rate of NK cells against target cells in each well.

[0293] Figure 6 (The results of the primary NK cell killing assay using nanobodies) show the results of the primary NK cell killing assay. It can be seen that both nanobodies and the positive control IgG4 isotype control can enhance the killing of A375 target cells by primary NK cells. The killing activity of nanobodies is better than or comparable to that of the positive control BND-22.

[0294] Example 8: Experiment to detect the effect of nanobodies on macrophage killing

[0295] The following steps were performed to detect the activity of selected nanobodies (2-D11, 2-E2, 3-A3, 3-A1, 3-D8, 3-E1, 3-C8, 3-C12, 3-G4, 3-H5, 3-A5) and the positive control BND-22 in promoting the killing of tumor cells by primary macrophages.

[0296] (1) PBMCs isolated from healthy individuals were sorted into mononuclear cells using CD14+ magnetic beads (purchased from Miltenyi). The cell density was adjusted to 1×10⁻⁶ cells using RPMI 1640 medium containing 1000 U / mL GM-CSF and 10% FBS. 6 The cells were cultured at 37°C and 5% CO2 for 5 days. After the culture was completed, RPMI 1640 medium containing 20 ng / mL IFN-γ, 50 ng / mL LPS, and 10% FBS was added to induce culture for 3 days to obtain M1 macrophages.

[0297] (2) Centrifuge 500g of induced macrophages for 5min, wash twice with PBS, and adjust the cell density to 2×10⁶ cells / cells with RPMI 1640 medium containing 10% FBS. 6 / mL, inoculate 50μL of 1×10⁶ mg / well in each well of a 96-well plate.5 Each cell.

[0298] (3) Take the positive control BND-22 and the nanobody and dilute them to 200 μg / mL. Then, perform a 5-fold serial dilution to antibody concentrations of 200 μg / mL, 40 μg / mL, and 8 μg / mL respectively. Add 100 μL to each well of a 96-well plate. Incubate at 37℃ and 5% CO2 for 1 h.

[0299] (4) Log-proliferating A375-HLA-G-Luciferase cells were taken, washed twice with PBS, and the cell density was adjusted to 4×10⁶. 5 / mL, inoculate 50μL of 2×10⁶ mg / well in each well of a 96-well plate. 4 Cells were selected to achieve an effector-to-target ratio of 5:1. The final antibody concentrations in the 96-well plates were 100 μg / mL, 20 μg / mL, and 4 μg / mL, respectively. Wells containing only target cells and wells containing only target cells and effector cells were set up as controls. The cells were co-cultured at 37°C and 5% CO2 for 6 hours.

[0300] (5) Add 20 μL of One-Glo luminescent substrate to each well, place at room temperature in the dark for 5 min, read the Luciferase fluorescence value using a Tecan M1000 multi-functional microplate reader, and calculate the killing rate of macrophages against target cells in each well.

[0301] Figure 7 (The results of the macrophage killing experiment with nanobody are shown in the figure. It can be seen that both nanobody and positive control can enhance the killing of A375 target cells by primary macrophages in a dose-dependent manner. The killing activity of nanobody is better than or comparable to that of positive control BND-22.

[0302] Example 9: Antibody Humanization

[0303] Nanobodies 3-A1 and 3-C8 were humanized using amino acid sequences of their VHH chains, while retaining the original antibody's CDR region sequence. Humanized antibody templates were selected based on germline alignment and antibody structure simulation results. Reversion mutations were then performed on the framework region after humanization to design candidate humanized antibody sequences. The VHH chains of the designed humanized antibodies were synthesized and subcloned into the pcDNA3.4-hIgG4 expression vector. After sequencing verification, the constructed vectors were transiently transfected into CHO-K1 cells according to the steps described in Example 4, and the humanized antibodies were purified using Protein A affinity chromatography. The VHH chain sequences of the humanized antibodies corresponding to nanobodies 3-A1 and 3-C8 are shown in Table 4.

[0304] Table 4. Humanized nanobody VHH chain sequence

[0305]

[0306]

[0307] Example 10: Reporter Gene Assay for Humanized Nanobody Activity

[0308] Following the steps described in Example 2, the activity of humanized nanobodies was detected using the reporter gene assay. The results showed that nanobodies 3-A1 and 3-C8 maintained high activity even after humanization, as shown in Table 5.

