CN117447593A

CN117447593A - anti-Siglec-15 monoclonal antibody

Info

Publication number: CN117447593A
Application number: CN202211463289.1A
Authority: CN
Inventors: 白义; 刘思; 裴爽
Original assignee: BEIJING JINGYI TAIXIANG TECHNOLOGY DEVELOPMENT CO LTD; Beijing Dongfang Baitai Biotechnology Co ltd
Current assignee: BEIJING JINGYI TAIXIANG TECHNOLOGY DEVELOPMENT CO LTD; Beijing Dongfang Baitai Biotechnology Co ltd
Priority date: 2022-07-26
Filing date: 2022-07-26
Publication date: 2024-01-26
Also published as: CN117447595A; CN117447594A; CN117447592A

Abstract

The invention relates to the field of biological medicine, and particularly provides an anti-Siglec-15 monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the monoclonal antibody is one of A-I, A-II, A-III or A-IV. The anti-Siglec-15 monoclonal antibody provided by the invention can be specifically combined with Siglec-15, can block the combination of the Siglec-15 and a cell surface receptor, can play an anti-tumor activity, and can be used for treating cancers or immune diseases, wherein the cancers comprise but are not limited to brain glioma, melanoma, colorectal cancer, renal cancer, lung cancer, lymphoma or leukemia and the like. Immune diseases include, but are not limited to, psoriasis, crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, and systemic lupus erythematosus and multiple sclerosis.

Description

anti-Siglec-15 monoclonal antibody

Technical Field

The invention relates to the technical field of biological medicines, in particular to an anti-Siglec-15 monoclonal antibody.

Background

According to the data of the 2020 world cancer report issued by the world health organization IARC, 1929 ten thousand new cancer cases are generated worldwide in 2020, 457 ten thousand people are only generated in China, 23.7% of the world is occupied, and the new people of the Chinese cancer are far beyond other countries in the world, so that the research and development of cancer medicaments are urgent.

Cancer cells can sometimes be prevented from being detected and destroyed by the immune system by reducing tumor antigen expression on their surface, making their detection by the immune system more difficult. Meanwhile, cancer cells may also express proteins on their surfaces that induce immune cell inactivation, or induce cells in the surrounding environment to release substances that inhibit immune responses and promote proliferation and survival of tumor cells. With the great success of anti-PD-1 antibodies in the field of tumor immunotherapy, how to change tumor microenvironment and activate the killing of the immune system to tumors has become a hotspot of research.

Sialic acid binding immunoglobulin-like lectin 15 (Siglec-15), a member of the SIGLEC gene family, is also a DAP 12-related immune receptor, belonging to the immunoglobulin superfamily and the Sialic acid binding Ig-like lectin family. The Siglec family is divided into two classes, one is a sequence-conserved Siglecs, including sialoadhesin, CD22, MAG, and Siglec-15; the other is a CD 33-related variable sequence Siglecs. Siglecs are sialic acid binding cell surface proteins which are predominantly present on the immune cell surface and are a subset of type I lectins, structurally very typical and conserved structural features, with a transmembrane region consisting of 2-17 extracellular Ig domains, the N-terminal consisting of a sialic acid binding V-set Ig domain and a number of C2-set Ig domains. Most of the Siglecs intracellular segments contain immunoreceptor tyrosine activation motifs (immunoreceptor tyrosine-based activation motifs, ITAMs) to exert immunosuppressive functions; a few Siglecs, such as Siglec-14-16 and Siglec-H, exert an immunomodulatory effect by binding arginine, which is positively charged in its transmembrane region, to the transformant DAP12 of ITAMs. Siglec-15 consists of an immunoglobulin (Ig) -like domain, a transmembrane domain, and a short cytoplasmic tail. Siglec-15 is classified as both exogenous and endogenous. Typically, exogenous Siglec-15 is highly expressed on tumor cell surfaces, while endogenous Siglec-15 is predominantly expressed on macrophage and dendritic cell surfaces, with minimal expression in human and mouse tissues and in various cell types. Research shows that Siglec-15 has the function of inhibiting T cell activity, and macrophage expressed Siglec-15 can inhibit proliferation and activity of human and mouse T cell directly and inhibit secretion of IFN-gamma and TNF-alpha; the gene knockout and antibody blocking of Siglec-15 in mice can enhance tumor immunity in microenvironment and inhibit tumor growth in certain mouse models. Thus, siglec-15 is a completely new immunosuppressive molecule that is widely present in many tumors, with potential clinical relevance.

Currently available tumor immunotherapy via the PD-1/PD-L1 pathway is effective only in about 40% of patients with solid tumors. In the course of actual therapy, when PD-L1 is up-regulated, many other molecular and cellular mechanisms, including lack of effective immune cell infiltration, lack of enrichment of regulatory T, lack of tumor-associated macrophages, and lack of bone marrow-derived suppressor cells, etc., lead to impaired immune function in the microenvironment, while the success of PD-1 therapy underscores the importance of repairing immunodeficiency and serves as an important criterion for restoring immune system normalization in cancer immunotherapy. Although Siglec-15 has certain homology with PD-L1, the expression of the two has no correlation, and the research and development of the drug targeting Siglec-15 is an immune checkpoint anticancer new drug complementary with the anti-PD-1 drug, so that the research and development of the anti-Siglec-15 monoclonal antibody drug has important clinical significance.

Disclosure of Invention

In order to solve the problems in the prior art and meet the domestic market demand, the invention obtains the anti-Siglec-15 monoclonal antibody which can specifically bind with Siglec-15 and block the combination of the Siglec-15 and a cell surface receptor through screening an immune library.

The specific technical scheme of the invention is as follows:

the invention provides an anti-Siglec-15 monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 heavy chain complementarity determining regions respectively represented by HCDR1, HCDR2 and HCDR3, the light chain variable region comprises 3 light chain complementarity determining regions respectively represented by LCDR1, LCDR2 and LCDR3, and the anti-Siglec-15 monoclonal antibody is selected from any one of the following:

A-I: the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No. 1, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No. 2, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No. 3, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No. 4, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No. 5, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No. 6;

A-II: the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No. 7, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No. 8, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No. 9, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No. 10, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No. 11, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No. 12;

A-iii: the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No. 13, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No. 14, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No. 15, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No. 16, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No. 17, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No. 18;

A-IV: the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No. 19, the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No. 20, the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No. 21, the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No. 22, the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No. 23, and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No. 24.

Further, the anti-Siglec-15 monoclonal antibody is a murine antibody molecule selected from any one of the following:

MA-I: the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 25, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 26;

MA-II: the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 27, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 28;

MA-III: the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 29, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 30;

MA-IV: the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 31, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 32.

