Antibody capable of binding to LILRB2 or antigen binding fragment thereof

Abstract

The present invention provides antibodies, or antigen binding fragments thereof, capable of binding to leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2). The invention also provides application of the antibody or the antigen binding fragment thereof in preparation of drugs for treating diseases related to high expression of LILRB2. The antibody or the antigen binding fragment thereof provided by the invention can bind to human LILRB2 with high affinity, block the binding of the LILRB2 and the ligand HLA-G thereof, and enhance the T cell function.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and relates to an antibody or an antigen-binding fragment thereof capable of binding to leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2). The present invention also relates to the application of the antibody or the antigen-binding fragment thereof. Background Art

[0002] LILRB2 is a member of the inhibitory leukocyte immunoglobulin-like receptor (LIR) family and belongs to the subfamily B of the LIR receptor, also known as ILT4. The protein has an immunoglobulin-like (Ig-like) domain in the extracellular region and an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular region. It has been found that in solid tumors, LILRB2 can interact with its ligand human leukocyte antigen G (HLA-G) to induce myeloid cells to produce a tolerant phenotype, thereby inhibiting T cell activation and leading to tumor immune escape. Therefore, blocking the binding of LILRB2 and HLA-G can enhance tumor immune response.

[0003] Merck has developed the LILRB2 blocking antibody MK-4830, which is in Phase II clinical trials to verify the therapeutic effect of MK-4830 on patients with various solid tumors who are ineffective with PD1 inhibitors. Currently, there are no antibodies targeting LILRB2 on the market. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide an anti-LILRB2 antibody or an antigen-binding fragment thereof, which can bind to LILRB2 with high affinity and specificity, thereby blocking the interaction of LILRB2 with ligands such as HLA-G associated with cancer immunosuppression in the tumor microenvironment. Furthermore, based on the binding effect of the antibody or its antigen-binding fragment on LILRB2, another purpose of the present invention is to provide its therapeutic use in tumors.

[0005] The present invention provides the following technical solutions.

[0006] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the fragment thereof can specifically bind to LILRB2, in particular human LILRB2. According to a specific embodiment of the present invention, the antibody of the present invention is a mouse antibody obtained by using the extracellular region of human LILRB2 as an immunogen, and a humanized antibody obtained by humanizing the mouse antibody.

[0007] Specifically, the antibodies or antigen-binding fragments thereof provided by the present invention comprise heavy chain complementary determining regions (HCDRs, i.e., HCDR1, HCDR2, HCDR3) and light chain complementary determining regions (LCDRs, i.e., LCDR1, LCDR2, LCDR3), and the heavy chain and light chain complementary determining regions are derived from the following amino acid sequence combinations:

[0008] (1) the amino acid sequence shown in SEQ ID NO: 1; and the amino acid sequence shown in SEQ ID NO: 2; or

[0009] (2) the amino acid sequence shown in SEQ ID NO: 3; and the amino acid sequence shown in SEQ ID NO: 4.

[0010] The amino acid sequences of the above items are respectively the amino acid sequences of the heavy chain variable region and the light chain variable region of the specific anti-LILRB2 antibodies (mouse antibodies and humanized antibodies) provided by the present invention; see the Examples section of the present invention. Using the well-known or conventional definition tools for the complementary determining regions in the heavy chain or light chain of antibodies (such as the Kabat scheme used in the examples of the present invention), those skilled in the art can easily determine the heavy chain and light chain CDRs contained when the two are combined to form an antibody. Antibodies or antigen-binding fragments thereof containing each of these heavy chain and light chain CDR combinations are within the scope of protection of the present invention.

[0011] Furthermore, the antibody or antigen-binding fragment thereof provided by the present invention comprises the following combination of heavy chain CDRs and light chain CDRs (HCDR1, HCDR2, HCDR3; and, LCDR1, LCDR2, LCDR3):

[0012] HCDR1, HCDR2, HCDR3 comprising the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, respectively; and LCDR1, LCDR2, LCDR3 comprising the amino acid sequences shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, respectively.

[0013] The antibody or antigen-binding fragment thereof provided by the present invention is an antibody or antigen-binding fragment thereof against LILRB2. Preferably, the LILRB2 is human LILRB2.

[0014] Preferably, the antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). In the antibody or antigen-binding fragment thereof provided by the present invention, preferably, the heavy chain variable region comprises a sequence selected from the following: an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or an amino acid sequence having at least 75% identity with the amino acid sequence; and / or, the light chain variable region comprises a sequence selected from the following: an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0015] More preferably, the heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment thereof provided by the present invention comprise:

[0016] (1) the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 1; and, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 2; or

[0017] (2) the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:3; and, the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:4.

