CN116143922A - anti-LILRB 4 monoclonal antibody - Google Patents

Abstract

The invention relates to the field of biological medicine, and in particular provides an anti-LILRB 4 monoclonal antibody, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.

Description

anti-LILRB 4 monoclonal antibody

Technical Field

The invention relates to the technical field of biological medicines, in particular to an anti-LILRB 4 monoclonal antibody.

Background

Acute myeloid leukemia (Acute myeloid leukemia, AML) is a hematological malignancy that affects the abnormal proliferation of myelogenous hematopoietic cells and the production of normal hematopoietic cells, and is one type of leukemia. AML forms in bone marrow, resulting in an increase in the number of abnormal leukocytes in blood and bone marrow, often worsening rapidly, and therefore AML is one of the most invasive and refractory hematological cancers, and treatment regimens are also very limited.

In recent years, with the continuous transformation and upgrading of the medical industry in China, a batch of imitation drugs and innovation drugs for AML treatment appear in China. Azacitidine (novel chemotherapeutic agent) for injection, which is useful for the treatment of AML, is produced in batches for the preparation of both sunny and tandem drugs, as in month 9 of 2019. Meanwhile, as the national government reform continues to advance, the medicine approval speed is continuously increased. The drug administration accepts the new acute myelogenous leukemia drug xoata (english generic name: gilsteritinib, chinese generic name: giritinib) for sale in month 4 of 2020.

With the continued development of biological medicine, immunotherapy, which re-activates immune system functions by blocking immunosuppressive signals, is considered as a cornerstone means for treating AML. Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB 4) is a LILRB family member that structurally comprises two extracellular immunoglobulin-like domains and three intracellular ITIM domains. LILRB4 is selectively expressed in myeloid Antigen Presenting Cells (APCs) such as monocytes, macrophages and dendritic cells. LILRB4 is specifically highly expressed on both M4 and M5 AML cells, and survival of patients with high LILRB4 expression is significantly reduced. Apolipoprotein E (ApoE) binds to and activates LILRB4, recruits SHP-2, regulates nuclear factor κB (NF- κB) signaling pathway, thereby stimulating the expression of urokinase receptor (uPAR) and arginase 1 (ARG 1), further inhibiting T cell proliferation and promoting tissue infiltration. anti-LILRB 4 antibodies are capable of blocking the interaction of LILRB4 with ApoE, inhibiting NF- κb signaling pathway, reducing ARG1 expression, thereby promoting T cell proliferation and reducing tissue infiltration.

At present, ming's organism, engram biopharmaceutical company and China English Tai (Beijing) biotechnology limited company are developing monoclonal antibody medicaments targeting LILRB4, and are in clinical stage I at present. In order to meet global AML and chronic myelomonocytic leukemia (CMML) patient needs, the present invention provides an anti-LILRB 4 monoclonal antibody.

Disclosure of Invention

According to the invention, through screening of an immune library, the anti-LILRB 4 monoclonal antibody which can be specifically combined with LILRB4 and has higher biological activity is obtained.

The specific technical scheme of the invention is as follows:

the invention provides an anti-LILRB 4 monoclonal antibody, comprising:

(a) The amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown in SEQ ID No. 1;

(b) The amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown in SEQ ID No. 2;

(c) The amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown in SEQ ID No. 3;

(d) The amino acid sequence of the light chain complementary determining region LCDR1 is shown as SEQ ID No. 4;

(e) The amino acid sequence of the light chain complementary determining region LCDR2 is shown as SEQ ID No. 5;

(f) The amino acid sequence of the light chain complementary determining region LCDR3 is shown in SEQ ID No. 6.

Further, the anti-LILRB 4 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 8.

Further, the anti-LILRB 4 monoclonal antibody also comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from one of murine IgG1 type, igG2a type, igG2b type or IgG3 type constant regions, and the light chain constant region is murine C with an amino acid sequence shown as SEQ ID No. 13 _k A constant region of the type; the amino acid sequence of the constant region of the IgG1 type is shown as SEQ ID No. 9, the amino acid sequence of the constant region of the IgG2a type is shown as SEQ ID No. 10, the amino acid sequence of the constant region of the IgG2b type is shown as SEQ ID No. 11, and the amino acid sequence of the constant region of the IgG3 type is shown as SEQ ID No. 12.

Further, the anti-LILRB 4 monoclonal antibody is one or a combination of more of Fab, F (ab) 2, fv or ScFv.

