WO2024022008A1 - Anti-siglec-15 monoclonal antibody, and antigen-binding fragment and use thereof - Google Patents

Abstract

The present invention relates to the field of biomedicine, and in particular, provided is an anti-Siglec-15 monoclonal antibody or an antigen-binding fragment thereof. The anti-Siglec-15 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and the monoclonal antibody or the antigen-binding fragment thereof is selected from one of A-I, A-II, A-III or A-IV. The anti-Siglec-15 monoclonal antibody or the antigen-binding fragment thereof provided in the present invention can specifically bind to Siglec-15, so as to block the binding of Siglec-15 to a cell surface receptor and exert anti-tumor activity, and is therefore used for treating cancers or immune diseases, the cancers including, but not being limited to, brain glioma, melanoma, colorectal cancer, kidney cancer, lung cancer, lymphoma, leukemia, etc. The immune diseases include, but are not limited to, psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, and multiple sclerosis.

Description

An anti-Siglec-15 monoclonal antibody, its antigen-binding fragment and its application

Cross-references to related applications

This application claims the rights and interests of Chinese patent applications 202210882813.2 submitted on July 26, 2022, Chinese patent applications 202211155583.6 submitted on September 22, 2022, and Chinese patent applications 202211531006.2 submitted on December 01, 2022. The contents of this application are incorporated by reference. are incorporated into this article.

Technical field

The present invention relates to the field of biomedicine technology, in particular to an anti-Siglec-15 monoclonal antibody, its antigen-binding fragment and its application.

Background technique

According to data from the "World Cancer Report 2020" released by the World Health Organization IARC, there were 19.29 million new cancer cases worldwide in 2020. There were 4.57 million new cancer cases in China alone, accounting for 23.7% of the world. The number of new cancer cases in China far exceeded In other countries around the world, the research and development of cancer drugs is particularly urgent for this reason.

Cancer cells can sometimes avoid detection and destruction by the immune system by reducing the expression of tumor antigens on their surface, making their detection more difficult. At the same time, cancer cells can also express proteins on their surface that induce the inactivation of immune cells, or induce cells in the surrounding environment to release substances that inhibit immune responses and promote tumor cell proliferation and survival. With the great success of anti-PD-1 antibodies in the field of tumor immunotherapy, how to change the tumor microenvironment and activate the immune system to kill tumors has become a hot research topic.

Sialic acid-binding Ig-like lectin-15 (Siglec-15) is a member of the SIGLEC gene family and a DAP12-related immune receptor, belonging to the immunoglobulin superfamily and sialic acid-binding Ig-like lectin family. The Siglec family is divided into two categories: one is sequence-conserved Siglecs, including sialyadhesin, CD22, MAG, and Siglec-15; the other is sequence-variable Siglecs related to CD33. Siglecs are cell surface proteins that bind sialic acid. They mainly exist on the surface of immune cells. They are a subset of type I lectins. They have very typical and conserved structural characteristics. Their transmembrane region consists of 2 to 17 cells. It consists of an outer Ig domain, and the N-terminus consists of a V-set Ig domain that binds sialic acid and a certain number of C2-set Ig domains. The intracellular segment of most Siglecs contains immunoreceptor tyrosine-based activation motifs (ITAMs), thereby exerting immunosuppressive functions; a few Siglecs, such as Siglec-14-16 and Siglec-H, function through their transmembrane regions The positively charged arginine combines with the transformant DAP12 of ITAMs to exert an immune regulatory effect. Siglec-15 consists of an immunoglobulin (Ig)-like domain, a transmembrane domain, and a short cytoplasmic tail. Siglec-15 is divided into two types: exogenous and endogenous. Generally, exogenous Siglec-15 is highly expressed on the surface of tumor cells, while endogenous Siglec-15 is mainly expressed on the surface of macrophages and dendritic cells, while it is minimally expressed in human and mouse tissues and various cell types. . Studies have found that Siglec-15 has the function of inhibiting T cell activity. Siglec-15 expressed by macrophages can directly inhibit the proliferation and activity of human and mouse T cells, and inhibit the secretion of IFN-γ and TNF-α; mice Both gene knockout and antibody blocking of Siglec-15 in vivo can enhance tumor immunity in the microenvironment and inhibit tumor growth in some mouse models. Therefore, Siglec-15 is a new immunosuppressive molecule that is widely present in a variety of tumors and has potential clinical relevance.

Currently, existing tumor immunotherapy through the PD-1/PD-L1 pathway is only effective for about 40% of patients with solid tumors. During actual treatment, when PD-L1 is upregulated, many other molecular and cellular mechanisms lead to weakened immune function in the microenvironment. Mechanisms include the lack of effective immune cell infiltration, lack of regulatory T enrichment, lack of tumor-associated macrophages, and lack of bone marrow-derived suppressor cells. At the same time, the success of PD-1 therapy emphasizes the need to repair immune deficiencies. importance and serve as an important guideline for restoring normal immune system function in cancer immunotherapy. Although Siglec-15 has certain homology with PD-L1, there is no correlation in the expression between the two, and the drug developed to target Siglec-15 is an immune checkpoint antibody that is complementary to anti-PD-1 drugs. New cancer drugs, therefore, the development of anti-Siglec-15 monoclonal antibody drugs has important clinical significance.

Contents of the invention

In order to solve the above-mentioned problems existing in the prior art and meet the needs of the domestic market, the present invention screened immune libraries and obtained anti-Siglec that can specifically bind to Siglec-15 and block the binding of Siglec-15 to cell surface receptors. -15 monoclonal antibodies and antigen-binding fragments thereof.

The specific technical solutions of the present invention are as follows:

The invention provides an anti-Siglec-15 monoclonal antibody or an antigen-binding fragment thereof, which includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes three regions represented by HCDR1, HCDR2 and HCDR3 respectively. The heavy chain complementarity determining region, the light chain variable region includes three light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3 respectively, the anti-Siglec-15 monoclonal antibody or its antigen-binding fragment is selected from any of the following :

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

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

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

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

Further, the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof is a murine antibody molecule, and the murine antibody molecule is selected from any of the following:

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

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

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

MA-IV: 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.

Further, the murine antibody molecule further includes a heavy chain constant region and a light chain constant region, and the heavy chain constant region is one of murine IgG1 type, IgG2a type, IgG2b type or IgG3 type constant region, and the The light chain constant region is a murine Ck type constant region with an amino acid sequence as shown in SEQ ID No: 33, the amino acid sequence of the IgG1 type constant region is as shown in SEQ ID No: 34, and the IgG2a type constant region The amino acid sequence is shown in SEQ ID No: 35, the amino acid sequence of the constant region of the IgG2b type is shown in SEQ ID No: 36, and the amino acid sequence of the constant region of the IgG3 type is shown in SEQ ID No: 37.