[0018] In the context of the present invention, "at least 75% identity" refers to any percentage number between 75% and 100% identity, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identity.

[0019] In particular, in the antibodies or antigen-binding fragments thereof of the present invention, the heavy chain variable region and the light chain variable region both include the above-mentioned CDRs and the framework regions in between, and the arrangement of each region is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further optionally, the at most 25% difference in amino acid sequence caused by the "at least 75% identity" may exist in any framework region in the heavy chain variable region or the light chain variable region, or in any domain or sequence other than the heavy chain variable region and the light chain variable region in the antibodies or antigen-binding fragments thereof of the present invention. The difference may be caused by deletion, addition or substitution of amino acids at any position.

[0020] In terms of antigen, the antibody or antigen-binding fragment thereof of the present invention is an antibody or antigen-binding fragment thereof against LILRB2. For example, the antibody may be in any form such as mouse antibody, rabbit antibody, monoclonal antibody, chimeric antibody, partially or fully humanized antibody, etc.; the antigen-binding fragment is any fragment of the antibody, which retains the antibody's ability to bind to the antigen and the corresponding biological activity. The fragment may be a half antibody or any antigen-binding fragment of an antibody or a half antibody, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.

[0021] More preferably, the antibody provided by the present invention is IgG. According to a specific embodiment of the present invention, the present invention provides an isolated and structurally characterized murine or humanized antibody, such as a monoclonal antibody, that specifically binds to LILRB2 (such as human LILRB2).

[0022] In addition to the variable region, the antibody or antigen-binding fragment thereof provided by the present invention further comprises a constant region, such as a human or mouse constant region, preferably comprising a human or mouse heavy chain constant region (CH) and / or a light chain constant region (CL); preferably, the antibody or fragment thereof comprises a heavy chain and a light chain; more preferably, the antibody or fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

[0023] According to a specific embodiment of the present invention, the antibody is a monoclonal antibody against LILRB2, preferably a mouse, chimeric or humanized monoclonal antibody. Preferably, the heavy chain constant region of the monoclonal antibody is of IgG1 or IgG4 subtype, and the light chain constant region is of κ type. Preferably, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 7; the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 8.

[0024] According to a specific embodiment of the present invention, the anti-human LILRB2 antibody of the present invention is a monoclonal antibody. Preferably, the anti-human LILRB2 antibody provided by the present invention is an immunoglobulin, for example, the type of the immunoglobulin is human IgA, IgD, IgE, IgG or IgM. Further preferably, the antibody is human IgG1 or IgG4 subtype.

[0025] On the other hand, the present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain CDR, light chain CDR, light chain variable region, heavy chain variable region, heavy chain or light chain contained in the antibody or antigen-binding fragment thereof provided by the present invention.

[0026] The nucleic acid molecules of the present invention can be cloned into a vector, and then transformed or transfected into a host cell. Therefore, in another aspect, the present invention also provides a vector, the vector comprises the nucleic acid molecules of the present invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome and a phage vector, etc. The vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell. Therefore, in another aspect, the present invention provides a host cell, the host cell comprises the nucleic acid molecules of the present invention and / or the vector, or the host cell is transformed or transfected by the nucleic acid molecules of the present invention and / or the vector. The host cell can be any prokaryotic or eukaryotic cell, for example a bacterium or an insect, fungus, plant or animal cell.

[0027] The antibodies or antigen-binding fragments thereof provided by the present invention can be obtained by any method known in the art. For example, the host cells provided by the present invention are allowed to express the heavy and light chains of the antibodies, and the host cells are cultured. Optionally, the method further comprises the step of recovering the produced antibodies.

[0028] The antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell and / or provided by the present invention can be included in a composition, more particularly in a pharmaceutical composition such as a pharmaceutical preparation, so as to be used for various purposes according to actual needs. Therefore, in another aspect, the present invention also provides a composition, such as a pharmaceutical composition, comprising the antibody or fragment thereof, nucleic acid molecule, vector and / or host cell described in the present invention, and optional pharmaceutically acceptable excipients.

[0029] In another aspect, the present invention also provides the use of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell and / or composition in the preparation of a medicament for treating a disease, wherein the disease is a tumor associated with high expression of LILRB2. Preferably, the disease is one or more selected from esophageal squamous cell carcinoma, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer and non-small cell lung cancer. Further preferably, the disease is non-small cell lung cancer.

[0030] Therefore, accordingly, the present invention also provides a method for treating a disease, the method comprising administering the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present invention to a subject in need thereof, wherein the disease is a tumor associated with high expression of LILRB2. Preferably, the disease is one or more selected from esophageal squamous cell carcinoma, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer and non-small cell lung cancer. Further preferably, the disease is non-small cell lung cancer. The subject is a mammal; preferably, the subject is a human.