Further, the murine antibody molecule further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is 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. 33 _k The amino acid sequence of the constant region of the IgG1 type is shown as SEQ ID No. 34, the amino acid sequence of the constant region of the IgG2a type is shown as SEQ ID No. 35, the amino acid sequence of the constant region of the IgG2b type is shown as SEQ ID No. 36, and the amino acid sequence of the constant region of the IgG3 type is shown as SEQ ID No. 37.

Further, the anti-Siglec-15 monoclonal antibody is a chimeric antibody molecule comprising a heavy chain variable region of the murine antibody molecule, a light chain variable region of the murine antibody molecule, and a humanized antibody constant region.

Further, the anti-Siglec-15 monoclonal antibody is a humanized antibody molecule selected from any one of the following:

HA-I: the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 42, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 43;

HA-II: the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 42, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 44.

Further, the humanized antibody molecules also include humanized antibody constant regions.

Further, the humanized antibody constant region comprises a humanized antibody heavy chain constant region and a humanized antibody light chain constant region, wherein the humanized antibody heavy chain constant region is one of human IgG1 type, igG2 type or IgG4 type constant regions, the amino acid sequence of the IgG1 type constant region is shown as SEQ ID No. 38, the amino acid sequence of the IgG2 type constant region is shown as SEQ ID No. 39, and the amino acid sequence of the IgG4 type constant region is shown as SEQ ID No. 39 The amino acid sequence is shown as SEQ ID No. 40, and the humanized antibody light chain constant region is human C with the amino acid sequence shown as SEQ ID No. 41 _k Constant region of the type.

Further, the humanized antibody molecule is a full-length antibody or an antibody fragment, and the humanized antibody molecule comprises one or a combination of a plurality of Fab, F (ab) 2, fv or ScFv.

The invention also provides a protein which comprises the anti-Siglec-15 monoclonal antibody.

The invention also provides a polynucleotide molecule which codes for the anti-Siglec-15 monoclonal antibody.

The invention also provides a recombinant DNA expression vector comprising the polynucleotide molecule.

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.

The invention also provides a medicine containing the anti-Siglec-15 monoclonal antibody.

The invention also provides application of the anti-Siglec-15 monoclonal antibody in preparing medicines for treating immune diseases or cancers;

Preferably, the cancer comprises brain glioma, melanoma, colorectal cancer, renal cancer, lung cancer, lymphoma or leukemia;

the immune disease comprises psoriasis, crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis or autoimmune hepatitis.

The invention further provides application of the anti-Siglec-15 monoclonal antibody and the anti-PD-1 monoclonal antibody in medicines for treating cancers or immune diseases.

The beneficial effects of the invention are as follows: the anti-Siglec-15 monoclonal antibody provided by the invention can be specifically combined with Siglec-15, can block the combination of Siglec-15 and a cell surface receptor, can inhibit the conduction of an intracellular signal path, can inhibit the growth of tumors, and can be used for treating cancers or immune diseases, wherein the cancers comprise but are not limited to brain glioma, melanoma, colorectal cancer, renal cancer, lung cancer, lymphoma or leukemia, and the like, and the immune diseases comprise but are not limited to psoriasis, crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis, autoimmune hepatitis, and the like.

Drawings

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

FIG. 2 is a graph comparing the affinity of the gradient dilution ELISA anti-Siglec-15 phage monoclonal antibody of example 3 of the invention;

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

FIG. 4 is a diagram showing the gel electrophoresis of a denatured polyacrylamide gel of a murine antibody molecule of example 5 of the present invention;

FIG. 5 is a graph showing the comparison of the binding capacity of murine antibody molecules to Siglec-15 in example 6 of the present invention;

FIG. 6 is a graph showing the comparison of the ability of murine antibodies of example 7 of the present invention to inhibit the binding of Siglec-15 to Jurkat cell surface receptors;

FIG. 7 is a comparative graph of the ability of murine antibody molecules of example 8 of the present invention to inhibit the binding of Siglec-15 to CHOSLV-LRRC4C cell surface receptor;

FIG. 8 is a graph showing the promotion of human TNF- α cytokine release by a murine antibody molecule of example 9 of the present invention;

FIG. 9 is a graph showing the promotion of human IFN-gamma cytokine release by murine antibody molecules of example 9 of the present invention;

FIG. 10 is a graph showing the promotion of T cell activation and proliferation by murine antibody molecules of example 9 of the present invention;

FIG. 11 is a graph showing the biological activity of murine antibody molecules in example 10 of the present invention;

FIG. 12 is a photograph showing a denaturing polyacrylamide gel electrophoresis of a humanized antibody molecule of example 15 of the present invention;

FIG. 13 is a graph showing the comparison of the binding capacity of humanized antibody molecules to Siglec-15 in example 18 of the present invention;

FIG. 14 is a graph showing the cross-binding of humanized antibodies of example 19 of the present invention to Siglec-15 of different species;

FIG. 15 is a graph of the binding ability of humanized antibody molecules of example 20 of the present invention to inhibit Siglec-15 from binding to Jurkat cell surface receptors;

FIG. 16 is a graph showing the detection of the biological activity of the humanized antibody molecules of example 21 of the present invention;

FIG. 17 is a graph showing tumor volume growth in a mouse MC38-Siglec-15 colorectal cancer model with anti-Siglec-15 monoclonal antibody HA-I of example 22 of the present invention;

FIG. 18 is a graph of the evaluation of the HA-I thermostability of the anti-Siglec-15 monoclonal antibody of example 23 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 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 "murine antibody molecule" as used herein, is derived from an antibody obtained after immunization of mice with Siglec-15 antigen.

The term "chimeric antibody molecule" as used herein is an antibody in which a variable region of a murine antibody is fused to a constant region of a humanized antibody, and which can reduce the immune response induced by the murine antibody in humans. The chimeric antibody is prepared by inserting the light and heavy chain variable region genes of murine monoclonal antibody into an expression vector containing a human antibody constant region by utilizing a DNA recombination technology, so that the variable region of the light and heavy chain in the expressed antibody molecule is murine, the constant region is human, and the near 2/3 part of the whole antibody molecule is human, thus the generated antibody reduces the immunogenicity of the murine antibody and simultaneously retains the specific antigen binding capability of the parent antibody.

The term "humanized antibody molecule" as used herein is a humanized antibody molecule in which CDRs of a murine mab are grafted onto a human antibody variable region, replacing human antibody CDRs, allowing the human antibody to acquire the antigen binding specificity of the murine mab while reducing its heterology.

The term "CHO cell" is a chinese hamster ovary cell (chinese hamster ovary cell); the term "HEK293E cell" is a human embryonic kidney 293E cell (human embryonic kidney 293E cell), and the term "NS0 cell" is a mouse NS0 thymoma cell.