The invention further provides a protein comprising the anti-LILRB 4 monoclonal antibody.

The invention further provides a nucleotide molecule which codes for the anti-LILRB 4 monoclonal antibody.

The invention further provides a recombinant DNA expression vector comprising the nucleotide molecule.

The present invention further provides a host cell comprising a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell transfected with the recombinant DNA expression vector;

preferably, the host cell is a mammalian cell, which is a HEK293 cell, CHO cell or NS0 cell.

The invention also provides a medicine which comprises the anti-LILRB 4 monoclonal antibody.

The invention also provides application of the anti-LILRB 4 monoclonal antibody in preparing a medicament for treating cancer;

the cancer comprises one or more of acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, multiple myeloma, blast plasmacytoid dendritic cell tumor, breast cancer, lung cancer or prostate cancer.

The beneficial effects of the invention are as follows: the anti-LILRB 4 monoclonal antibody provided by the invention has higher binding capacity with LILRB4 antigen, and can effectively inhibit the binding of LILRB4 antigen and ligand complex thereof, thereby preventing downstream NF- κB signal path activation and ARG1 release, promoting T cell proliferation and inhibiting tissue infiltration; in addition, the anti-LILRB 4 monoclonal antibodies screened in the present invention can be used to treat cancers including, but not limited to, acute Myelogenous Leukemia (AML), acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL), multiple Myeloma (MM), blast plasmacytoid dendritic cell tumor (BPDCN), breast cancer, lung cancer or prostate cancer.

Drawings

FIG. 1 is a plasmid map of pScFv-Disb-HS vector in example 2 of the present invention;

FIG. 2 is a graph showing the relative affinities of gradient dilution ELISA anti-LILRB 4 phage monoclonal antibodies in example 3 of the invention;

FIG. 3 is a map of vector pTSE in example 5 of the invention;

FIG. 4 is a denaturing polyacrylamide gel electrophoresis of an anti-LILRB 4 monoclonal antibody according to example 5 of the present invention;

FIG. 5 is a graph showing the binding capacity of anti-LILRB 4 monoclonal antibody molecules to LILRB4 in example 6 of the present invention;

FIG. 6 is a graph showing the binding capacity of anti-LILRB 4 monoclonal antibody molecules to human monocytic leukemia cell (THP-1) surface LILRB4 in example 8 of the present invention;

FIG. 7 is a diagram showing the competition between anti-LILRB 4 monoclonal antibody molecules and ApoE for LILRB4 binding in example 9 of the present invention;

FIG. 8 is a graph showing the inhibition of ARG1 secretion by THP-1 by anti-LILRB 4 monoclonal antibody molecules of example 10 of the present invention.

Detailed Description

For easier understanding of the present invention, the following description will be given with respect to certain technical and scientific terms of the present invention, before describing the embodiments:

the term "antibody" as used herein, includes whole antibodies and any antigen-binding fragment thereof, including murine, humanized, bispecific or chimeric antibodies, which may also be Fab, F (ab) 2, fv or ScFv (single chain antibody), which may be naturally occurring or altered (e.g., mutated, deleted, substituted, etc.).

The terms "variable region" and "constant region" as used herein mean that the regions of the heavy and light chains adjacent to the N-segment of an antibody are variable regions (v regions), the remaining amino acid sequences adjacent to the C-segment are relatively stable, and are constant regions (C regions), the variable regions comprise 3 Complementarity Determining Regions (CDRs) and 4 Framework Regions (FRs), each of the light chain variable regions and heavy chain variable regions consists of 3 CDR regions and 4 FR regions, the 3 CDR regions of the heavy chain are represented by HCDR1, HCDR2 and HCDR3, respectively, and the 3 CDR regions of the light chain are represented by LCDR1, LCDR2 and LCDR3, respectively.

The term "CHO cell" is a chinese hamster ovary cell (chinese hamster ovary cell); the term "HEK293 cells" is human embryonic kidney 293 cells (human eMDryonic kidney 293 cells), and the term "NS0 cells" is mouse NS0 thymoma cells.

The invention will be described in further detail with reference to the following examples.