Further, the anti-Siglec-15 monoclonal antibody or its antigen-binding fragment is a chimeric antibody molecule, and the chimeric antibody molecule includes the heavy chain variable region of the murine antibody molecule, the Light chain variable regions and humanized antibody constant regions.

Further, the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof is a humanized antibody molecule, and the humanized antibody molecule is selected from any of the following:

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

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

Furthermore, the humanized antibody molecule also includes a humanized antibody constant region.

Further, the humanized antibody constant region includes a humanized antibody heavy chain constant region and a humanized antibody light chain constant region, and the humanized antibody heavy chain constant region is human IgG1 type, IgG2 type or IgG4 type. A kind of constant region, the amino acid sequence of the constant region of the IgG1 type is shown in SEQ ID No: 38, the amino acid sequence of the constant region of the IgG2 type is shown in SEQ ID No: 39, and the amino acid sequence of the IgG4 type constant region is shown in SEQ ID No: 38. The amino acid sequence of the constant region is as shown in SEQ ID No: 40, and the humanized antibody light chain constant region is a human Ck type constant region with the amino acid sequence as shown in SEQ ID No: 41.

Furthermore, the humanized antibody molecule is a full-length antibody or an antibody fragment, and the humanized antibody molecule includes one or several combinations of Fab, F(ab)2, Fv or ScFv.

The present invention also provides a protein comprising the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof.

The invention also provides a polynucleotide molecule encoding the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof.

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

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

Preferably, the host cell is a mammalian cell, and the mammalian cell is HEK293 cell, CHO cell or NSO cell.

The present invention also provides a medicament comprising the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof.

The invention also provides the use of the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof in the preparation of drugs for the treatment of immune diseases or cancer;

Preferably, the cancer includes glioma, melanoma, colorectal cancer, kidney cancer, lung cancer, lymphoma or leukemia;

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

The present invention further provides the use of the anti-Siglec-15 monoclonal antibody or its antigen-binding fragment in combination with an anti-PD-1 monoclonal antibody for the treatment of cancer or immune diseases.

The beneficial effects of the present invention are as follows: the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof provided by the present invention can specifically bind to Siglec-15, block the binding of Siglec-15 to cell surface receptors, and inhibit intracellular signaling pathways. conduction, and achieve the effect of inhibiting tumor growth, used to treat cancer or immune diseases. Cancers include but are not limited to brain glioma, melanoma, colorectal cancer, kidney cancer, lung cancer, lymphoma or leukemia, etc., immune Diseases include, but are not limited to, psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis, and autoimmune hepatitis.

Description of drawings

Figure 1 is the plasmid map of the pScFv-Disb-HS vector in Example 2 of the present invention;

Figure 2 is a comparative diagram of the affinity of gradient dilution ELISA anti-Siglec-15 phage monoclonal antibodies in Example 3 of the present invention;

Figure 3 is a map of the vector pTSE in Example 5 of the present invention;

Figure 4 is a denaturing polyacrylamide gel electrophoresis pattern of mouse antibody molecules in Example 5 of the present invention;

Figure 5 is a comparison diagram of the binding capabilities of mouse antibody molecules and Siglec-15 in Example 6 of the present invention;

Figure 6 is a comparative diagram of the binding ability of mouse-derived antibodies to inhibit Siglec-15 and Jurkat cell surface receptors in Example 7 of the present invention;

Figure 7 is a comparative diagram of the binding ability of mouse-derived antibody molecules to inhibit Siglec-15 and CHOSLV-LRRC4C cell surface receptors in Example 8 of the present invention;

Figure 8 is a graph showing the promotion of human TNF-α cytokine release by mouse antibody molecules in Example 9 of the present invention;

Figure 9 is a graph showing the promotion of human IFN-γ cytokine release by mouse antibody molecules in Example 9 of the present invention;

Figure 10 is a graph showing the promotion of T cell activation and proliferation by mouse antibody molecules in Example 9 of the present invention;

Figure 11 is a graph showing the molecular biological activity detection of mouse-derived antibodies in Example 10 of the present invention;

Figure 12 is a denaturing polyacrylamide gel electrophoresis pattern of the humanized antibody molecule in Example 15 of the present invention;

Figure 13 is a comparison chart of the binding abilities of humanized antibody molecules and Siglec-15 in Example 18 of the present invention;

Figure 14 is a diagram of cross-binding experiments between humanized antibodies and Siglec-15 of different species in Example 19 of the present invention;

Figure 15 is a diagram showing the ability of humanized antibody molecules to inhibit the binding of Siglec-15 to Jurkat cell surface receptors in Example 20 of the present invention;

Figure 16 is a biological activity detection chart of the humanized antibody molecule in Example 21 of the present invention;

Figure 17 is a graph showing the tumor volume growth curve of the anti-Siglec-15 monoclonal antibody HA-I in the MC38-Siglec-15 colorectal cancer model in mice in Example 22 of the present invention;

Figure 18 is a thermal stability evaluation chart of anti-Siglec-15 monoclonal antibody HA-I in Example 23 of the present invention.

Detailed ways

In order to make it easier to understand the present invention, before describing the embodiments, some technical and scientific terms of the present invention are explained as follows:

The term "antibody" used herein includes whole antibodies and any antigen-binding fragments thereof. Antibodies include murine antibodies, humanized antibodies, bispecific antibodies or chimeric antibodies. Antibodies can also be Fab, F(ab)2 , Fv or ScFv (single chain antibody), the antibody can be a naturally occurring antibody or an antibody that has been modified (such as mutation, deletion, substitution, etc.).

The terms "variable region" and "constant region" used herein refer to the sequence region of the antibody heavy chain and light chain near the N segment as the variable region (V region), and the remaining amino acid sequences near the C segment are relatively stable and are The constant region (C region) and variable region include 3 complementarity determining regions (CDR) and 4 framework regions (FR). Each light chain variable region and heavy chain variable region have 3 CDR regions and 4 It consists of three FR regions. The three CDR regions of the heavy chain are represented by HCDR1, HCDR2 and HCDR3 respectively. The three CDR regions of the light chain are represented by LCDR1, LCDR2 and LCDR3 respectively.

The term "mouse-derived antibody molecule" used herein is derived from antibodies obtained after immunizing mice with Siglec-15 antigen.