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-Siglec-15 monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 heavy chain complementarity determining regions respectively represented by HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises 3 light chain complementarity determining regions respectively represented by LCDR1, LCDR2 and LCDR3, and the anti-Siglec-15 monoclonal antibody is selected from any one of the following.

EXAMPLE 2 murine antibody molecular screening

According to the invention, a mouse is immunized by using a Siglec-15 antigen (the extracellular segment of the Siglec-15 protein, and the Siglec-15 antigen and the protein used in subsequent experiments are all the extracellular segment of the Siglec-15), an immunization method is optimized, a phage display library is created, an antigen site screening method is established, and the construction and screening identification of a specific phage display library are as follows:

Step one: siglec-15 antigen immunized mice

1. Experimental animals:

species strain: BALB/c, female, mouse;

weight of: 18-20g;

experimental animal provider: beijing Fukang biotechnology Co., ltd.

2. Immunization: mice were immunized with human Siglec-15 (a synthetic gene from Nanjing Jinsri Biotechnology Co., ltd., vector constructed by this company and expressed and purified).

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 Ambion, 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 gene library (VH) and a light chain variable region gene library (VL), respectively carrying out double enzyme digestion on the light chain and heavy chain, connecting the heavy chain and the light chain variable region gene library and the light chain gene library to a vector subjected to the same step-by-step enzyme digestion, and constructing a pScFv-Disb-VL gene library, wherein the pScFv-Disb-HS vector is obtained by modifying a vector pComb3 (purchased from a Chinese plasmid vector strain gene collection center) by adopting a series of gene cloning methods, so that the vector pComb3 vector is 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: coating immune tube with Siglec-15 as antigen in an amount of 5 μg/500 μl/tube, coating overnight at 4deg.C, and sealing the immune tube and the immune tube with 4% skimmed milk powder/PBST respectivelyThe phage antibody library was blocked at room temperature for 1h. 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 1h of reaction at room temperature, unbound phage was washed off with PBST-PBS, eluted with 0.1M Glycine-HCl at pH2.2, and the eluted phage antibody solution was finally neutralized to about pH7.0 with 1.5M Tris-HCl at pH 8.8.

Step four: 10ml of TG1 bacterial liquid growing to logarithmic phase after the neutralization of phage infection is placed in an incubator at 37 ℃ for 30min, part of bacterial liquid is taken out for gradient dilution and coated on a 2YTAG plate, and the phage yield is calculated. 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 Siglec-15 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 statically infected with helper phage M13KO7 at 37℃for 30min.4000rpm, centrifuging for 15min, discarding the supernatant, re-suspending the pellet with 2YTAK, and culturing overnight at 28deg.C and 220 rpm. After centrifugation at 4000rpm and 4 ℃ for 15min, the amplified phage supernatant is sucked for ELISA identification, and finally four murine antibody molecules with higher affinity and against Siglec-15 are obtained through screening, which are named MA-I, MA-II, MA-III and MA-IV respectively, the obtained monoclonal antibodies are subjected to gene sequencing to determine correct antibody sequences, and the sequences of the 4 monoclonal antibodies obtained through screening are as follows:

specifically, SEQ ID No. 25 (amino acid sequence of the heavy chain variable region of MA-I)

EVKLEQSGPELVKPGASVKISCKASGYTFTDYNMFWVKQSHGKTLEWIGYIYPDNGGTGYNQNFKSKATLTVDNSSSSAYMELRSLTSEDSAVYYCARSEYDYFDYWGQGTTLTVSS；

SEQ ID No. 26 (amino acid sequence of the light chain variable region of MA-I)

DIVLTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPLTFGAGTKLELK；

SEQ ID No. 27 (amino acid sequence of the heavy chain variable region of MA-II)

EVKLEQSGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQSPEKRLEWVAEIISGGSHTYYPDTVTGRFTISRDDAKNTLYLEMSSLRSEDTAMYYCARDGNYGYAMDYWGQGTSVTVSS；

SEQ ID No. 28 (amino acid sequence of the light chain variable region of MA-II)

DIVITQTPAIMSASPGEKVTMTCRASSSVSSSYLHWYQQKSGASPKLWIYSTSNLASGVPARFSGGGSGTSYSLTISSVEAEDDATYYCQQWSGYPWTFGGGTKLEIK；

SEQ ID No. 29 (amino acid sequence of the heavy chain variable region of MA-III)

QVKLEESGPELVKPGASVKMSCKASGYIFTSYVMHWVRQKPGQGLEWIGYIDPYNDRTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARSGDYGSSFDYWGQGTTLTVSS；

SEQ ID No. 30 (amino acid sequence of the light chain variable region of MA-III)

DIVMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPYTFGGGTKLEIK；

SEQ ID No. 31 (amino acid sequence of the heavy chain variable region of MA-IV)

EVKLQESGAELVKPGASVKLSCIASGFNIKDTFIHWVKQRPEQGLEWIGRIDPASGYTKYDPKFQGKATITTDTSSNTAYLQLSSLTSEDTAVYYCTRSGSYVSFVYWGQGTLVTVSA；

SEQ ID No. 32 (amino acid sequence of the light chain variable region of MA-IV)

DIVMTQTPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK。

Example 3 gradient dilution ELISA comparing affinity of anti-Siglec-15 phage monoclonal antibodies

The 4 murine antibody molecules (MA-I, MA-II, MA-III and MA-IV) obtained in example 2 were subjected to the display and purification of monoclonal phages, and then subjected to phage gradient dilution ELISA experiments to identify affinities, the control antibody being a monoclonal antibody against SIGLEC-15 of the Nescol company (also known as 5G12, patent application number 201780067999.3, patent name being an antibody against SIGLEC-15 and methods of use thereof), in particular as follows:

the Siglec-15 antigen was coated with a carbonate buffer at pH9.6, 100 ng/well/100. Mu.L, coated overnight at 4℃and washed three times with PBST, and the 4 phage monoclonal antibodies selected in example 2 were each diluted with a five-fold gradient of PBST, 100. Mu.L of the 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 1h. TMB chromogenic kit developed, developed at room temperature for 10 min, with 2M H ₂ SO ₄ After termination, the microplate reader reads at 450nm/630nm and calculates the corresponding EC50 value as follows:

through the data and as shown in FIG. 2, 4 different murine antibody molecules screened in example 2 can be combined with Siglec-15, and the monoclonal antibodies provided by the invention and Siglec-15 have higher affinity.