Example 1

The embodiment 1 of the invention provides an anti-LILRB 4 monoclonal antibody, which comprises:

(a) Heavy chain complementarity determining region HCDR1, its amino acid sequence is shown in SEQ ID No:1 is shown in the specification;

(b) Heavy chain complementarity determining region HCDR2 having the amino acid sequence set forth in SEQ ID No:2 is shown in the figure;

(c) Heavy chain complementarity determining region HCDR3 having the amino acid sequence set forth in SEQ ID No:3 is shown in the figure;

(d) The amino acid sequence of the light chain complementary determining region LCDR1 is shown in SEQ ID No:4 is shown in the figure;

(e) Light chain complementarity determining region LCDR2, its amino acid sequence is shown in SEQ ID No:5 is shown in the figure;

(f) Light chain complementarity determining region LCDR3, its amino acid sequence is shown in SEQ ID No: shown at 6.

Heavy chain complementarity determining region HCDR1	Heavy chain complementarity determining region HCDR2	Heavy chain complementarity determining region HCDR3	Light chain complementarity determining region LCDR1	Light chain complementarity determining region LCDR2	Light chain complementarity determining region LCDR3
						NYWIH	EINPTNGHTNYNEKFKS	TYDQDAMDYWGQG	RASQDISNYLNWYQQK	HSGVPSR	LPPTFGGGT
(SEQ ID NO：1)	(SEQ ID NO：2)	(SEQ ID NO：3)	(SEQ ID NO：4)	(SEQ ID NO：5)	(SEQ ID NO：6)

EXAMPLE 2 screening of anti-LILRB 4 monoclonal antibody molecules

According to the invention, a phage display library is created by immunizing a mouse with an LILRB4 antigen (an extracellular segment of LILRB4 protein, and the LILRB4 antigen and the LILRB4 protein are both extracellular segments of LILRB4 used in subsequent experiments), and an immune method is optimized, and the construction and screening identification of the phage display library are as follows:

step one: LILRB4 antigen immunized mice

1. Experimental animals:

species strain: BALB/c, female, mouse;

weight of: 18-20g;

experimental animal provider: also kang (Beijing) pharmaceutical technology Co., ltd.

2. Immunization: mice were immunized with human LILRB4 (a synthetic gene from south kyo gold sri biotechnology limited, the company constructs a vector and expresses and purifies it).

Step two: construction of phage antibody library

The method comprises the steps of taking mouse spleen cells with higher titer, extracting total RNA in the mouse spleen cells by using Trizol reagent (purchased from AMDion, cat# 15596026), obtaining cDNA by RT-PCR, carrying out PCR amplification by using the cDNA as a template and degenerate primers (used in degenerate primer reference: journal of Immunological Methods (2000) 167-177) so as to obtain an immune mouse antibody heavy chain variable region (VH) gene library and a light chain variable region (VL) gene library, respectively carrying out double enzyme digestion on the light chain and heavy chain, connecting the heavy chain gene library and the light chain gene library to a vector subjected to enzyme digestion in the same step by step, and constructing pScFv-Disb-HS-VL gene library, wherein the pScFv-Disb-HS vector is a series of gene cloning methods for modifying a vector pCoMD3 vector (purchased from a Chinese plasmid vector strain gene collection center) so as to be used for constructing and expressing a phage single chain antibody library. The transformed vector is named pScFv-Disb-HS vector, the plasmid map of which is shown in figure 1 is obtained, and a mouse immune phage antibody library is constructed based on the vector.

Step three: the immune tube was coated with LILRB4 as antigen in an amount of 5. Mu.g/500. Mu.L/tube, coated overnight at 4℃and the immune tube and immune phage antibody library were blocked with 4% nonfat milk powder/PBST, respectively, for 1 hour at room temperature. Adding the blocked immune phage antibody library into immune tube to combine antigen and antibody, and adding phage with input of about 10 ⁹ ～10 ¹² After 1 hour of reaction at room temperature, unbound phage was washed off with PBST-PBS, eluted with 0.1M Glycine-HCl at pH 2.2, and the eluted phage antibody solution was finally neutralized to pH 7.0 with 1.5M Tris-HCl at pH 8.8.

Step four: the neutralized phage was infected with 10mL of TG1 bacterial liquid grown to log phase, allowed to stand in an incubator at 37℃for 30 minutes, and then a part of bacterial liquid was taken out for gradient dilution and plated on a 2YTAG plate for calculating phage yield. The remaining bacterial liquid was centrifuged to discard the supernatant, the bacterial pellet was resuspended in a small amount of medium, aspirated and spread on a 2YTAG large plate, ready for the next round of screening.