The term "chimeric antibody molecule" used herein is an antibody formed by fusing the variable region of a murine antibody with the constant region of a humanized antibody, which can reduce the immune response induced by the murine antibody in the human body. Chimeric antibodies use DNA recombinant technology to insert the light and heavy chain variable region genes of mouse monoclonal antibodies into an expression vector containing the human antibody constant region. The variable regions of the light and heavy chains in the expressed antibody molecules are mouse-derived. , while the constant region is of human origin, nearly 2/3 of the entire antibody molecule is of human origin. The antibody produced in this way reduces the immunogenicity of the mouse-derived antibody while retaining the ability of the parent antibody to specifically bind to the antigen. .

The term "humanized antibody molecule" used herein refers to transplanting the CDR of a mouse monoclonal antibody into the variable region of a human antibody, replacing the CDR of the human antibody, so that the human antibody obtains the antigen-binding specificity of the murine monoclonal antibody. sex while reducing its heterogeneity.

The term "CHO cells" refers to Chinese hamster ovary cells; the term "HEK293E cells" refers to human embryonic kidney 293E cells (human embryonic kidney 293E cells); and the term "NS0 cells" refers to mouse NS0 thymoma cells.

The present invention will be further described in detail below in conjunction with the following examples.

Example 1

Embodiment 1 of the present invention provides an anti-Siglec-15 monoclonal antibody or an antigen-binding fragment thereof, including a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three HCDR1 and HCDR2 and the heavy chain complementarity determining region represented by HCDR3, the light chain variable region includes three light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3 respectively. Specifically, the anti-Siglec-15 monoclonal antibody or its antigen-binding fragment is selected from Any of the following.

Example 2 Screening of mouse antibody molecules

The present invention immunizes mice with Siglec-15 antigen (Siglec-15 protein extracellular segment, the Siglec-15 antigen and proteins used in subsequent experiments are all Siglec-15 extracellular segment), optimizes the immunization method, creates a phage display library and Establish an antigen site screening method. The construction, screening and identification of specific phage display libraries are as follows:

Step 1: Siglec-15 antigen immunization of mice

1. Experimental animals:

Species and strain: BALB/c, female, mouse;

Weight: 18-20g;

Experimental animal provider: Beijing Huafukang Biotechnology Co., Ltd.

2. Immunization: Mice were immunized. The immune antigen was human Siglec-15 (a gene synthesized by Nanjing Genscript Biotechnology Co., Ltd., and our company constructed the vector and expressed and purified it).

Step 2: Construction of phage antibody library

Take the mouse splenocytes with higher titers, use Trizol reagent (purchased from Ambion, product number: 15596026), extract the total RNA in the mouse splenocytes, obtain cDNA by RT-PCR, use cDNA as the template, and use degenerate primers ( The degenerate primers used (reference: Journal of Immunological Methods 233 (2000) 167-177) were used for PCR amplification to obtain the immunized mouse antibody heavy chain variable region gene library (VH) and light chain variable region gene library (VL). ), the light and heavy chains were double-digested respectively, and connected to the vector that had been digested in the same step-by-step manner to construct the pScFv-Disb-HS-VH-VL gene library. The pScFv-Disb-HS vector was cloned using a series of gene cloning methods. The vector pComb3 vector (purchased from China Plasmid Vector, Strain and Cell Line Gene Collection Center) was modified and used for the construction and expression of phage single chain antibody library. The modified vector was named pScFv-Disb-HS vector, and its plasmid map was obtained as shown in Figure 1. Based on this vector, a mouse immune phage antibody library was constructed.

Step 3: Use Siglec-15 as the antigen to coat the immune tube. The antigen coating amount is 5 μg/500 μL/tube. Coat overnight at 4°C. Then use 4% skim milk powder/PBST to seal the immune tube and the immune phage antibody library at room temperature. Closed for 1 hour. The blocked immune phage antibody library is added to the immune tube for antigen-antibody binding. The input amount of phage is about 10 ⁹ to 10 ^12. After reacting at room temperature for 1 hour, use PBST-PBS to wash away the unbound phage, and pass it through 0.1M pH2.2 Glycine-HCl elution, and finally use 1.5M Tris-HCl pH 8.8 to neutralize the eluted phage antibody solution to about pH 7.0.

Step 4: Infect 10 ml of TG1 bacterial liquid grown to the logarithmic phase with the above neutralized phage, let it stand for 30 minutes in a 37°C incubator, take out part of the bacterial liquid for gradient dilution, and spread it on a 2YTAG plate for counting phages. output. Centrifuge the remaining bacterial liquid and discard the supernatant, resuspend the bacterial pellet in a small amount of culture medium, aspirate and spread on a 2YTAG large plate to prepare for the next round of screening.

Step 5: Scrape the bacteria plated after the above infection from the large plate, inoculate them into 2YTAG liquid culture medium, shake to the logarithmic phase, add M13KO7 helper phage for superinfection, and cultivate overnight at 28°C and 220rpm for preparation. Phages, PEG/NaCl sedimentation purified phages were used for the next round of screening, and a total of one round of phage library enrichment screening was performed.

Step 6: Screening of Siglec-15 phage single-chain antibody positive clones: After one round of screening, select well-separated single clone colonies and inoculate them into a 96-well deep-well plate with 2YTAG liquid medium and incubate at 37°C. The cells were cultured to the logarithmic growth phase at 220 rpm. About 10 ¹⁰ helper phages M13KO7 were added to each well, and the infection was carried out statically at 37°C for 30 min. Centrifuge at 4000 rpm for 15 min, discard the supernatant, resuspend the bacterial pellet in 2YTAK, and culture overnight at 28°C and 220 rpm. After centrifugation for 15 minutes at 4000 rpm and 4°C, the amplified phage supernatant was collected for ELISA identification and final screening. Four mouse-derived antibody molecules with higher affinity against Siglec-15 were obtained, named MA-I, MA-II, MA-III and MA-IV respectively. The monoclonal antibodies obtained above were genetically sequenced to confirm that they were correct. Antibody sequences, after sequencing, the sequences of the four monoclonal antibodies screened above are as follows:

Specifically, SEQ ID No: 25 (amino acid sequence of heavy chain variable region of MA-I)

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

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

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

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

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

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

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

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

The four mouse antibody molecules (MA-I, MA-II, MA-III and MA-IV) obtained in Example 2 were displayed and purified by monoclonal phage, and then a phage gradient dilution ELISA experiment was performed to determine the affinity. Control Antibodies are from Nescor Anti-SIGLEC-15 monoclonal antibody (also known as 5G12, patent application number is 201780067999.3, patent name is antibody against SIGLEC-15 and its use method), the specific method is as follows:

Coat Siglec-15 antigen with pH 9.6 carbonate buffer, 100ng/well/100μL, coat overnight at 4°C, wash three times with PBST, and combine the four phage singles screened in Example 2. The cloned antibodies were diluted five-fold in PBST, and 100 μl of the diluted sample was added to each well and left to stand at room temperature for 1 hour. Wash the ELISA plate with PBST, add HRP-anti-M13 (purchased from Bio-viewshine, product number: GE27-9421-01) monoclonal antibody diluted in PBST to the ELISA plate, and leave it at room temperature for 1 hour. TMB color development kit develops color at room temperature for 10 minutes. After terminating with 2M H ₂ SO ₄ , the microplate reader reads at 450nm/630nm and calculates the corresponding EC50 value. The specific data are as follows:

According to the above data and as shown in Figure 2, the four different mouse antibody molecules screened in Example 2 can all bind to Siglec-15, and the monoclonal antibodies provided by the present invention all have high affinity with Siglec-15.