Example 4

Example 4 of the present invention further defines on the basis of example 2 that the murine antibody molecule further comprises a heavy chain constant region which is one of the murine IgG1, igG2a, igG2b or IgG3 type constant regions and a light chain constant region which is murine C having the amino acid sequence shown in SEQ ID No. 33 _k Constant volumeThe amino acid sequence of the constant region of the IgG1 type is shown as SEQ ID No. 34, the amino acid sequence of the constant region of the IgG2a type is shown as SEQ ID No. 35, the amino acid sequence of the constant region of the IgG2b type is shown as SEQ ID No. 36, and the amino acid sequence of the constant region of the IgG3 type is shown as SEQ ID No. 37; the specific sequence is as follows:

SEQ ID No. 33 (mouse C) _k Light chain constant region amino acid sequence):

ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC；

SEQ ID No. 34 (murine heavy chain constant region amino acid sequence of IgG1 type):

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG；

SEQ ID No. 35 (murine heavy chain constant region amino acid sequence of IgG2a type):

AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK；

SEQ ID No. 36 (murine heavy chain constant region amino acid sequence of IgG2b type):

AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK；

SEQ ID No. 37 (murine heavy chain constant region amino acid sequence of IgG3 type):

ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA。

EXAMPLE 5 preparation of murine antibody molecules as anti-Siglec-15 monoclonal antibodies

Example 5 of the invention on the basis of example 4 it is preferred to define murine 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: 34) and murine C _k A light chain constant region of the type (the amino acid sequence of which is shown as SEQ ID No. 33). The preparation method of the antibody specifically comprises the following steps:

1. in the case where the genes encoding the heavy chain VH and the light chain VL of the 4 monoclonal antibodies selected in example 2 were cloned into the vector pTSE (shown in FIG. 3) harboring the heavy chain and light chain constant region genes, respectively, the preferred heavy chain constant region was a murine IgG1 type constant region (amino acid sequence shown in SEQ ID No: 34) and the light chain constant region was a murine C _k The strand (amino acid sequence shown in SEQ ID No. 33) 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. HEK293E cells (purchased from basic medical institute of China medical sciences, cat# GNHu 43) were transiently transfected, antibody expression was performed, 4 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 Ten eastern technology Co., cat# BCA 0020), and then protein sizes were identified by SDS-PAGE, and as a result, non-reduced MA-I, MA-II, MA-III and MA-IV were sequentially performed from left to right, protein molecular weight Marker1, protein molecular weight Marker2, and reduced MA-I, MA-II, MA-III and MA-IV murine anti-Siglec-15 monoclonal antibodies were obtained, and the molecular weight sizes of each of the bands were consistent with theory.

EXAMPLE 6 binding experiments of murine antibody to Siglec-15

Siglec-15 antigen was coated with a carbonate buffer at pH9.6, 100 ng/well/100. Mu.L, and at a temperature of 4℃overnight. The mixture was washed five times with 300. Mu.L/well PBST, then 1% BSA-PBST was added and blocked for 1 hour at 37℃and MA-I, MA-II, MA-III and MA-IV murine antibody molecules were added at different dilution concentrations, the initial maximum concentration of the 4 antibody molecules was 5. Mu.g/ml, each was diluted with 5-fold gradients, 8 gradients per antibody were used and incubated for 1 hour at 37 ℃. 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-PBST 1:2000 was added thereto and incubated for 1h at 37 ℃. TMB chromogenic kit, 100. Mu.L/well, room temperature for 8min, 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:

by the above data and as shown in FIG. 5, 4 different murine antibody molecules were screened to bind Siglec-15.

Example 7 murine antibody inhibits Siglec-15 binding to Jurkat cell surface receptor

First, four kinds of murine antibodies (MA-I, MA-II, MA-III, MA-IV) and control antibody 5G12 were prepared as protein solutions at a concentration of 600. Mu.g/mL, respectively, and added to a 96-well plate at a concentration of 25. Mu.L per well. Next, siglec-15 ligand protein was formulated at a concentration of 200. Mu.g/mL, 25. Mu.L per well, and added to a 96-well plate. Again, jurkat cell lines were counted, a number of cells were taken, centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 2e+6cells/mL, 50 μl per well was added to a 96-well plate. All samples and protein dilutions were performed using PBS buffer. Finally, the 96-well plate after the sample addition is placed at 4 ℃ and incubated for 1h. After removal, 100. Mu.L of LPBS buffer was added to each well, and the cells were washed once by centrifugation at 3000rpm, and cell pellets were collected. A pre-formulated dilution of AF488-anti human IgG-Fc antibody (purchased from southern Biotech, accession number 2048-30) was added to the cell pellet and incubated at 4℃for 1h. After removal, the sample was washed once at 3000rpm, resuspended at 200. Mu.LPBS, and then run on the flow machine to collect the fluorescent signal in the FL1-A channel.

As shown in FIG. 6, the four murine antibody molecules screened in example 2 of the present invention all inhibited the binding of Siglec-15 to Jurkat cell surface receptors and were comparable to control antibody 5G12 at the same concentration of action.

Example 8 murine antibody inhibits Siglec-15 binding to CHOSLV-LRRC4C cell surface receptor

Four murine antibody molecules (MA-I, MA-II, MA-III, MA-IV) and control antibody 5G12 were gradient diluted to a concentration of 200. Mu.g/mL, 3 Xgradient diluted, total of 8 gradients, 25. Mu.L per well added to the corresponding position in 96-well plates. Siglec-15-Fc ligand protein was diluted and formulated at a concentration of 40. Mu.g/mL, 25. Mu.L per well was added to the corresponding location in the 96-well plate. The CHOSLV-LRRC4C cell line was counted, a certain number of cells were collected, centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 2E+6cells/mL, and 50. Mu.L per well was added to a 96-well plate. All samples and protein dilutions were performed using PBS buffer. The 96-well plate after the sample addition was placed at 4℃and incubated for 1h. After removal, 100. Mu.L of LPBS buffer was added to each well, and the cells were washed once by centrifugation at 3000rpm, and cell pellets were collected. Add pre-formulated AF488-anti human IgG-Fc antibody (purchased from southern Biotech, accession number 2048-30) to the cell pellet, incubate at 4℃for 1h and wash at 3000rpm once, after 200 μLPBS resuspension, flow-on-machine detection, collect fluorescent signal in FL1-A channel. Dose-response curves were plotted and candidate molecules were calculated to inhibit the binding of Siglec-15 ligand protein to the cell surface LRRC4C receptor.

Conclusion: as can be seen from the above data and FIG. 7, four murine candidate molecules (MA-I, MA-II, MA-III, MA-IV) each block the binding of Siglec-15 to its receptor.