[0031] In another aspect, the present invention also provides the use of the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell and / or composition in the preparation of a reagent for diagnosing a disease, wherein the disease is a tumor associated with high expression of LILRB2. Preferably, the disease is one or more selected from esophageal squamous cell carcinoma, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer and non-small cell lung cancer. Further preferably, the disease is non-small cell lung cancer. The diagnosis is achieved by detecting the presence (e.g., high expression) of the antigen LILRB2 in a biological sample from a subject. The subject is a mammal; preferably, the subject is a human.

[0032] In another aspect, the present invention provides a kit comprising the antibody or fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present invention. The kit can be used for the above-mentioned treatment or diagnosis. Depending on the intended application, the kit also comprises other reagents for treatment or diagnosis. For example, the kit is a kit for detecting the presence (e.g., high expression) of LILRB2 in any biological sample using ELISA or FACS.

[0033] The present invention provides an antibody or an antigen-binding fragment thereof that can bind to leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2). Experiments have shown that the anti-LILRB2 antibody provided by the present invention can bind to human LILRB2 with high affinity, thereby blocking the binding of LILRB2 to its ligand HLA-G, blocking LILRB2 downstream signals, and enhancing T cell function; in particular, compared with the known anti-LILRB2 antibody MK-4830, the anti-LILRB2 antibody provided by the present invention has a stronger T cell enhancement effect. In addition, tumor transplantation animal experiments have shown that the anti-LILRB2 antibody provided by the present invention can significantly inhibit the growth of tumor cells in animals, and can play a synergistic anti-tumor effect with anti-PD1 antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0035] Figure 1The results of FACS detection of the binding of hybridoma cell culture supernatant to human LILRB2-expressing cells are shown.

[0036] Figure 2 The figure shows the FACS detection results of hybridoma cell culture supernatant blocking the binding of HLA-G protein to human LILRB2-expressing cells.

[0037] Figure 3 The FACS detection results of the binding of anti-LILRB2 antibodies to human LILRB2-expressing cells are shown; wherein, 3A: MK-4830; 3B: 11C6-8.

[0038] Figure 4 The FACS detection results of the binding of anti-LILRB2 antibodies to monkey LILRB2-expressing cells are shown; wherein, 4A: MK-4830; 4B: 11C6-8.

[0039] Figure 5 The FACS detection results of anti-LILRB2 antibodies blocking the binding of HLA-G protein to human LILRB2-expressing cells are shown; among them, 5A: MK-4830; 5B: 11C6-8.

[0040] Figure 6 The FACS detection results of the binding of anti-LILRB2 antibodies to human LILRB2-expressing cells and monkey LILRB2-expressing cells are shown.

[0041] Figure 7 The results of FACS detection showing that anti-LILRB2 antibodies blocked the binding of HLA-G protein to human LILRB2-expressing cells.

[0042] Figure 8 Shown are the results of testing that anti-LILRB2 antibodies blocked LILRB2 downstream signaling.

[0043] Fig. 9 Shown are the results of detection of surface molecule expression on macrophages after incubation with anti-LILRB2 antibody.

[0044] Fig.10 Shown are the results of detecting T cell proliferation after incubation with anti-LILRB2 antibody.

[0045] Fig.11 Tumor growth curves after anti-LILRB2 antibody administration are shown. DETAILED DESCRIPTION

[0046] The present invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0047] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0048] The positive control antibody used in the example is antibody MK-4830, and the variable region sequence is:

[0049] >MK-4830 heavy chain variable region sequence (SEQ ID NO: 5)

[0050] EVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIG

[0051] EINHAGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLP

[0052] TRWVTTRYFDLWGRGTLVTVSS

[0053] >MK-4830 light chain variable region sequence (SEQ ID NO: 6)

[0054] ESVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIY

[0055] GDSNRPSGVPDRFSVSKSGASASLAITGLQAEDEADYYCQSFDNSLSAYV

[0056] FGGGTQLTVL

[0057] The above variable region sequence and the following heavy chain and light chain constant region sequences were used to recombinantly express the antibody MK-4830 (see Example 10):

[0058] >hIgG4-S228P heavy chain constant region sequence (SEQ ID NO:7)

[0059] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0060] >hkappa light chain constant region sequence (SEQ ID NO:8)

[0061] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0062] Example 1 Preparation of hybridoma cells

[0063] The mice were immunized with a fusion protein of the extracellular region of human LILRB2 (NCBI Reference Sequence: NM_005874.4, AA Gln 22-Val461) and mouse IgG2a-FC (Genbank Accession No.: AAH31470.1) as an immunogen.