Example 4

On the basis of Example 2, Example 4 of the present invention further defines that the murine antibody molecule also includes a heavy chain constant region and a light chain constant region, and the heavy chain constant region is murine IgG1 type, IgG2a type, IgG2b type or IgG3 type. A type of constant region, the light chain constant region is a murine C _k type constant region with an amino acid sequence shown in SEQ ID No: 33, and an IgG1 type constant region has an amino acid sequence shown in SEQ ID No: 34, IgG2a type The amino acid sequence of the constant region of IgG2b is shown in SEQ ID No: 35, the amino acid sequence of the constant region of IgG2b is shown in SEQ ID No: 36, and the amino acid sequence of the constant region of IgG3 is shown in SEQ ID No: 37; specifically The sequence is as follows:

SEQ ID No: 33 (light chain constant region amino acid sequence of mouse C _k -type):

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

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

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

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

Example 5 Preparation of anti-Siglec-15 monoclonal antibody murine antibody molecules

On the basis of Example 4, Example 5 of the present invention preferably defines that the murine antibody molecules include the heavy chain constant region of murine IgG1 type (the amino acid sequence of which is shown in SEQ ID No: 34) and the light chain of murine C _k type. chain constant region (the amino acid sequence of which is shown in SEQ ID No: 33). The antibody preparation method is as follows:

1. Clone the heavy chain VH and light chain VL coding genes of the four monoclonal antibodies screened out in Example 2 respectively into the vector pTSE containing the heavy chain and light chain constant region genes (as shown in Figure 3), The preferred heavy chain constant region is a murine IgG1 type constant region (the amino acid sequence is shown in SEQ ID No: 34), the light chain constant region is a murine C _k chain (the amino acid sequence is shown in SEQ ID No: 33), pTSE The vector structure is shown in Figure 3 (for the preparation process of pTSE vector, please refer to paragraph [0019] on page 3 of the CN103525868A specification).

2. Transiently transfect HEK293E cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Cat. No. GNHu43) for antibody expression. Use AKTA instrument to purify through protein A affinity column to obtain 4 monoclonal antibodies. At the same time, use BCA kit ( Purchased from: Beijing Huitian Oriental Technology Co., Ltd., Catalog No.: BCA0020), the protein concentration was measured, and then the protein size was identified by SDS-PAGE. The results are shown in Figure 4, from left to right, non-reducing MA-I, MA-II, MA-III, and MA-IV, protein molecular weight Marker1, protein molecular weight Marker2, and reduced MA-I, MA-II, MA-III, and MA-IV mouse anti-Siglec-15 monoclonal antibodies, each band The molecular weight is consistent with theory.

Example 6 Binding experiment of mouse antibody and Siglec-15

Coat Siglec-15 antigen with pH 9.6 carbonate buffer, 100ng/well/100μL, and coat overnight at 4°C. Wash five times with 300 μL/well PBST, then add 1% BSA-PBST and block for 1 hour at 37°C. Add MA-I, MA-II, MA-III and MA-IV mouse antibody molecules at different dilution concentrations. The initial maximum concentration of the four antibody molecules was 5 μg/ml. Each antibody was diluted 5 times in a gradient. Each antibody was diluted in a total of 8 gradients and incubated at 37°C for 1 hour. Wash five times with 300 μL/well PBST, then add Goat Anti-Mouse IgG-HRP (purchased from solarbio, product number: SE131) diluted with 1% BSA-PBST 1:2000, and incubate at 37°C for 1 hour. Develop color with TMB color development kit, 100 μL/well, develop color at room temperature for 8 minutes, then use 2M H ₂ SO ₄ to terminate color development. The microplate reader reads at 450nm/630nm and calculates the corresponding EC50 value. The specific data are as follows:

Based on the above data and as shown in Figure 5, the four different mouse antibody molecules screened out can all bind to Siglec-15.

Example 7 Murine-derived antibodies inhibit the binding of Siglec-15 to Jurkat cell surface receptors

First, four types of mouse antibodies (MA-I, MA-II, MA-III, MA-IV) and control antibody 5G12 were prepared into a protein solution with a concentration of 600 μg/mL, and added to a 96-well plate. 25μL. Secondly, prepare Siglec-15 ligand protein at a concentration of 200 μg/mL, 25 μL per well, and add it to a 96-well plate. Again, count the Jurkat cell line, take a certain number of cells, centrifuge and resuspend in PBS buffer, adjust the cell density to 2E+6 cells/mL, and add 50 μL per well to a 96-well plate. All sample and protein dilutions were performed using PBS buffer. Finally, place the loaded 96-well plate at 4°C and incubate for 1 hour. After removal, add 100 μL PBS buffer to each well, centrifuge at 3000 rpm to wash the cells once, and collect the cell pellet. Add the pre-prepared dilution of AF488-anti human IgG-Fc antibody (purchased from SouthemBiotech, product number 2048-30) to the cell pellet, and incubate at 4°C for 1 hour. After taking it out, wash it once at 3000 rpm, resuspend it in 200 μL PBS, and put it on a flow cytometer for detection, and collect the fluorescence signal in the FL1-A channel.

The results are shown in Figure 6. The four mouse-derived antibody molecules screened in Example 2 of the present invention can all inhibit the binding of Siglec-15 to Jurkat cell surface receptors, and are equivalent to the control antibody 5G12 at the same concentration.