Example 9 murine antibodies promote T cell activation and proliferation

Human PBMC cells (peripheral blood mononuclear cell, PBMC) were recovered, cell pellet collected after centrifugation, resuspended and counted in RPMI1640 complete medium and cell density adjusted to 2e+6cells/mL, 50 μl per well added to 96 well plates. Siglec-15 ligand protein was formulated at a concentration of 20. Mu.g/mL, and 50. Mu.L per well was added to the corresponding location in the 96-well plate. The final concentration of Anti-CD3 antibody was 0.5. Mu.g/well, and 10. Mu.g/mL was prepared and 50. Mu.L of antibody per well was added to the corresponding position in the 96-well plate. Four murine molecules (MA-I, MA-II, MA-III, MA-IV) and control antibody 5G12 were formulated at an initial concentration of 100 μg/mL, diluted 3 Xgradient, for a total of 8 gradients. 50 μl per well was added to the corresponding location in the 96-well plate. The dilution of the protein and the antibody is carried out by using RPMI1640 complete culture medium, evenly mixing the mixture with a 96-well plate and incubating the mixture for 3 days at 37 ℃ in a dark place. Cell culture supernatants were taken, diluted 10-fold, and used for cytokine detection. Activation of native T cells by anti-Siglec-15 murine antibody molecules was evaluated from three perspectives: human TNF- α cytokine release, human IFN- γ cytokine release and T cell proliferation are performed as follows:

Human IFN-gamma detection kit (purchased from Ekesai Biotechnology Co., ltd., product number H008-96): the diluted supernatant and standard were added to the sample wells, 100 μl per well, covered with a sealing plate membrane and incubated for 1.5h at room temperature. After incubation, the plates were washed 3 times. Add Biotinylated antibody dilution in human IFN-gamma detection kit, 100. Mu.L per well, cover the sealing plate membrane and incubate for 1h at room temperature. After incubation, the plates were washed 3 times. Adding strepitavidin-HRP working solution, covering a sealing plate membrane at 100 mu L of each hole, and incubating for 30min at room temperature. After incubation, the plates were washed 3 times. The reaction was stopped by adding 100. Mu.L of TMB chromogenic solution per well, incubating at room temperature for about 15 minutes in the dark, and adding 100. Mu.L of Stop solution per well. After the OD value is read by the enzyme labeling instrument, a dose response curve is drawn.

Human TNF-alpha assay kit (purchased from Ekesai Biotechnology Co., ltd., cat. No. EM 008-96): the diluted supernatant and standard were added to the sample wells, 100 μl per well, covered with a sealing plate membrane and incubated for 1.5h at room temperature. After incubation, the plates were washed 3 times. Add Biotinylated antibody dilution, 100. Mu.L per well, cover the sealing plate membrane and incubate for 1h at room temperature. After incubation, the plates were washed 3 times. Adding Strepitavidin-HRP working solution in the human IFN-gamma detection kit, covering a sealing plate film at 100 mu L of each hole, and incubating for 30min at room temperature. After incubation, the plates were washed 3 times. The reaction was stopped by adding 100. Mu.L of TMB chromogenic solution per well, incubating at room temperature for about 15 minutes in the dark, and adding 100. Mu.L of Stop solution per well. After the OD value is read by the enzyme labeling instrument, a dose response curve is drawn.

Cell pellet was collected, stained with CD3e Monoclonal Antibody (purchased from Semer Feiche technologies Co., ltd., cat No. MA 1-10177), incubated at room temperature for 15min in the dark, washed once with 100. Mu.LPBS buffer, resuspended, counted in flow-through machine, and dose-response curves were drawn.

Human TNF-alpha cytokine release

Human IFN-gamma cytokine release

T cell proliferation (absolute count)

From the above data and FIGS. 8-10, it can be seen that all four murine antibody molecules (MA-I, MA-II, MA-III, MA-IV) can block the binding of Siglec-15 ligand protein to the native T cell surface receptor, block intracellular inhibitory signaling pathways, activate T cells, promote T cell proliferation and activation (release of TNF- α and IFN- γ) by binding to Siglec-15.

EXAMPLE 10 detection of biological Activity of murine antibody molecules (reporter Gene method)

Counting Jurkat-NFAT-Luc engineering cell strain, adjusting cell density to 2E+6cells/mL with sample dilution (its components include 90% RPM 1640, 10% FBS, 0.5 μg/mL Puromycin), mixing gently, adding cell solution into 96Well plate, 50 μl/well. Four murine molecules (MA-I, MA-II, MA-III and MA-IV) are diluted to an initial concentration of 800 mug/mL respectively by using sample diluents, 5-time gradient dilution is carried out, 8 gradients are added, 50 mug/hole is added to the corresponding position of a 96-well plate, and two compound holes are arranged for each sample concentration. Siglec-15 antigen was formulated and 50. Mu.L per well was added to 96-well plates to give final concentrations of action of 16. Mu.g/mL. Human CD3 anti-body (purchased from Yiqiao China Biotechnology Co., ltd., product No. 10977-H001) was prepared and 50. Mu.L of each well was added to a 96-well plate so that the concentration of the active substance was 1. Mu.g/mL. Gently mixing cell culture plate, placing in CO at 37deg.C ₂ Incubate for 6h. The supernatant was centrifuged off, the lysate was added, 10. Mu.L of the lysate was added to 384-well plates per well, an equal amount of luciferase reaction substrate (purchased from Promega Biotechnology Co., ltd., product No. E2610) was added, the reaction was carried out at room temperature for 5min, the fluorescence value was read under a microplate reader, and the corresponding IC50 value was calculated as follows:

through the data and as shown in FIG. 11, the 4 different murine antibodies and control antibodies screened each bind to Siglec-15 and inhibit Siglec-15 binding to Jurkat cell surface receptors, blocking intracellular inhibitory signaling pathways, and reactivating T cells. Construction of the engineered cell line Jurkat-NFAT-Luc can simulate T cells. Siglec-15 inhibits and down regulates the intracellular activation signaling pathway (NFAT-Luc) by binding to T cell surface receptors. These 4 murine antibody molecules can effectively block the binding of Siglec-15 to cell surface receptors, reactivating T cell signaling pathways.

Example 11

Example 11 of the present invention further defines the anti-Siglec-15 monoclonal antibody as a chimeric antibody molecule comprising the heavy chain variable region of the murine antibody molecule of example 2, the light chain variable region of the murine antibody molecule, and the humanized antibody constant region. The humanized antibody constant region includes a humanized antibody heavy chain constant region and a humanized antibody light chain constant region, the humanized antibody heavy chain constant region being human One of constant regions of IgG1 type, igG2 type or IgG4 type, the amino acid sequence of the constant region of IgG1 type is shown as SEQ ID No. 38, the amino acid sequence of the constant region of IgG2 type is shown as SEQ ID No. 39, the amino acid sequence of the constant region of IgG4 type is shown as SEQ ID No. 40, and the light chain constant region of humanized antibody is human C with the amino acid sequence shown as SEQ ID No. 41 _k A constant region of the type;

SEQ ID No. 38 (heavy chain constant region amino acid sequence of human IgG1 type):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；

SEQ ID No. 39 (heavy chain constant region amino acid sequence of human IgG2 type):

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；

SEQ ID No. 40 (heavy chain constant region amino acid sequence of human IgG4 type):

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK；

SEQ ID No. 41 (human C _k Light chain constant region amino acid sequence of chain):

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

EXAMPLE 12 preparation of chimeric antibody molecule antibody

Example 12 of the present invention further defines on the basis of example 7 that the humanized antibody constant region comprises a heavy chain constant region of human IgG1 type (having an amino acid sequence as shown in SEQ ID No: 38) and human C _k A light chain constant region of the type (the amino acid sequence of which is shown as SEQ ID No. 41).