[0309] Table 5. Activity detection results of humanized nanobody reporter gene assay

[0310] Antibody No. IC 50 (μg / mL) BND-22 7.549 3-A1-VHH1 0.0453 3-A1-VHH2 0.1419 3-A1-VHH3 0.1797 3-A1-VHH4 0.1497 3-A1-VHH5 0.103 3-A1-VHH6 0.1557 3-C8-VHH1 1.89 3-C8-VHH2 1.015 3-C8-VHH3 2.576 3-C8-VHH5 1.255 3-C8-VHH6 1.538 3-C8-VHH7 1.656

[0311] Example 11: Experiment to detect the effect of humanized nanobodies on primary NK cell killing

[0312] The activity of humanized nanobodies and the positive control BND-22 in promoting the killing of tumor cells by primary NK cells was detected according to the steps described in Example 7. Figure 8 (Figure showing the detection results of the primary NK cell killing experiment using humanized nanobodies) The results of the primary NK cell killing experiment are shown. It can be seen that both humanized nanobodies and positive controls can enhance the killing of A375 target cells by primary NK cells in a dose-dependent manner. In terms of the effect of promoting killing at high concentrations, the humanized nanobodies 3-C8-VHH7, 3-A1-VHH1, 3-C8-VHH6, and 3-C8-VHH1 have better killing activity than the positive control BND-22.

[0313] The sequence of this application is listed in Table 6 below.

[0314] Table 6 Sequence of this application

[0315]

[0316]

[0317]

[0318]

[0319] For purposes of description and disclosure, all patents, patent applications, and other publications are expressly incorporated herein by reference. These publications are provided solely because their publication predates the filing date of this application. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or contents of these documents. Furthermore, in any country, any reference to these publications herein does not constitute an endorsement that such publication is part of the general knowledge in the art.

[0320] Those skilled in the art will recognize that the scope of this application is not limited to the various specific implementations and embodiments described above, but rather that various modifications, substitutions, or recombinations can be made without departing from the spirit of this application, all of which fall within the protection scope of this application.

Claims

1. An anti-LILRB1 Nanobody, whose VHH chain comprises the following complementarity determining regions CDR1, CDR2 and CDR3: (1) CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, CDR3 as shown in SEQ ID NO: 3; or (2) CDR1 as shown in SEQ ID NO: 4, CDR2 as shown in SEQ ID NO: 5, CDR3 as shown in SEQ ID NO:

6.

2. The anti-LILRB1 nanobody of claim 1, wherein, said VHH chain has an amino acid sequence as shown in SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 or 82, or an amino acid sequence which is at least 95% identical to the amino acid sequence as shown in SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 or 82.

3. The anti-LILRB1 nanobody of claim 1, wherein, said VHH chain has an amino acid sequence as shown in SEQ ID NO: 71, 77, 81 or 82.

4. The anti-LILRB1 nanobody of claim 1 or 2, wherein, said anti-LILRB1 Nanobody is a camelid Nanobody, a humanized Nanobody.

5. The anti-LILRB1 nanobody of claim 1 or 2, wherein, said antibody is selected from a monovalent antibody, a multivalent antibody.

6. The anti-LILRB1 nanobody of claim 5, wherein, said antibody is a multivalent antibody, which comprises at least 2 of said VHH chains.

7. A nucleic acid molecule encoding the anti-LILRB1 Nanobody of any one of claims 1-6.

8. A recombinant vector comprising the nucleic acid molecule of claim 7.

9. A recombinant cell comprising the nucleic acid molecule of claim 7 or the recombinant vector of claim 8.

10. A kit comprising the anti-LILRB1 Nanobody of any one of claims 1-6.

11. Use of the anti-LILRB1 Nanobody of any one of claims 1-6, the nucleic acid molecule of claim 7, the recombinant vector of claim 8, the recombinant cell of claim 9 or the kit of claim 10 for the manufacture of a medicament for the diagnosis, prevention and / or treatment of melanoma.

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