Example 8 Murine antibodies inhibit the binding of Siglec-15 to CHOSLV-LRRC4C cell surface receptors

Gradient dilution of four mouse antibody molecules (MA-I, MA-II, MA-III, MA-IV) and control antibody 5G12, the preparation concentration is 200μg/mL, 3x gradient dilution, a total of 8 gradients, 25μL is added to each well The corresponding position of the 96-well plate. Dilute the Siglec-15-Fc ligand protein to prepare a concentration of 40 μg/mL, and add 25 μL to each well into the corresponding position of the 96-well plate. Count the CHOSLV-LRRC4C cell line, take a certain number of cells, centrifuge and resuspend in PBS buffer, adjust the cell density to 2E+6 cells/mL, and add 50 μL per well to a 96-well plate. All sample and protein dilutions were performed using PBS buffer. Place the loaded 96-well plate at 4°C and incubate for 1 hour. After removal, add 100 μL PBS buffer to each well, centrifuge at 3000 rpm to wash the cells once, and collect the cell pellet. Add the pre-prepared AF488-anti human IgG-Fc antibody (purchased from SouthernBiotech, product number 2048-30) to the cell pellet, incubate at 4°C for 1 hour, wash once at 3000 rpm, resuspend in 200 μL PBS, perform flow cytometry detection, and collect FL1 Fluorescence signal in -A channel. A dose-effect curve was drawn to calculate the candidate molecule's ability to inhibit the binding of Siglec-15 ligand protein to the LRRC4C receptor on the cell surface.

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

Example 9 Mouse-derived antibodies promote T cell activation and proliferation

Resuscitate human PBMC cells (peripheral blood mononuclear cell, PBMC), collect the cell pellet after centrifugation, resuspend and count in RPMI1640 complete medium, adjust the cell density to 2E+6 cells/mL, and add 50 μL per well to a 96-well plate. Siglec-15 ligand protein was prepared at a concentration of 20 μg/mL, and 50 μL per well was added to the corresponding position of the 96-well plate. The final concentration of Anti-CD3 antibody is 0.5 μg/well, the preparation concentration is 10 μg/mL, and 50 μL per well is added to the corresponding position in the 96-well plate. Four mouse-derived molecules (MA-I, MA-II, MA-III, MA-IV) and control antibody 5G12 were prepared with a starting concentration of 100 μg/mL and 3x gradient dilution, with a total of 8 gradients. 50 μL per well was added to the corresponding position in the 96-well plate. Proteins and antibodies were diluted using RPMI1640 complete medium, mixed in a 96-well plate, and incubated at 37°C in the dark for 3 days. Take the cell culture supernatant, dilute it 10 times, and use it for cytokine detection. The activation of natural T cells by anti-Siglec-15 mouse antibody molecules is evaluated and considered from the following three perspectives: human TNF-α cytokine release, human IFN-γ cytokine release, and T cell proliferation. The specific operations are as follows:

Human IFN-γ detection kit (purchased from Ecosai Biotechnology Co., Ltd., product number: H008-96): Combine the diluted supernatant and standard Add the standard product into the sample wells, 100 μL per well, cover with sealing film, and incubate at room temperature for 1.5 hours. After incubation, wash the plate 3 times. Add the Biotinylated antibody diluent in the human IFN-γ detection kit, 100 μL per well, cover with sealing film and incubate at room temperature for 1 hour. After incubation, wash the plate 3 times. Add Streptavidin-HRP working solution, 100 μL per well, cover with sealing film, and incubate at room temperature for 30 minutes. After incubation, wash the plate 3 times. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for about 15 minutes. Add 100 μL of Stop solution to each well to terminate the reaction. After reading the OD value with a microplate reader, draw a dose-effect curve.

Human TNF-α detection kit (purchased from Ecosai Biotechnology Co., Ltd., product number: EM008-96): Add the diluted supernatant and standard to the sample wells, 100 μL per well, cover with sealing film, and incubate at room temperature for 1.5 h. After incubation, wash the plate 3 times. Add Biotinylated antibody diluent, 100 μL per well, cover with sealing film and incubate at room temperature for 1 hour. After incubation, wash the plate 3 times. Add 100 μL of Streptavidin-HRP working solution in the human IFN-γ detection kit to each well, cover with sealing film, and incubate at room temperature for 30 minutes. After incubation, wash the plate 3 times. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for about 15 minutes. Add 100 μL of Stop solution to each well to terminate the reaction. After reading the OD value with a microplate reader, draw a dose-effect curve.

Collect the cell pellet, stain the cells with CD3e Monoclonal Antibody (purchased from Thermo Fisher Scientific Co., Ltd., product number MA1-10177), incubate at room temperature for 15 minutes in the dark, wash the plate once, resuspend in 100 μL PBS buffer, and perform flow cytometry Perform absolute counting on the machine and draw a dose-effect curve.

Human TNF-alpha cytokine release

Human IFN-γ cytokine release

From the above data and Figures 8-10, it can be seen that the four mouse antibody molecules (MA-I, MA-II, MA-III, and MA-IV) can block the interaction between Siglec-15 ligand protein and Siglec-15. The binding of natural T cell surface receptors blocks intracellular inhibitory signaling pathways, activates T cells, and promotes T cell proliferation and activation (release of TNF-α and IFN-γ).

Example 10 Detection of molecular biological activity of mouse-derived antibodies (reporter gene method)

Count the Jurkat-NFAT-Luc engineered cell line, use sample diluent (its ingredients include 90% RPMI1640, 10% FBS, 0.5 μg/ml Puromycin) to adjust the cell density to 2E+6 cells/mL, mix gently and then divide the cells The solution was added to a 96-well plate, 50 μL/well. Four mouse-derived molecules (MA-I, MA-II, MA-III, MA-IV) were diluted to an initial concentration of 800 μg/mL using sample diluent, 5-fold gradient dilution, a total of 8 gradients, 50 μL/well, Add to the corresponding position of the 96-well plate, and set two duplicate wells for each sample concentration. Prepare Siglec-15 antigen, 50 μL per well, was added to the 96-well plate to make the final concentration reach 16 μg/mL. Prepare Human CD3antibody (purchased from Yiqiao Shenzhou Biotechnology Co., Ltd., product number: 10977-H001), add 50 μL per well into a 96-well plate, and adjust the concentration to 1 μg/mL. Gently mix the cell culture plate and place it in a 37°C _CO2 incubator for 6 hours. Centrifuge and discard the supernatant, add lysis solution, add 10 μL from each well to a 384-well plate, add an equal amount of luciferase reaction substrate (purchased from Promega Biotechnology Co., Ltd., product number: E2610), and react at room temperature for 5 minutes. Read the fluorescence value under a microplate reader and calculate the corresponding IC50 value. The specific data are as follows:

Based on the above data and shown in Figure 11, the 4 different mouse antibodies and control antibodies screened out can all bind Siglec-15, inhibit the binding of Siglec-15 to Jurkat cell surface receptors, and block intracellular inhibitory signals. pathway, reactivating T cells. The construction of the engineered cell line Jurkat-NFAT-Luc can simulate T cells. Siglec-15 inhibits and downregulates the intracellular activation signaling pathway (NFAT-Luc) by binding to T cell surface receptors. These four mouse-derived antibody molecules can effectively block the binding of Siglec-15 to cell surface receptors and reactivate signaling pathways in T cells.