[0064] Select mice with high serum antibody titers, take serum and spleen, separate spleen cells from them, fuse spleen cells with cultured myeloma cells, and obtain hybridoma cells that can secrete monoclonal antibodies. Expand the cells and culture.

[0065] Example 2 ELISA detection of the binding between hybridoma cell culture supernatant and human LILRB2 protein

[0066] Dilute the fusion protein of the extracellular region of human LILRB2 and human IgG1-FC (Genbank registration number: CAC20454.1) to 1-2 μg / ml with coating solution, add the diluent to the ELISA plate at 50-100 μl / well, and then place the ELISA plate at 4°C overnight or at 37°C for 2 hours. Discard the liquid in the wells, wash the plate 3 times with washing solution, 3-5 minutes each time, and pat dry. Add 200 μl of blocking solution to each well, and then place the ELISA plate at 4°C overnight or at 37°C for 2 hours. Discard the liquid in the wells, wash 3 times with washing solution, and then store the ELISA plate at -20°C or 4°C for use.

[0067] Add 50-100 μl / well of the culture supernatant of the hybridoma cells to be tested to the above-mentioned ELISA plate, and set up a positive control (adding serum from fusion mice), a negative control (adding serum from normal mice), and a blank control (adding cell culture medium). Incubate at 37°C for 1-2 hours, wash, and pat dry. Then add enzyme-labeled secondary antibody, which is 1:10000 diluted horseradish peroxidase-labeled goat anti-mouse IgG (SIGMA, Cat No.: A9044-2ml), 50-100 μl per well, incubate at 37°C for 1-2 hours, wash, and pat dry. Add 50-100 μl of freshly prepared substrate colorimetric solution TMB to each well and incubate at 37°C for 10-30 minutes.

[0068] The reaction was terminated by adding 2 mol / L H2SO4 and the OD value was read on a microplate reader.

[0069] Result determination: P / N>2:1 (P represents the positive value, N represents the normal mouse serum value) is considered positive. If the negative control well is colorless or nearly colorless, and the positive control well is clearly colored, the result can be directly observed with the naked eye.

[0070] Example 3 FACS detection of the binding between the culture supernatant of hybridoma cells and human LILRB2 expressing cells

[0071] The full-length human LILRB2 gene was constructed into a pLVX virus packaging vector (clontech, virus packagemix, Cat No.: 631275), and the constructed vector was transfected into 293T cells to harvest the virus. The virus was then used to transfect 293T cells, and puromycin was used to screen for drug-resistant cell lines, namely 293T cells stably transfected with human LILRB2 (293T-LILRB2; Kyinno Biotechnology Co. Ltd. (hereinafter referred to as "Kyinno"), Cat No.: KC-1472). 293T-LILRB2 cells were prepared in PBS containing 2% FBS to a cell concentration of 10 7cells / ml of cell suspension.

[0072] Place 50 μl of cell suspension in each flow sample tube, then add 50 μl of culture supernatant of hybridoma cells to be tested, and incubate at 4°C for 60 minutes. Add 1 ml of flow buffer to each flow tube, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, and repeat washing three times. At the same time, set up control tube 1 (no culture supernatant and the secondary antibody below, only cell suspension) and control tube 2 (no culture supernatant, only cell suspension and the secondary antibody below).

[0073] Then, 100 μl of flow buffer was added to each flow tube for resuspending, and then 5 μl of PE anti-mouse IgG Fc (Biolegend, Cat No.: 405307) was added, and incubated at 4°C in the dark for 30 minutes. Then, 1 ml of flow buffer was added, and centrifuged at 1200 rpm for 5 minutes at room temperature, and the supernatant was discarded. This operation was repeated three times for washing.

[0074] Add 250 μl of flow buffer to each flow tube again, resuspend the cells, and detect using flow cytometer. The test results are shown in Table 1 and Figure 1 .

[0075] Table 1. FACS detection results of the binding between the culture supernatant of hybridoma cells and human LILRB2 expressing cells

[0076]

[0077] Example 4 FACS detection of the blocking effect of hybridoma cell culture supernatant on the binding of HLA-G protein to human LILRB2 expressing cells

[0078] 293T-LILRB2 cells were washed once with FACS buffer and centrifuged at 2-5 × 10 5 Cells / well were plated in a 96-deep-well plate, and 50 μl of hybridoma cell culture supernatant and 500 ng of human B2M-HLAG-hFC protein (Kyinno, Cat No.: KP-1090; NCBI Reference Sequence of B2M: NM_004048.2; NCBI Reference Sequence of HLA-G: NM_002127.5; GenBank of hFC: CAC20454.1) premix were added to each well and incubated for 2 hours. 400 μl of FACS buffer was added to each well for washing twice, PE anti-human IgG Fc secondary antibody was added, incubated for 1 hour, and then 400 μl of FACS buffer was added to each well for washing twice.