Step five: scraping the infected bacteria coated on the plate from a large plate, inoculating the bacteria to a 2YTAG liquid culture medium, shaking to a logarithmic phase, adding M13KO7 auxiliary phage to perform superinfection, culturing overnight at 220rpm at 28 ℃ to prepare phage, and carrying out PEG/NaCl sedimentation to purify phage for the next round of screening, thereby carrying out a round of phage library enrichment screening.

Step six: screening of LILRB4 phage single-chain antibody positive clones: after one round of screening, selecting well-separated monoclonal colonies, inoculating to a 96-well deep-hole plate with 2YTAG liquid culture medium, culturing at 37deg.C and 220rpm to logarithmic phase, and adding about 10 per well ¹⁰ Is the helper phage M13 of (2)KO7, at a temperature of 37 ℃ for 30 minutes. 4000rpm, centrifuging for 15 minutes, discarding the supernatant, re-suspending the pellet with 2YTAK, and culturing overnight at 28℃and 220 rpm. Centrifuging at 4000rpm and 4 ℃ for 15 minutes, absorbing amplified phage supernatant, performing ELISA identification, finally screening to obtain an anti-LILRB 4 antibody candidate molecule with higher affinity, namely MD-I, performing gene sequencing on the obtained monoclonal antibody to determine the correct antibody sequence, and sequencing, wherein the sequence of the screened monoclonal antibody is as follows:

monoclonal antibody molecules	Heavy chain variable region sequences	Light chain variable region sequences
			MD-Ⅰ	SEQ ID No:7	SEQ ID No:8

Specifically, SEQ ID No. 7 (amino acid sequence of the heavy chain variable region of MD-I):

EVQLQESGPQLVRPGTSVKLSCKASGYTFTNYWIHWVKQRPGQGLEWIGEINPTNGHTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARYTYDQDAMDYWGQGTSVTVSS；

SEQ ID No. 8 (amino acid sequence of the light chain variable region of MD-I):

DIVITQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPPTFGGGTKLEIK。

EXAMPLE 3 gradient dilution ELISA detection of affinity of anti-LILRB 4 monoclonal antibodies

The anti-LILRB 4 monoclonal antibody molecule MD-I obtained in example 2 was subjected to monoclonal phage display and purification, and then subjected to phage gradient dilution ELISA assay to identify affinity, as follows:

the anti-LILRB 4 monoclonal antibody molecule MD-I selected in example 2 was diluted with a four-fold gradient of PBST, 100. Mu.L of diluted sample was added to each well, and allowed to stand at room temperature for 1 hour. The ELISA plate was washed with PBST, and the HRP-anti-M13 (purchased from Bio-view stone, cat# GE 27-9421-01) monoclonal antibody diluted with PBST was added to the ELISA plate and left at room temperature for 1 hour. TMD color development kit developed, developed at room temperature for 10 min, and developed with 2M H ₂ SO ₄ After termination, the microplate reader reads at 450nm/630nm and calculates the corresponding half maximal effect concentration (EC 50) values as follows:

cloning	MD-Ⅰ
		EC50	14.41

By the above data and as shown in FIG. 2, the anti-LILRB 4 monoclonal antibody molecule MD-I screened in example 2 can bind to LILRB4.

Example 4

Example 4 of the present invention further defines, based on example 2, that the anti-LILRB 4 monoclonal antibody further comprises a heavy chain constant region selected from one of murine IgG1, igG2a, igG2b, or IgG3 constant regions, and a light chain constant region that is murine C having an amino acid sequence as shown in SEQ ID No. 13 _k A constant region of the type; igG1 typeThe amino acid sequence of the constant region of the IgG2a type is shown as SEQ ID No. 9, the amino acid sequence of the constant region of the IgG2b type is shown as SEQ ID No. 10, the amino acid sequence of the constant region of the IgG2b type is shown as SEQ ID No. 11, and the amino acid sequence of the constant region of the IgG3 type is shown as SEQ ID No. 12.