Example 11

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

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

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

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

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

Example 12 Preparation of chimeric antibody molecules

On the basis of Example 11, Example 12 of the present invention further defines that the humanized antibody constant region includes a human IgG1 type heavy chain constant region (the amino acid sequence of which is shown in SEQ ID No: 38) and a human _Ck type. Light chain constant region (the amino acid sequence of which is shown in SEQ ID No: 41).

Specific preparation method:

The heavy chain variable region VH (SEQ ID No: 25) and light chain variable region VL gene (SEQ ID No: 26) of the antibody molecule MA-I obtained by screening the immune phage antibody library in Example 2 maintain the mouse sequence. unchanged, cloned into the vector pTSE (shown in Figure 3) containing heavy chain constant region and light chain constant region genes respectively. The heavy chain constant region is human IgG1 type (the amino acid sequence is shown in SEQ ID NO: 38) , the light chain constant region is human C _k type (the amino acid sequence is shown in SEQ ID NO: 41). HEK293E cells (purchased from: Institute of Basic Medicine, Chinese Academy of Medical Sciences, product number: GNHu43) were transiently transfected, and antibody expression was performed to obtain chimeric antibody CA-I.

Example 13 Humanization of mouse antibody molecules

First, select the sequence of the mouse antibody molecule MA-1 in Example 2 and compare it with the human antibody germline database (v-base) to find the human antibody light and heavy chain germline with higher homology as candidate sequences, and then The CDR sequence of the mouse antibody molecule MA-I was transplanted to the human candidate sequence for homology modeling. Then three-dimensional structural simulation is used to calculate the key framework amino acid residues that may play an important role in maintaining the CDR loop structure, thereby designing back mutations of the humanized antibody. The designed light and heavy chain variable region sequences of humanized antibodies containing reverse mutations were optimized and synthesized by Nanjing Genscript Biotechnology Co., Ltd., and then connected to a transient expression vector. Chain combination analysis resulted in the following humanized antibody molecules: HA-I, HA-II. The sequences of the two monoclonal antibodies screened above are as follows:

Specifically, SEQ ID No: 42 (amino acid sequence of the heavy chain variable region of HA-I and HA-II):

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

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

Example 14

On the basis of Example 13, Example 14 of the present invention further defines that the humanized antibody molecule also includes a humanized antibody constant region; the humanized antibody constant region includes a humanized IgG1 type, an IgG2 type, or an IgG4 type. The heavy chain constant region and the light chain constant region of the human C _k type, the amino acid sequence of the heavy chain constant region of the IgG1 type is shown in SEQ ID No: 38, and the amino acid sequence of the heavy chain constant region of the IgG2 type is shown in SEQ ID No: 39 , the amino acid sequence of the heavy chain constant region of the IgG4 type is shown in SEQ ID No: 40, and the amino acid sequence of the light chain constant region of the human C _k type is shown in SEQ ID No: 41.

The specific sequence of the constant region of the above-mentioned humanized antibody is the same as that in Example 11.

Example 15 Preparation of humanized antibody molecules

On the basis of Example 10, Example 15 of the present invention further defines that the humanized antibody constant region includes the human IgG1 type heavy chain constant region (the amino acid sequence of which is shown in SEQ ID No: 38) and the human C _k type. The light chain constant region (the amino acid sequence of which is shown in SEQ ID No: 41).

The heavy chain VH and light chain VL coding genes of the two humanized antibody molecules obtained by humanization in Example 13 were cloned into the vector pTSE containing the heavy chain constant region and light chain constant region genes respectively (as shown in Figure 3 ), the heavy chain constant region is human IgG1 type (the amino acid sequence is shown in SEQ ID NO: 38), and the light chain constant region is the C _k chain (the amino acid sequence is shown in SEQ ID NO: 41).

The humanized antibody molecules HA-I and HA-II were transiently transfected into HEK293 cells (purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences, Cat. No. GNHu43), and the antibodies were expressed and purified through protein A affinity columns using AKTA instruments. Monoclonal antibodies were used to measure the protein concentration using a BCA kit (purchased from: Beijing Huitian Oriental Technology Co., Ltd., Cat. No.: BCA0020), and then the protein size was identified by SDS-PAGE. The results are shown in Figure 12, from left to On the right side are the non-reduced protein molecular weight HA-I, HA-II, the chimeric antibody CA-I prepared in Example 12, the non-reduced protein molecular weight Marker1 and the reduced protein molecular weight Marker2, HA-I, HA-II, chimeric Antibody CA-I, the molecular weight of each band is consistent with the theory.

Example 16

Embodiment 16 of the present invention further defines that the humanized antibody molecule is a full-length antibody or an antibody fragment, and the humanized antibody molecule includes one or more combinations of Fab, F(ab)2, Fv or ScFv.

Example 17

The present invention also provides a protein comprising the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof as defined in the above embodiment.

The present invention also provides a polynucleotide molecule encoding the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof as defined in the above embodiments.

The invention also provides a recombinant DNA expression vector, which contains the above-mentioned polynucleotide molecule.

The invention also provides a host cell transfected with the above recombinant DNA expression vector. The host cell includes prokaryotic cells, yeast cells, insect cells or mammalian cells;

Preferably, the host cell is a mammalian cell, and the mammalian cell is HEK293 cells, CHO cells or NSO cells.

The present invention also provides a medicine, which contains the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof as defined in the above embodiments of the present invention.

The invention also provides anti-Siglec-15 monoclonal antibodies or antigen-binding fragments thereof for preparing drugs for treating immune diseases or cancer. Applications in;

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

Preferably, the above-mentioned cancer includes brain glioma, melanoma, colorectal cancer, kidney cancer, lung cancer, lymphoma or leukemia, etc.;

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

The present invention further provides the use of anti-Siglec-15 monoclonal antibodies or antigen-binding fragments thereof in combination with anti-PD-1 monoclonal antibodies for the treatment of cancer or immune diseases.

The anti-PD-1 monoclonal antibody is selected from Nivolumab, Pembrolizumab, toripalimab, sintilimab, tislelizumab, camrelizumab or the patent number is ZL201510312910.8, and the patent name is DFPD1-9, DFPD1-10, DFPD1-11, DFPD1-12 or DFPD1-13 anti-PD-1 monoclonal antibodies disclosed in patent documents of an anti-PD-1 monoclonal antibody and a method for obtaining the same, here are not limited to The above restrictions on anti-PD-1 monoclonal antibodies can also apply to other commercialized anti-PD-1 monoclonal antibodies used in experiments. As long as the target is PD-1, any other monoclonal antibodies are not specifically limited here. of anti-PD-1 monoclonal antibodies.