[0079] Flow cytometry was used for detection. The detection results are shown in Table 2 and Figure 2 .

[0080] Table 2. FACS detection results of hybridoma cell culture supernatant blocking the binding of HLA-G protein to human LILRB2 expressing cells

[0081]

[0082] The corresponding mouse antibodies were obtained from the above hybridoma cells. The variable region sequences of mouse antibody 11C6-8 (named according to the hybridoma cells) are as follows (wherein the heavy chain and light chain CDRs are underlined, which are obtained according to the Kabat definition method):

[0083] >VH(VH / HCDR1 / HCDR2 / HCDR3:SEQ ID NOs:1 / 9 / 10 / 11)

[0084] QVQLKESGPGLVQPSQSLSITCTVSGCSLT TYG WVRQSPGKGLEWLG VIWSGGNTDYNAAFIS RLSINKDNSKSQVFFKMNSLQANDTAIYYCAR NGYGAMDY WGQGTSVTVSS

[0085] >VL(VL / LCDR1 / LCDR2 / LCDR3:SEQ ID NO:2 / 12 / 13 / 14)

[0086] DIVMTQAHKFMSTSLGDRVSITC KASHDVSIAVA WYQQKPGQSPKLLIY SASYRYT GVPDRFTGSGSGTDFIFIISSVQAEDLAVYYC QQYYSTPRT FGGGTTLDIK

[0087] Example 5 Affinity test of mouse antibody binding to human LILRB2 protein

[0088] ForteBio Octet was used to detect the binding affinity of mouse anti-11C6-8 to human LILRB2 protein. AMC chip was selected, the concentration of immobilized antibody was 10 μg / mL, and LILRB2-His (Kyinno, Cat No.: KP-1033) was used as the mobile phase, and the concentration was diluted to 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, and 3.12nM. The test results are shown in Table 3.

[0089] Table 3. Affinity test results of mouse antibody binding to human LILRB2 protein

[0090] Mouse Antibody KD(M) kon(1 / Ms) kdis(1 / s) RMax FullR2 11C6-8 1.02E-09 5.66E+05 5.78E-04 0.6421 0.9839

[0091] Example 6 FACS detection of mouse antibody binding to human LILRB2 expressing cells

[0092] Mouse anti-11C6-8 and antibody MK-4830 were serially diluted and incubated with 293T cells expressing human LILRB2, i.e., 293T-LILRB2 cells, and then incubated with PE anti-mouse IgG Fc secondary antibody (Biolegend, Cat No.: 405307) or PE anti-human IgG Fc secondary antibody (SouthernBiotech, Cat No.: 2010-09). The secondary antibody signal bound to the cells was detected using a flow cytometer.

[0093] The test showed that the EC50 of antibody 11C6-8 was 0.2μg / ml, and the EC50 of antibody MK-4830 was 0.07μg / ml. Figure 3 .

[0094] Example 7 FACS detection of mouse antibody binding to monkey LILRB2 expressing cells

[0095] Mouse anti-11C6-8 and antibody MK-4830 were serially diluted and incubated with 293T cells expressing monkey LILRB2 (293T-cynoLILRB2) (Kyinno, Cat No.: KC-1461), and then incubated with PE anti-mouse IgG Fc (Biolegend, Cat No.: 405307) or PE anti-human IgG Fc secondary antibody (SouthernBiotech, Cat No.: 2010-09). The secondary antibody signal bound to the cells was detected by flow cytometry.

[0096] The test showed that the EC50 of antibody 11C6-8 was 0.72ug / ml, and the EC50 of antibody MK-4830 was 9.6μg / ml. Figure 4 .

[0097] Example 8 FACS detection of mouse antibody blocking the binding of HLA-G protein to human LILRB2 expressing cells

[0098] Mouse anti-11C6-8 and antibody MK-4830 were diluted in series, incubated with 293T cells expressing human LILRB2, i.e., 293T-LILRB2 cells, and then incubated with 3ug / ml B2M-HLAG-hFc protein, and finally incubated with anti-human Goat anti-human IgG-PE secondary antibody (Southern Biotech, 2010-09). The B2M-HLAG-hFc signal bound to the cells was detected by flow cytometry.

[0099] The test showed that the IC50 of antibody 11C6-8 was 0.133ug / ml, and the IC50 of antibody MK-4830 was 0.045μg / ml. Figure 5 .