SEQ ID No. 9 (amino acid sequence of heavy chain constant region of murine IgG1 type):

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG；

SEQ ID No. 10 (amino acid sequence of heavy chain constant region of murine IgG2a type):

AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK；

SEQ ID No. 11 (amino acid sequence of heavy chain constant region of murine IgG2b type):

AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK；

SEQ ID No. 12 (amino acid sequence of heavy chain constant region of murine IgG3 type):

ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA；

SEQ ID No. 13 (murine C _k Amino acid sequence of the light chain constant region):

ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

EXAMPLE 5 preparation of anti-LILRB 4 monoclonal antibody molecules

Example 5 of the invention on the basis of example 4 it is preferred to define monoclonal antibody molecules comprising a murine heavy chain constant region of the IgG1 type (the amino acid sequence of which is shown in SEQ ID No: 9) and murine C _k A light chain constant region of the type (the amino acid sequence of which is shown as SEQ ID No. 13). The preparation method of the antibody specifically comprises the following steps:

1. the genes encoding VH and VL of the anti-LILRB 4 monoclonal antibody molecule MD-I selected in example 2 were cloned into vector pTSE containing heavy and light chain constant region genes (shown in FIG. 3), respectively, the preferred heavy chain constant region being a murine IgG1 type constant region (amino acid sequence shown in SEQ ID No: 9), the light chain constant region being murine C _k The strand (amino acid sequence shown in SEQ ID No. 13) of which the pTSE vector structure is shown in FIG. 3 (see page 3 [0019 ] of the description of CN103525868A for the preparation of pTSE vector)]Segments).

2. HEK293 cells (purchased from basic medical institute of China medical sciences, cat# GNHu 43) were transiently transfected, antibody expression was performed, MD-I monoclonal antibodies were obtained by protein A affinity column purification using an AKTA instrument, protein concentration was measured using a BCA kit (purchased from Beijing Hui Tian Oriental science and technology Co., ltd., cat# BCA 0020), and then protein sizes were identified by SDS-PAGE, as shown in FIG. 4, non-reducing anti-LILRB 4 monoclonal antibodies MD-I, protein molecular weight Marker and reducing MD-I anti-LILRB 4 monoclonal antibodies were sequentially arranged from left to right, and the molecular weight of each band was consistent with theory.

EXAMPLE 6 binding experiments of anti-LILRB 4 monoclonal antibody molecules to LILRB4

Coating of LILR with carbonate buffer pH 9.6B4 100 ng/well/100. Mu.L, coated overnight at a temperature of 4 ℃. Washing with 300. Mu.L/well PBST five times, adding 1% BSA-PBST, blocking at 37deg.C for 1 hr, adding anti-LILRB 4 monoclonal antibody molecule MD-I with different dilution concentration, initial maximum concentration of 50. Mu.g/mL, diluting with 5-fold gradient, diluting for 12 gradients, and incubating at 37deg.C for 1 hr. Five washes with 300. Mu.L/well PBST were performed, and Goat Anti-Mouse IgG-HRP (purchased from solabio, cat# SE 131) diluted with 1% BSA-PBST1:2000 was added thereto and incubated at 37℃for 1 hour. TMD chromogenic kit, 100. Mu.L/well, room temperature for 8 min, then 2M H ₂ SO ₄ The color development was terminated. The microplate reader reads at 450nm/630nm and calculates the corresponding EC50 value, the specific data are as follows:

cloning	MD-I
		EC50(ng/mL)	221

Through the above data and as shown in FIG. 5, the screened anti-LILRB 4 monoclonal antibody molecule MD-I can bind to LILRB4.

EXAMPLE 7 binding experiments with anti-LILRB 4 monoclonal antibody molecules and LILR family proteins

The LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, 100 ng/well/100 μl were coated with carbonate buffer at pH 9.6 overnight at a temperature of 4 ℃. Five washes with 300. Mu.L/well PBST, 1% BSA-PBST was added and blocked at 37℃for 1 hour, followed by 100. Mu.L of anti-LILRB 4 monoclonal antibody MD-I at a concentration of 50. Mu.g/mL. Incubate at 37℃for 1 hour. With 300mu.L/well PBST was washed five times and added with Goat Anti-Mouse IgG-HRP (purchased from solabio, cat# SE 131) diluted 1:2000 with 1% BSA-PBST and incubated for 1 hour at 37 ℃. TMD chromogenic kit, 100. Mu.L/well, room temperature for 8 min, then 2M H ₂ SO ₄ The color development was terminated. The microplate reader reads at 450nm/630nm, and the specific data are as follows:

	LILRA1	LILRA2	LILRA3	LILRA4	LILRA5	LILRA6	LILRB1	LILRB2	LILRB3	LILRB4	LILRB5
												MD-I	0.075	0.054	0.028	0.156	0.078	0.045	0.047	0.014	0.015	2.421	0.056

as shown by the data above, the screened anti-LILRB 4 monoclonal antibody MD-I can specifically bind to LILRB4 and does not bind to other proteins of the LILR family.