Example 18 Binding experiment of humanized antibody molecules and Siglec-15

Coat Siglec-15 antigen with pH 9.6 carbonate buffer, 200ng/well/100μL, and coat overnight at 4°C. Wash five times with 300 μL/well PBST, then add 1% BSA-PBST and block for 1 hour at 37°C. Add different dilution concentrations of humanized antibodies HA-I, HA-II and the chimeric antibody prepared in Example 12. For antibody CA-I, the initial maximum concentration of the three antibodies was 50 μg/mL. After 3-fold dilution, 10 gradients were made for each antibody and incubated at 37°C for 1 hour. Wash five times with 300 μL/well PBST, then add Goat Anti Human IgG-HRP (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., product number: ZB-2304) diluted with 1% BSA-PBST 1:5000, and incubate at 37°C. Incubate under conditions for 1 hour. Develop color with TMB color development kit, 100 μL/well, develop color at room temperature for 5 minutes, then use 2M H2SO4 to terminate color development. The microplate reader reads at 450nm/630nm and calculates the corresponding EC50 value. The specific data are as follows:

Based on the above data and experimental results, as shown in Figure 13, two different humanized antibody molecules can bind to Siglec-15, and the EC50 values of the two humanized antibody molecules are close to those of the chimeric antibody CA-I. This shows that the humanized antibody molecule retains the high binding ability of the mouse parent antibody MA-I and Siglec-15.

Example 19 Cross-binding experiments between humanized antibodies and Siglec-15 of different species

Human Siglec-15, mouse Siglec-15-His (purchased from Nearshore Protein Technology Co., Ltd., product number: CW71), and cynomolgus monkey Siglec-15-His (purchased from Nearshore Protein Technology Co., Ltd., Cat. No.: CW70) 100ng/well/100μL, coated overnight at 4°C. Wash five times with 300 μL/well PBST, then add 1% BSA-PBST and block for 1 hour at 37°C. Add different dilution concentrations of humanized antibodies HA-I and HA-II. The starting point of 2 humanized antibodies The highest concentration was 25 μg/mL. After 5-fold dilution, each antibody was divided into 8 gradients and incubated at 37°C for 1 hour. Wash five times with 300 μL/well PBST, then add Goat Anti Human IgG-HRP diluted 1:5000 with 1% BSA-PBST, and incubate at 37°C for 1 hour. Develop color with TMB color development kit, 100 μL/well, develop color at room temperature for 5 minutes, then use 2M H ₂ SO ₄ to terminate color development. The microplate reader reads at 450nm/630nm and calculates the corresponding EC50 value. The specific data are as follows:

Based on the above data and as shown in Figure 14, the two different humanized antibody molecules screened out can bind to human Siglec-15, cynomolgus monkey Siglec-15, and mouse Siglec-15.

Example 20 Humanized antibody molecules inhibit the binding of Siglec-15 to Jurkat cell surface receptors

Gradient dilution of two humanized antibody molecules (HA-I, HA-II) and control antibody 5G12, the preparation concentration is 200 μg/mL, 5 gradient dilutions, a total of 8 gradients, 25 μL per well is added to the corresponding position of the 96-well plate. Siglec-15 protein was fluorescently labeled using a FITC fluorescent labeling protein kit (purchased from Thermo Fisher Scientific Co., Ltd., product number: F6434) to prepare Siglec-15-FITC protein. Use PBS buffer to adjust the Siglec-15-FITC protein concentration. The prepared concentration is 40 μg/mL. Add 25 μL per well to the corresponding position of the 96-well plate. Count the Jurkat cell line, take a certain number of cells, centrifuge and resuspend in PBS buffer, adjust the cell density to 2E+6 cells/mL, and add 50 μL per well to a 96-well plate. All sample and protein dilutions were performed using PBS buffer. Place the loaded 96-well plate at 4°C and incubate for 1 hour. After removal, add 100 μL PBS buffer to each well, centrifuge and wash the cells once at 3000 rpm, collect the cell pellet, resuspend it in 200 μL PBS, run it on a flow cytometer for detection, and collect the fluorescence signal in the FL1-A channel. A dose-effect curve was drawn to calculate the candidate molecule's ability to inhibit the binding of Siglec-15 ligand protein to Jurkat cell surface receptors.

It can be seen from the above data and Figure 15 that both humanized candidate molecules (HA-I and HA-II) can block the binding of Siglec-15 to receptors on the surface of Jurkat cells.

Example 21 Biological activity detection of humanized antibody molecules (reporter gene)

Count the Jurkat-NFAT-Luc engineered cell line, use sample diluent (its ingredients include 90% RPMI1640, 10% FBS, 0.5 μg/ml Puromycin) to adjust the cell density to 2E+6 cells/mL, mix gently and then divide the cells The solution was added to a 96-well plate, 50 μL/well. Two humanized antibody molecules (HA-I, HA-II) were diluted with sample diluent to an initial concentration of 800 μg/ml, 5-fold gradient dilution, a total of 8 gradients, 50 μL/well, and added to the corresponding position of the 96-well plate. , set two duplicate wells for each sample concentration. Prepare Siglec-15 antigen and add 50 μL per well into a 96-well plate to reach a final concentration of 16 μg/mL. Prepare anti-CD3 antibody (purchased from Yiqiao Shenzhou Biotechnology Co., Ltd., product number: 10977-H001), add 50 μL per well into a 96-well plate, and adjust the concentration to 1 μg/mL. Gently mix the cell culture plate and place it in a 37°C _CO2 incubator for 6 hours. Centrifuge and discard the supernatant, add lysis solution, add 10 μL from each well to a 384-well plate, add an equal amount of luciferase reaction substrate (purchased from Promega Biotechnology Co., Ltd., product number: E2610), and react at room temperature for 5 minutes. Read the fluorescence value under a microplate reader and calculate the corresponding IC50 value. The specific data are as follows:

Based on the above data and shown in Figure 16, the two screened humanized antibody molecules can both bind Siglec-15 and inhibit the binding of Siglec-15 to Jurkat cell surface receptors, blocking the intracellular inhibitory signaling pathway and reactivating it. T cells.

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

1. Experimental animals:

Species and strain: C57BL/6JGpt, mouse;

Age: 6-8 weeks;

Experimental animal provider: Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

2. Cell culture:

MC38 tumor cells (YK-CL-256-02) (purchased from: BiovectorNTCC Inc., product number: NTCC-MC38) are original cells to construct MC38-Siglec-15 tumor cells strain. The serum containing inactivated 10% fetal calf serum (ExCell Bio, Cat. No.: FND500), 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 μg/mL Hygromycin B (purchased from: Thermo Fisher Scientific ( China) Co., Ltd. (Gibco), Catalog No.: 10687010) and 2mM glutamine DMEM medium (purchased from: Thermo Fisher Scientific (China) Co., Ltd. (Gibco), Catalog No.: 10566-016) at 37°C, Tumor cells were cultured in an incubator with 5% _CO2 . The cells were divided into bottles and passaged every 3 to 4 days after the cells had reached full growth. The tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.