[0100] Example 9 Humanization of mouse antibodies

[0101] For mouse antibody 1C6-8, the following human germline sequences IGHV4-39 and IGKV1-39 were selected as templates for humanization of the heavy chain and light chain, respectively. Homology modeling was performed on the antibody, and structural simulation of the Fab region was performed. After homology modeling calculations, the predicted Fab structure of the antibody was finally obtained.

[0102] By comparing and analyzing the predicted Fab structure and heavy chain with the IGHV4-39 sequence, several amino acids that affect the CDR structure were retained as the original mouse amino acids, and all other mouse amino acids were replaced with the corresponding IGHV4-39 template human amino acids.

[0103] By comparing the predicted Fab structure and light chain with the IGKV1-39 sequence, except for the CDR region which retains the original mouse amino acids, all other mouse amino acids are replaced with the corresponding IGKV1-39 template human amino acids.

[0104] The resulting humanized sequences are as follows (wherein the heavy and light chain CDRs are underlined and obtained according to the KABAT definition method):

[0105] >11C6-8 heavy chain variable region humanized sequence (VH / HCDR1 / HCDR2 / HCDR3: SEQ ID NOs: 3 / 9 / 10 / 11)

[0106] QVQLQESGPGLVKPSETLSLTCTVSGSSLT TYG WVRQPPGKGLEWLG VIWSGGNTDYNAAFIS RLTISKDNSKSQVSLKLSSVTAADTAVYYCAR NGYGAMDY WGQGTTVTVSS

[0107] >11C6-8 light chain variable region humanized sequence (VL / LCDR1 / LCDR2 / LCDR3: SEQ ID NOs: 4 / 12 / 13 / 14)

[0108] DIQMTQSPSSLSASVGDRVTITC KASHDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYYSTPRT FGQGTRLEIK

[0109] Example 10 Expression and purification of humanized antibodies

[0110] According to the variable region sequence obtained by humanization provided in Example 9 and the constant region sequence shown in SEQ ID NO: 7 and SEQ ID NO: 8, the coding genes of the heavy chain and light chain of the humanized antibody were constructed and then cloned into the eukaryotic expression vector pcDNA3.4, respectively, to obtain two recombinant expression plasmids.

[0111] HEK293F expression system was used to transiently co-transfect two plasmids to produce LILRB2 humanized antibody. Briefly, HEK293F cells were cultured at 1×10 6 The cells were inoculated into 250 mL of culture medium at a density of 10 / mL and incubated at 110 rpm and 5% CO2. The next day, the two expression vectors prepared in advance were mixed with the transfection reagent in a certain proportion and the cells were inoculated at a density of 2×10 6 The cell density was 100 / mL, and the transfection complex was added to the cells. Nutrients and DNA inhibitors were added after 24 hours. After 5-7 days of cell culture, the expression supernatant was collected, centrifuged, filtered, and purified using a MabSelectSure affinity chromatography column (GE Healthcare). The purity of the purified antibodies was detected by SDS-PAGE electrophoresis, and the antibody concentration was detected using Nanodrop.

[0112] The obtained humanized antibody is referred to as "11C6-8 (humanized)" in the present invention.

[0113] Embodiment 11 Affinity test of humanized antibody binding to LILRB2 protein

[0114] ForteBio Octet was used to detect the binding affinity of humanized antibody 11C6-8 (humanized) to human LILRB2 protein. AMC chip was selected, the concentration of immobilized antibody was 10 μg / mL, LILRB2-His (Kyinno, CatNo.: KP-1033) was used as mobile phase, and the concentration was diluted to 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.12nM. The antibodies to be tested include the humanized antibody 11C6-8 (humanized) of the present invention, an unrelated antibody (hIgG1) and the antibody MK-4830. The test results are shown in Table 4.

[0115] Table 4. Affinity test results of humanized antibodies binding to human LILRB2 protein

[0116] Antibody KD(M) kon(1 / Ms) kdis(1 / s) RMax FullR2 MK-4830 7.39E-09 1.77E+05 1.30E-03 0.8929 0.9983 11C6-8 (humanized) 6.73E-10 2.85E+05 1.92E-04 0.9647 0.998

[0117] Example 12 FACS detection of humanized antibody binding to human LILRB2 expressing cells

[0118] The humanized antibody 11C6-8 (humanized) and the antibody MK-4830 were serially diluted and incubated with 293T cells expressing human LILRB2 (293T-LILRB2) and 293T cells expressing monkey LILRB2 (293T-cynoLILRB2), respectively, and then incubated with the anti-human secondary antibody Goat anti-human IgG-PE (SouthernBiotech, Cat No.: 2010-09). The secondary antibody signal bound to the cells was detected using a flow cytometer.