The specific preparation method comprises the following steps:

the heavy chain variable region VH (SEQ ID NO: 25) and the light chain variable region VL gene (SEQ ID NO: 26) of the antibody molecule MA-I obtained by screening the immune phage antibody library of example 2 were kept unchanged in murine sequences, cloned into vector pTSE (shown in FIG. 3) harboring the heavy chain constant region and the light chain constant region gene, respectively, the heavy chain constant region being of human IgG1 type (amino acid sequence shown in SEQ ID NO: 38) and the light chain constant region being of human C _k Type (amino acid sequence shown as SEQ ID NO: 41). HEK293E cells (purchased from the institute of basic medicine of the national academy of sciences of medicine, cat# GNHu 43) were transiently transfected and antibody expression was performed to obtain chimeric antibody CA-I.

EXAMPLE 13 humanization of murine antibody molecules

Firstly, the sequence of the murine antibody molecule MA-1 in example 2 is selected and compared with a human antibody germline database (v-base), a human antibody light chain germline and a human antibody heavy chain germline with higher homology are searched as candidate sequences, and then the sequences of CDRs of the murine antibody molecule MA-I are transplanted onto the human candidate sequences for homologous modeling. The back mutations of the humanized antibodies were then designed by three-dimensional structure modeling to calculate key framework amino acid residues that might play an important role in maintaining the CDR loop structure. The light chain variable region sequences and the heavy chain variable region sequences of the designed humanized antibody containing the back mutation are respectively synthesized by Nanjing Jinsri biotechnology limited company in an optimized mode, then are connected to a transient expression vector, and the light chain and heavy chain combination analysis obtained by humanization is carried out to obtain the following humanized antibody molecules: the sequences of the 2 monoclonal antibodies selected above were as follows:

specifically, SEQ ID No. 42 (amino acid sequences of the heavy chain variable regions of HA-I and HA-II):

METDTLLLWVLLLWVPGSTGQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMFWVRQAPGQRLEWIGYIYPDNGGTGYNQNFKSKATLTVDNSASTAYMELSSLRSEDTAVYYCARSEYDYFDYWGQGTLVTVSS；

SEQ ID No. 43 (amino acid sequence of the light chain variable region of HA-I):

DIVLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPLTFGQGTKVELK；

SEQ ID No. 44 (amino acid sequence of the light chain variable region of HA-II):

DIQLTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQGNTLPLTFGQGTKVELK。

example 14

Example 14 of the present invention further defines on the basis of example 13 that the humanized antibody molecule further comprises a humanized antibody constant region; the humanized antibody constant region comprises a heavy chain constant region selected from the group consisting of human IgG1, igG2, or IgG4 and human C _k The amino acid sequence of the light chain constant region of the type IgG1 and the heavy chain constant region of the type IgG2 is shown as SEQ ID No. 38, the amino acid sequence of the heavy chain constant region of the type IgG2 is shown as SEQ ID No. 39, the amino acid sequence of the heavy chain constant region of the type IgG4 is shown as SEQ ID No. 40, and the amino acid sequence of the heavy chain constant region of the type IgG2 is human C _k The amino acid sequence of the light chain constant region of the type is shown as SEQ ID No. 41.

The specific sequence of the above humanized antibody constant region was the same as that of example 11.

EXAMPLE 15 preparation of humanized antibody molecules

Example 15 of the present invention further defines on the basis of example 10 that the humanized antibody constant region comprises a heavy chain constant region of human IgG1 type (having an amino acid sequence as shown in SEQ ID No: 38) and human C _k A light chain constant region of the type (the amino acid sequence of which is shown as SEQ ID No. 41).

The genes encoding the heavy chain VH and the light chain VL of the 2 humanized antibody molecules obtained in example 13 were cloned into vector pTSE (shown in FIG. 3) harboring the heavy chain constant region and the light chain constant region genes, respectively, the heavy chain constant region being of human IgG1 type (amino acid sequence shown in SEQ ID NO: 38) and the light chain constant region being C _k The chain (amino acid sequence shown in SEQ ID NO: 41).

Humanized antibody molecule HA-I, HA-II was transiently transfected into HEK293 cells (purchased from the institute of basic medicine of the national academy of medicine, cat# GNHu 43) respectively, antibody expression was performed, monoclonal antibodies were obtained by protein A affinity column purification using an AKTA instrument, protein concentration was measured using a BCA kit (purchased from the company BCA0020, eastern technology, beijing) and then the size of the protein was identified by SDS-PAGE, and as a result, non-reduced protein molecular weight HA-I, HA-II, chimeric antibody CA-I prepared in example 12, non-reduced protein molecular weight Marker1 and reduced protein molecular weight Marker2, HA-I, HA-II, chimeric antibody CA-I were sequentially obtained from the left side to the right side, and the molecular weight of each band was consistent with theory.

Example 16

Example 16 of the present invention further defines the humanized antibody molecule as a full length antibody or antibody fragment, the humanized antibody molecule comprising one or a combination of several of Fab, F (ab) 2, fv, or ScFv.

Example 17

The invention also provides a protein comprising the anti-Siglec-15 monoclonal antibody defined in the above examples.

The invention also provides a polynucleotide molecule which codes for the anti-Siglec-15 monoclonal antibody defined in the above examples.

The invention also provides a recombinant DNA expression vector, which comprises the polynucleotide molecule.

The invention also provides a medicament comprising the anti-Siglec-15 monoclonal antibody defined in the above embodiment of the invention.

the invention also provides a method of treating a subject suffering from an immune disorder or cancer, the method comprising administering to the subject a therapeutically effective amount of an anti-Siglec-15 monoclonal antibody;

preferably, the cancer includes brain glioma, melanoma, colorectal cancer, renal cancer, lung cancer, lymphoma or leukemia;

the above immune diseases include psoriasis, crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis or autoimmune hepatitis.

The anti-PD-1 monoclonal antibody is selected from Nivolumab, pembrolizumab, teleplon Li Shan, xindi Li Shan, tilapia monoclonal antibody, carrilizumab or ZL201510312910.8, and the patent name is a monoclonal antibody against PD-1, such as DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 or DFPD1-13, disclosed in the patent document of the patent name and the obtaining method thereof, and is not limited to the above limitation of the anti-PD-1 monoclonal antibody, but can be any other monoclonal antibody against PD-1 commercially used in experiments, so long as the target is a monoclonal antibody against PD-1, and other anti-PD-1 monoclonal antibodies are not particularly limited herein.