EXAMPLE 8 binding experiments of anti-LILRB 4 monoclonal antibody molecules to human monocytic leukemia cell (THP-1) surface LILRB4

50 mu L of anti-LILRB 4 monoclonal antibody MD-I with different dilution concentrations is taken, the initial working concentration is 30 mu g/mL, 3-time gradient dilution is carried out, 10 gradients are totally diluted, and the mixture is added into a V bottom plate of a 96-well plate. Subsequently 50. Mu.L of THP-1 cell suspension was added to the wells at a concentration of 2X 10 ⁶ And mixing the mixture with cells/mL. Incubate at 4℃for 1 hour. Subsequently, 100. Mu.L of PBS buffer was added to each well, and the supernatant was discarded by centrifugation at 3000rpm for 5 minutes. 100. Mu.L/well Fluorescein Isothiocyanate (FITC) labeled goat anti-mouse IgG (purchased from Meter Cunninghamia sinensis Biotechnology Co., ltd., product number: ZF-0312) was again added (1:100 dilution). After mixing, incubate at 4℃for 30 min in the dark. 100. Mu.L of PBS buffer was added to each well and centrifuged at 3000rpm for 5 minutes to discard the supernatant. The cells were resuspended by adding 100. Mu.L of PBS buffer again and detected on-line by flow cytometry. Data were collected and corresponding EC50 values calculated as follows:

cloning	MD-Ⅰ
		EC50(ng/mL)	5091

Through the above data and as shown in FIG. 6, the screened anti-LILRB 4 monoclonal antibody MD-I can bind to LILRB4 on the surface of THP-1 cells.

EXAMPLE 9 anti-LILRB 4 monoclonal antibody molecules competing for ApoE binding to LILRB4 experiments

THP-1 cells were collected at a concentration of 2X 10 ⁶ cells/mL, plated in V-bottom 96-well plates, and 50. Mu.L of cell suspension was added to each well. Subsequently 50. Mu.L of anti-LILRB 4 monoclonal antibody MD-I at various dilutions was added to the wells, starting at 400. Mu.g/mL, 5-fold gradient dilution, 10 gradients total. Additionally, 100. Mu.L of 0.4. Mu.g/mL FITC-labeled ApoE protein was added to the wells. Incubate at 4℃for 1.5 hours in the absence of light. 200. Mu.L of PBS buffer was then added to each well and centrifuged at 3000rpm for 5 minutes to discard the supernatant. The cells were resuspended by adding 100. Mu.L of PBS buffer again and detected on-line by flow cytometry. Data were collected and corresponding IC50 values calculated as follows:

cloning	MD-Ⅰ
		IC50(μg/mL)	8.618

By the above data and as shown in FIG. 7, the screened anti-LILRB 4 monoclonal antibody MD-I competes with ApoE for binding to LILRB4 on the surface of THP-1 cells.

EXAMPLE 10 inhibition of THP-1 secretion of ARG1 by anti-LILRB 4 monoclonal antibody molecules

THP-1 cells were collected at a concentration of 1X 10 ⁷ cells/mL, plated in V-bottom 96-well plates, and 50. Mu.L of cell suspension was added to each well. The anti-LILRB 4 monoclonal antibody MD-I and ApoE with different concentrations are uniformly mixed according to a ratio of 1:1. The initial working concentration of the anti-LILRB 4 monoclonal antibody molecule MD-I was 400 μg/mL, 2-fold gradient dilution, 8 gradients total. The working concentration of ApoE was 0.5. Mu.g/mL. Subsequently 50. Mu.L of a mixture of anti-LILRB 4 monoclonal antibody MD-I and ApoE was added to the wells. After incubation at 37℃for 20 hours, centrifugation at 3000rpm for 5 minutes. 40. Mu.L of the supernatant was removed from each well and added to a 96-well plate, followed by preparation and preheating of the reaction substrate according to an arginase activity assay kit (purchased from Sigma-Aldrich, cat# MAK112-1 KT), adding 10. Mu.L of the reaction substrate to each well, mixing, and reacting at 37℃for 1 hour. 200. Mu.L of reaction termination solution was added again to each well, and absorbance was read at 450nm using a multifunctional microplate reader. Data were collected and corresponding IC50 values calculated as follows:

cloning	MD-Ⅰ
		IC50(μg/mL)	309.8

Through the above data and as shown in FIG. 8, the screened anti-LILRB 4 monoclonal antibody MD-I can inhibit the secretion of ARG1 by THP-1 cells.