[0119] The test showed that in the case of human LILRB2 expressing cells, the EC50 of humanized antibody 11C6-8 and antibody MK-4830 were 0.007 μg / ml and 0.01 μg / ml, respectively. Figure 6 .

[0120] Example 13 FACS detection of humanized antibody blocking the binding of HLA-G protein to human LILRB2 expressing cells

[0121] Humanized antibody 11C6-8 (humanized) and antibody MK-4830 were serially diluted and incubated with THP1 cells (THP1-LILRB2) overexpressing human LILRB2 (Kyinno, Cat No.: KC-1605), then incubated with 3 μg / ml B2M-HLAG-hFc protein, and finally incubated with anti-human Goat anti-human IgG-PE secondary antibody (SouthernBiotech, Cat No.: 2010-09). The B2M-HLAG-hFc signal bound to the cells was detected by flow cytometry.

[0122] The test showed that the IC50 of humanized antibody 11C6-8 and antibody MK-4830 were 0.059μg / ml and 0.062μg / ml respectively. Figure 7 .

[0123] Embodiment 14 Detection of humanized antibodies blocking LILRB2 downstream signaling

[0124] B2M-HLAG-hFc was coated on the cell culture plate at a concentration of 2 μg / ml overnight. Humanized antibody 11C6-8 (humanized) and antibody MK-4830 were serially diluted and co-incubated with Jurkat-NFAT-Luc2-LILRB2-PILRB cell line (Kyinno, Cat No.: KC-2094; luciferase is expressed after NFAT signal activation), and then the activated luciferase signal was detected.

[0125] The activation signal and antibody concentration were plotted as correlation curves, and the IC50 values ​​of humanized antibody 11C6-8 and antibody MK-4830 were calculated to be 0.002 μg / ml and 0.008 μg / ml, respectively. Figure 8 .

[0126] Embodiment 15 Detection of humanized antibodies inhibiting M2 macrophage differentiation and promoting M1 macrophage differentiation

[0127] CD14 positive cells (Meitianni, Cat No.: 130-050-201) separated from PBMC were added to a U-bottom 96-well plate at 100,000 cells / well, and 200 μl of culture medium (RPMI 1640+10% FBS+50 ng / ml M-CSF) was added.

[0128] Humanized antibody 11C6-8 (humanized) or antibody MK-4830 was added to each well of the 96-well plate to a final concentration of 1 μg / ml. After culturing for 5 days, the supernatant was harvested to detect the TNF-α concentration therein. And harvest the cells for flow cytometry analysis: divide the cells into two parts, add 100 μl of PBS containing 2% FBS to resuspend, add PE-anti-CD86 (1:100) and FITC-anti-HLA-DR (1:100) to the first part; add FITC-anti-CD14 (1:100), PE-anti-CD163 (1:100) and APC-cy7-anti-CD206 (1:100) to the second part, incubate at room temperature for about 30 minutes, wash with PBS containing 2% FBS, add 200 μl of PBS containing 2% FBS to resuspend, add 7-AAD, and use flow cytometer to analyze the mean fluorescence intensity MFI of 7-AAD-cell population in FITC, PE, and APC-cy7 channels.

[0129] The results showed that after adding humanized antibody 11C6-8 (humanized), the expression of CD86 and TNF-α increased, and the expression of CD14, CD206 and CD163 decreased, indicating that humanized antibody 11C6-8 (humanized) inhibited the differentiation of immunosuppressive M2 macrophages and promoted the differentiation of immune-enhancing M1 macrophages. Fig. 9 .

[0130] Example 16 Humanized antibodies enhance T cell function

[0131] CD14 positive cells (Meitianni, Cat No.: 130-050-201) separated from PBMC were added to a U-bottom 96-well plate at 50,000 cells / well, and 200 μl of culture medium (RPMI 1640 + 10% FBS + 50ng / ml M-CSF) was added. After 5 days of culture, the culture medium was carefully replaced with a new culture medium (RPMI 1640 + 10% FBS + 10ng / ml IL-10 + 50ng / ml M-CSF), and culture was continued for 2 days to obtain macrophages.

[0132] On the 7th day, PBMCs from the same donor as the purchased CD14 positive cells were taken, T cells were isolated, and CFSE with a final concentration of 2 μM was added for labeling. The T cell density was adjusted to about 5×10 using culture medium (RPMI 1640+10% FBS+10ng / ml IL-2+50ng / ml M-CSF). 6 / ml (a small portion of cells was left without CD3 / CD8 stimulation and cultured as a non-proliferated CFSE T cell control). Humanized antibody 11C6-8 (humanized) or antibody MK-4830 was diluted to 4 concentrations in a 10-fold gradient using culture medium (RPMI 1640 + 10% FBS + 10ng / ml IL-2 + 50ng / ml M-CSF) at a starting concentration of 20μg / ml.