EXAMPLE 18 humanized antibody molecules and Siglec-15 binding experiments

Siglec-15 antigen was coated with carbonate buffer at pH9.6, 200 ng/well/100. Mu.L, at a temperature of 4℃overnight. Wash five times with 300. Mu.L/well PBST and add 1%BSA-PBST was blocked for 1h at 37℃and humanized antibody HA-I, HA-II at different dilution concentrations and chimeric antibody CA-I prepared in example 12 were added, the initial maximum concentration of 3 antibodies was 50. Mu.g/mL, 10 gradients were made for each antibody after 3-fold dilution, and incubated for 1h at 37 ℃. Five washes with 300. Mu.L/well PBST were performed, and Goat Anti Human IgG-HRP (purchased from Abies media Biotechnology Co., ltd., product number ZB-2304) diluted with 1% BSA-PBST 1:5000 was added thereto and incubated at 37℃for 1 hour. TMB chromogenic kit, 100. Mu.L/well, 5min at room temperature, 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:

as shown in FIG. 13, the above data and experimental results show that 2 different humanized antibody molecules can be combined with Siglec-15, and the EC50 values of the 2 humanized antibody molecules are similar to those of the chimeric antibody CA-I, which indicates that the humanized antibody molecules retain the high combining ability of the murine parent antibody MA-I and Siglec-15.

EXAMPLE 19 Cross-binding experiments of humanized antibodies with Siglec-15 of different species

Human Siglec-15, murine Siglec-15-His (purchased from offshore protein technologies Co., ltd., cat# 71) and cynomolgus monkey Siglec-15-His (purchased from offshore protein technologies Co., ltd., cat# CW 70) were coated with 100 ng/well/100. Mu.L, respectively, in carbonate buffer at pH9.6, and coated overnight at a temperature of 4 ℃. Washing with 300. Mu.L/well PBST for five times, adding 1% BSA-PBST, blocking at 37deg.C for 1 hr, adding humanized antibody HA-I, HA-II with different dilution concentration, and incubating at 37deg.C for 1 hr with 8 gradients of each antibody after 5-fold dilution for 2 humanized antibodies each with 25. Mu.g/mL of initial maximum concentration. Five washes with 300. Mu.L/well PBST and additional Goat Anti Human IgG-HRP diluted with 1% BSA-PBST 1:5000 were added and incubated for 1h at 37 ℃. TMB chromogenic reagent Developing the cartridge, developing at room temperature for 5min at 100. Mu.L/well, and then using 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:

through the above data and as shown in FIG. 14, 2 different humanized antibody molecules were screened to bind to human Siglec-15, cynomolgus monkey Siglec-15, and murine Siglec-15.

EXAMPLE 20 humanized antibody molecules inhibit Siglec-15 binding to Jurkat cell surface receptors

Two humanized antibody molecules (HA-I, HA-II) and control antibody 5G12 were gradient diluted to a concentration of 200. Mu.g/mL, 5 gradient dilutions were performed for a total of 8 gradients, and 25. Mu.L per well was added to the corresponding position of the 96-well plate. Siglec-15-FITC protein was prepared by fluorescent labeling of Siglec-15 protein using FITC fluorescent labeling protein kit (purchased from Semer FireWipe technologies Co., ltd., product number F6434). Siglec-15-FITC protein concentration was adjusted using PBS buffer to give a concentration of 40. Mu.g/mL, and 25. Mu.L per well was added to the corresponding position in the 96-well plate. The Jurkat cell line was counted, a number of cells were taken, centrifuged, resuspended in PBS buffer, and the cell density was adjusted to 2E+6cells/mL, and 50. Mu.L per well was added to a 96-well plate. All samples and protein dilutions were performed using PBS buffer. The 96-well plate after the sample addition was placed at 4℃and incubated for 1h. After removal, 100. Mu.LPBS buffer was added to each well, the cells were washed once by centrifugation at 3000rpm, cell pellet was collected, and after 200. Mu.LPBS was resuspended, the flow-on-machine detection was performed and fluorescence signals in FL1-A channel were collected. And (3) drawing a dose response curve, and calculating the binding of the candidate molecule inhibition Siglec-15 ligand protein and the Jurkat cell surface receptor.

From the above data and FIG. 15, it can be seen that both humanized candidate molecules (HA-I, HA-II) were able to block the binding of Siglec-15 to the receptor on the Jurkat cell surface.

Example 21 detection of biological Activity of humanized antibody molecules (reporter Gene)

The Jurkat-NFAT-Luc engineered cell lines were counted, the cell density was adjusted to 2E+6cells/mL using a sample dilution (the composition of which included 90% RPM 1640, 10% FBS, 0.5. Mu.g/mL Puromycin), and the cell solution was added to a 96-well plate, 50. Mu.L/well after gentle mixing. Two humanized antibody molecules (HA-I, HA-II) were diluted with sample dilutions to an initial concentration of 800. Mu.g/ml, 5-fold gradient dilution, 8 gradients total, 50. Mu.L/well, and two multiplex wells were placed in the 96-well plate at the corresponding positions for each sample concentration. Siglec-15 antigen was formulated and 50. Mu.L per well was added to 96-well plates to give final concentrations of action of 16. Mu.g/mL. anti-CD3 antibody (purchased from Yinqiao Shenzhou Biotechnology Co., ltd., product No. 10977-H001) was prepared and added to a 96-well plate at a concentration of 50. Mu.L per well to effect 1. Mu.g/mL. Gently mixing cell culture plate, placing in CO at 37deg.C ₂ Incubate for 6h. The supernatant was centrifuged off, the lysate was added, 10. Mu.L of the lysate was added to 384-well plates per well, an equal amount of luciferase reaction substrate (purchased from Promega Biotechnology Co., ltd., product No. E2610) was added, the reaction was carried out at room temperature for 5min, the fluorescence value was read under a microplate reader, and the corresponding IC50 value was calculated as follows:

Through the data and as shown in FIG. 16, the 2 humanized antibody molecules screened all bind to Siglec-15 and inhibit the binding of Siglec-15 to Jurkat cell surface receptors, block intracellular inhibition signal pathways, and reactivate T cells.

EXAMPLE 22 inhibition experiment of anti-Siglec-15 monoclonal antibody HA-I on MC38-Siglec-15 colorectal cancer in mice

1. Experimental animals:

species strain: c57BL/6JGpt, mice;

week-old: for 6-8 weeks;

experimental animal provider Jiangsu Ji Yikang Biotechnology Co., ltd.