Example 11

Example 11 of the present invention defines the anti-LILRB 4 monoclonal antibody as one or a combination of several of Fab, F (ab) 2, fv, or ScFv based on examples 1-10 above.

The present invention provides a protein comprising an anti-LILRB 4 monoclonal antibody MD-i.

The invention further provides a nucleotide molecule which codes for the anti-LILRB 4 monoclonal antibody MD-I provided in the above examples.

The invention also provides a recombinant DNA expression vector, which comprises nucleotide molecules.

The invention also provides a host cell transfected with the recombinant DNA expression vector, wherein the host cell comprises a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell;

preferably, the host cell is a mammalian cell, which is a HEK293 cell, CHO cell or NS0 cell.

Example 12

Example 12 of the present invention provides a medicament comprising the anti-LILRB 4 monoclonal antibody MD-i provided in examples 1-10.

The invention also provides application of the anti-LILRB 4 monoclonal antibody MD-I in preparing a medicament for treating cancer;

the cancer comprises one or more of acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, multiple myeloma, blast plasmacytoid dendritic cell tumor, breast cancer, lung cancer or prostate cancer.

The present invention is not limited to the above-described preferred embodiments, and any person who can obtain other various products under the teaching of the present invention, however, any change in shape or structure of the product is within the scope of the present invention, and all the products having the same or similar technical solutions as the present application are included.

Claims (10)

1. An anti-LILRB 4 monoclonal antibody comprising:

(a) The amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown in SEQ ID No. 1;

(b) The amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown in SEQ ID No. 2;

(c) The amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown in SEQ ID No. 3;

(d) The amino acid sequence of the light chain complementary determining region LCDR1 is shown as SEQ ID No. 4;

(e) The amino acid sequence of the light chain complementary determining region LCDR2 is shown as SEQ ID No. 5;

(f) The amino acid sequence of the light chain complementary determining region LCDR3 is shown in SEQ ID No. 6.

2. The anti-LILRB 4 monoclonal antibody of claim 1, comprising a heavy chain variable region having an amino acid sequence shown in SEQ ID No. 7 and a light chain variable region having an amino acid sequence shown in SEQ ID No. 8.

3. The anti-LILRB 4 monoclonal antibody according to claim 2, further comprising a heavy chain constant region selected from one of murine IgG1, igG2a, igG2b or IgG3 constant regions and a light chain constant region of murine C having the amino acid sequence shown in SEQ ID No. 13 _k A constant region of the type; the amino acid sequence of the constant region of the IgG1 type is shown as SEQ ID No. 9, the amino acid sequence of the constant region of the IgG2a type is shown as SEQ ID No. 10, the amino acid sequence of the constant region of the IgG2b type is shown as SEQ ID No. 11, and the amino acid sequence of the constant region of the IgG3 type is shown as SEQ ID No. 12.

4. The anti-LILRB 4 monoclonal antibody of claim 1, wherein the anti-LILRB 4 monoclonal antibody is one or a combination of several of Fab, F (ab) 2, fv, or ScFv.

5. A protein comprising the anti-LILRB 4 monoclonal antibody of any one of claims 1-4.

6. A nucleotide molecule encoding the anti-LILRB 4 monoclonal antibody of any one of claims 1-4.

7. A recombinant DNA expression vector comprising the nucleotide molecule of claim 6.

8. A host cell transfected with the recombinant DNA expression vector of claim 7, wherein said host cell comprises a prokaryotic cell, a yeast cell, an insect cell, or a mammalian cell;

preferably, the host cell is a mammalian cell, which is a HEK293 cell, CHO cell or NS0 cell.

9. A medicament comprising the anti-LILRB 4 monoclonal antibody of any one of claims 1-4.

10. Use of an anti-LILRB 4 monoclonal antibody as claimed in any one of claims 1 to 4 in the manufacture of a medicament for the treatment of cancer;

the cancer comprises one or more of acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, multiple myeloma, blast plasmacytoid dendritic cell tumor, breast cancer, lung cancer or prostate cancer.

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