[0133] Carefully remove the culture medium of the macrophages induced above, add the same number of CFSE-labeled T cells and gradient dilutions of the test antibody to each well, so that each well has 50,000 T cells and 50,000 macrophages. At the same time, add CD3 / CD28 magnetic beads to each well at a ratio of 1:1 between magnetic beads and total number of cells, carefully mix and continue to culture, culture medium (RPMI 1640+10% FBS+10ng / ml IL-2+50ng / ml M-CSF), culture for 2.5 days, after cell centrifugation, stain with APC-anti-human CD8 (1:100) and PE-anti-human CD4 (1:100), use flow cytometry to analyze CFSE in CD8+ and CD4+ cells respectively. low The percentage is the percentage of T cells that proliferate, and the differences between different antibodies under different concentration conditions are compared.

[0134] Comprehensive analysis of the results of different concentrations showed that humanized antibody 11C6-8 significantly accelerated the proliferation of T cells, and its effect on enhancing T cell proliferation was stronger than that of MK-4830. Fig.10 .

[0135] Embodiment 17 Humanized antibodies inhibit tumor growth in animal models

[0136] Resuscitate mouse lung cancer LLC cells approximately 2 weeks before tumor cell inoculation. Collect LLC cells and adjust to 5x10 6 Each mouse was injected with 0.2 ml (i.e., 1 million cells / mouse) of PBS. The tumor size and mouse weight were measured twice a week (once every 3-4 days), and the tumor volume was calculated as = 0.5 x length x width x width.

[0137] On day 7, mice were intraperitoneally injected with different antibodies:

[0138] PD1 antibody RMP114-mPD1M (Kyinno, Cat No.: KA-1239), 10 mg / kg;

[0139] Anti-LILRB2 humanized antibody 11C6-8 (humanized), 10 mg / kg;

[0140] Unrelated antibody hIgG4; or

[0141] PD1 antibody RMP114-mPD1M+11C6-8 (humanized), 10 mg / kg each.

[0142] Inject twice a week; measure body weight twice a week, i.e. every 3 to 4 days, and draw a tumor growth curve.

[0143] The results showed that after injection of humanized antibody 11C6-8, LLC tumor growth was significantly inhibited. Fig.11 .

[0144] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of the claims attached to the present invention.

Claims

1. An antibody or antigen-binding fragment thereof, comprising the following combination of heavy chain CDRs and light chain CDRs (HCDR1, HCDR2, HCDR3; and, LCDR1, LCDR2, LCDR3): HCDR1, HCDR2, HCDR3 comprising the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, respectively; and LCDR1, LCDR2, LCDR3 comprising the amino acid sequences shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising a sequence selected from the following: an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence that has at least 75% identity with the amino acid sequence; and / or, the light chain variable region comprising a sequence selected from the following: an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or an amino acid sequence that has at least 75% identity with the amino acid sequence.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment thereof include: (1) the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 1; and, the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 2; or (2) the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:3; and, the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:

4.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof against LILRB2, and the LILRB2 is preferably human LILRB2; Preferably, the antibody is in any form such as mouse antibody, rabbit antibody, monoclonal antibody, chimeric antibody, partially or fully humanized antibody, etc.; or, the antigen-binding fragment is any fragment of the antibody that retains the antibody's ability to bind to the antigen and the corresponding biological activity, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv; More preferably, the antibody is IgG.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region; Preferably, the antibody or fragment thereof comprises a heavy chain and a light chain; More preferably, the antibody or fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a light chain constant region of kappa or lambda type.

6. A nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain CDR, a light chain CDR, a light chain variable region, a heavy chain variable region, a heavy chain or a light chain contained in the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. A vector comprising the nucleic acid molecule according to claim 6 .

8. A host cell comprising the nucleic acid molecule according to claim 6 and / or the vector according to claim 7, or transformed or transfected with the nucleic acid molecule according to claim 6 and / or the vector according to claim 7.

9. A composition comprising the antibody or fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the vector according to claim 7 and / or the host cell according to claim 8; Preferably, the composition is a pharmaceutical composition, which further comprises optional pharmaceutically acceptable excipients.

10. Use of the antibody or fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the host cell according to claim 8 and / or the composition according to claim 9 in the preparation of a medicament for treating a disease, wherein the disease is a tumor associated with high expression of LILRB2; Preferably, the disease is one or more selected from esophageal squamous cell carcinoma, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer and non-small cell lung cancer; Further preferably, the disease is non-small cell lung cancer.

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