2. Cell culture:

MC38 tumor cells (YK-CL-256-02) (purchased from general Biovector NTCC Inc. of Beijing Co., ltd., product number NTCC-MC 38) were used as primordial cells to construct MC38-Siglec-15 tumor cell lines. With DMEM medium (available from Sieimer Feier technologies (China) Co., ltd. (Gibco), cat# 10687010) containing inactivated 10% fetal bovine serum (Excell Bio, cat# FND 500), 100U/mL penicillin, 100 μg/mL streptomycin, 250 μg/mL Hygromycin B (available from Sieimer Feier technologies (China) Co., ltd., cat# 10566-016) and 2mM glutamine (available from Sieimer Feier technologies (China) Co., ltd.). Times.37℃5% CO ₂ Tumor cells were cultured in the incubator of (2) and were passaged in flasks after cells were grown to completion every 3 to 4 days, and tumor cells in the logarithmic growth phase were used for in vivo tumor inoculation.

Bone marrow derived macrophages (Bone marrow-derived macrophage, BMDM) were isolated from C57BL/6 mice. RPMI 1640 medium (available from Simer Feishan technologies (China) Co., ltd., product No. A10491-01) containing inactivated 10% fetal bovine serum (Excell Bio, product No.: FND 500), 100U/mL penicillin and 100 μg/mL streptomycin and 20ng/mL mouse M-CSF (available from Simer Feishan technologies Co., ltd., product No. 51112-MNAH) and 20ng/mL mouse IL-10 (available from Simer Feishan technologies Co., ltd., product No. 50245-MNAE) was used at 37℃and 5% CO ₂ After 4 days of culture in an incubator, can be used for in vivo tumor models.

3. Inoculation and grouping of tumor cells:

PBS resuspended MC38-Siglec-15 tumor cells at a cell density of 1.0X10 ⁶ Uniformly mixing/mL with a certain amount of BMDMs cell suspension, inoculating under the right flank of experimental animal, 100 μl/animal, and growing to 43mm in tumor ³ The left and right groups were administered in groups of 2, 5 each, each Vehicle control group (Vehicle, i.p., tiw.times.3w) and HA-1 (10 mg/kg, i.p., tiw.times.3w).

4. Detecting the index: tumor volume was measured 2 times per week using vernier calipers The calculation formulas of the length and the short diameter of the tumor volume are as follows: volume=0.5×long diameter×short diameter ² The method comprises the steps of carrying out a first treatment on the surface of the The change in tumor volume was recorded as a function of time of administration, and the experimental results are shown in fig. 17.

The data in FIG. 17 shows that the anti-Siglec-15 monoclonal antibody HA-1 was able to inhibit tumor growth and exhibited a dose-dependent response.

Example 23 evaluation of thermal stability of anti-Siglec-15 monoclonal antibody HA-I

The thermostability against Siglec-15 monoclonal antibody HA-I was evaluated using a multifunctional protein thermostability analysis system (purchased from Unchained Labs). Protein conformational stability was assessed by monitoring protein endogenous fluorescence over temperature (starting at 25 ℃ C., increasing temperature to 95 ℃ C. At a rate of 0.3 ℃ C./min) to detect changes in protein conformation, thereby determining the protein melting temperature Tm. When the sample is aggregated, the scattered light waves interfere, the scattered light signals increase, and the colloidal stability (characterized by Tagg) of the protein is measured by static light scattering, and the results are shown in the following table and fig. 18.

The temperature of the anti-Siglec-15 monoclonal antibody HA-I is 72.9 ℃, the average Tagg is 72.0 ℃, and the anti-Siglec-15 monoclonal antibody HA-I shows better conformational stability and colloid stability.

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

1. An anti-Siglec-15 monoclonal antibody comprising a heavy chain variable region comprising 3 heavy chain complementarity determining regions represented by HCDR1, HCDR2 and HCDR3, respectively, and a light chain variable region comprising 3 light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3, respectively, wherein the heavy chain complementarity determining region HCDR1 has the amino acid sequence of SEQ ID No. 19, the heavy chain complementarity determining region HCDR2 has the amino acid sequence of SEQ ID No. 20, the heavy chain complementarity determining region HCDR3 has the amino acid sequence of SEQ ID No. 21, the light chain complementarity determining region LCDR1 has the amino acid sequence of SEQ ID No. 22, the light chain complementarity determining region LCDR2 has the amino acid sequence of SEQ ID No. 23, and the light chain complementarity determining region LCDR3 has the amino acid sequence of SEQ ID No. 24.

2. The anti-Siglec-15 monoclonal antibody of claim 1, wherein the anti-Siglec-15 monoclonal antibody is a murine antibody molecule, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 32.

3. The anti-Siglec-15 monoclonal antibody of claim 2, wherein the murine antibody molecule further comprises a heavy chain constant region that is one of the murine IgG1, igG2a, igG2b, or IgG3 constant regions and a light chain constant region that is murine C having the amino acid sequence shown in SEQ ID No. 33 _k The amino acid sequence of the constant region of the IgG1 type is shown as SEQ ID No. 34, the amino acid sequence of the constant region of the IgG2a type is shown as SEQ ID No. 35, the amino acid sequence of the constant region of the IgG2b type is shown as SEQ ID No. 36, and the amino acid sequence of the constant region of the IgG3 type is shown as SEQ ID No. 37.

4. The anti-Siglec-15 monoclonal antibody of claim 2, wherein the anti-Siglec-15 monoclonal antibody is a chimeric antibody molecule comprising a heavy chain variable region of the murine antibody molecule, a light chain variable region of the murine antibody molecule, and a humanized antibody constant region.

5. The anti-Siglec-15 monoclonal antibody of claim 4, wherein the humanized antibody constant region comprises a humanA humanized antibody heavy chain constant region and a humanized antibody light chain constant region, wherein the humanized antibody heavy chain constant region is one of constant regions of human IgG1 type, igG2 type or IgG4 type, the amino acid sequence of the constant region of the IgG1 type is shown as SEQ ID No. 38, the amino acid sequence of the constant region of the IgG2 type is shown as SEQ ID No. 39, the amino acid sequence of the constant region of the IgG4 type is shown as SEQ ID No. 40, and the amino acid sequence of the constant region of the humanized antibody light chain is human C with the amino acid sequence shown as SEQ ID No. 41 _k Constant region of the type.

6. A polynucleotide molecule encoding the anti-Siglec-15 monoclonal antibody of any one of claims 1-5.

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

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

9. A medicament comprising the anti-Siglec-15 monoclonal antibody of any one of claims 1-5.

10. Use of an anti-Siglec-15 monoclonal antibody of any one of claims 1-5 in the manufacture of a medicament for the treatment of an immune disorder or cancer;