Bone marrow-derived macrophages (BMDM) were isolated from C57BL/6 mice. Using inactivated 10% fetal bovine serum (ExCell Bio, Cat. No.: FND500), 100 U/mL penicillin, 100 μg/mL streptomycin and 20 ng/mL mouse M-CSF (purchased from: Beijing Yiqiao Shenzhou Technology Co., Ltd. Company, Catalog No.: 51112-MNAH) and 20ng/mL mouse IL-10 (Purchased from: Beijing Yiqiao Shenzhou Technology Co., Ltd., Catalog No.: 50245-MNAE) in RPMI 1640 culture medium (Purchased from: Thermo Fisher Scientific (China) Co., Ltd. (Gibco, Cat. No.: A10491-01) can be used for in vivo tumor models after culturing for 4 days in an incubator at 37°C and 5% _CO2 .

3. Inoculation and grouping of tumor cells:

MC38-Siglec-15 tumor cells resuspended in PBS at a cell density of 1.0×10 ⁶ /mL were mixed evenly with a certain amount of BMDMs cell suspension, and inoculated subcutaneously into the right flank of experimental animals, 100 μL/animal, in When the tumors grow to about ^43mm3 , they are divided into two groups, with 5 animals in each group, namely vehicle control group (Vehicle, ip, tiw×3w) and HA-1 (10mg/kg, ip, tiw×3w).

4. Detection indicators: Use vernier calipers to measure the tumor volume twice a week to measure the long and short diameters of the tumors. The volume calculation formula is: volume = 0.5 × long diameter × short diameter ² ; record the changes in tumor volume and The experimental results of the relationship between administration time are shown in Figure 17.

The data in Figure 17 show that anti-Siglec-15 monoclonal antibody HA-1 can inhibit tumor growth in a dose-dependent manner.

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

The thermal stability of anti-Siglec-15 monoclonal antibody HA-I was evaluated using a multifunctional protein thermal stability analysis system (purchased from Unchained Labs). By monitoring the change of protein endogenous fluorescence with temperature (starting from 25°C and heating up to 95°C at a heating rate of 0.3°C/min), the changes in protein conformation are detected to determine the protein melting temperature Tm and evaluate the protein conformational stability. When the sample aggregates, the scattered light waves will interfere and the scattered light signal will increase. The colloidal stability of the protein (characterized by Tagg) is measured through static light scattering. The results are shown in the table below and Figure 18.

The temperature of anti-Siglec-15 monoclonal antibody HA-I is 72.9°C, and the average Tagg is 72.0°C, showing good conformational stability and colloidal stability.

The present invention is not limited to the above-mentioned best embodiment. Anyone can produce various other forms of products under the inspiration of the present invention. However, regardless of any changes in its shape or structure, any product with the same or similar properties as the present invention can be made. Similar technical solutions all fall within the protection scope of the present invention.

Claims (18)

1. An anti-Siglec-15 monoclonal antibody or an antigen-binding fragment thereof, characterized in that it includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes three heavy chain complementarity determining regions HCDR1 and HCDR2 and HCDR3, the light chain variable region includes three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, and the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof is selected from any of the following:

   A-I: The amino acid sequence of the HCDR1 is shown in SEQ ID No: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID No: 2, the amino acid sequence of the HCDR3 is shown in SEQ ID No: 3, the LCDR1 The amino acid sequence of LCDR2 is shown in SEQ ID No: 4, the amino acid sequence of LCDR2 is shown in SEQ ID No: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID No: 6;

   A-II: The amino acid sequence of HCDR1 is shown in SEQ ID No: 7, the amino acid sequence of HCDR2 is shown in SEQ ID No: 8, and the amino acid sequence of HCDR3 is shown in SEQ ID No: 9, so The amino acid sequence of LCDR1 is shown in SEQ ID No: 10, the amino acid sequence of LCDR2 is shown in SEQ ID No: 11, and the amino acid sequence of LCDR3 is shown in SEQ ID No: 12;

   A-III: The amino acid sequence of HCDR1 is shown in SEQ ID No: 13, the amino acid sequence of HCDR2 is shown in SEQ ID No: 14, and the amino acid sequence of HCDR3 is shown in SEQ ID No: 15, so The amino acid sequence of LCDR1 is shown in SEQ ID No: 16, the amino acid sequence of LCDR2 is shown in SEQ ID No: 17, and the amino acid sequence of LCDR3 is shown in SEQ ID No: 18;

   A-IV: The amino acid sequence of HCDR1 is shown in SEQ ID No: 19, the amino acid sequence of HCDR2 is shown in SEQ ID No: 20, and the amino acid sequence of HCDR3 is shown in SEQ ID No: 21, so The amino acid sequence of LCDR1 is shown in SEQ ID No: 22, the amino acid sequence of LCDR2 is shown in SEQ ID No: 23, and the amino acid sequence of LCDR3 is shown in SEQ ID No: 24.
2. The anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof is a mouse-derived antibody molecule, and the mouse-derived antibody molecule Choose from any of the following:

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

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

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

   MA-IV: 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 or antigen-binding fragment thereof according to claim 2, wherein the murine antibody molecule further includes a heavy chain constant region and a light chain constant region, and the heavy chain constant region is murine IgG1 type, IgG2a type, A kind of constant region of IgG2b type or IgG3 type. The light chain constant region is a murine Ck type constant region with an amino acid sequence as shown in SEQ ID No: 33. The amino acid sequence of the IgG1 type constant region is as SEQ. ID No: 34, the amino acid sequence of the constant region of the IgG2a type is shown in SEQ ID No: 35, the amino acid sequence of the constant region of the IgG2b type is shown in SEQ ID No: 36, the IgG3 type The amino acid sequence of the constant region is shown in SEQ ID No: 37.
4. The anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody molecule, and the chimeric antibody molecule It includes the heavy chain variable region of the murine antibody molecule, the light chain variable region of the murine antibody molecule and the humanized antibody constant region.
5. The anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof is a humanized antibody molecule, and the humanized antibody Molecules are selected from any of the following:

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

   HA-II: The amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 42, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 44.
6. The anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to claim 5, wherein the humanized antibody molecule further includes a humanized antibody constant region.
7. The anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to claim 4 or 6, wherein 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 is one of human IgG1 type, IgG2 type or IgG4 type constant region, the amino acid sequence of the IgG1 type constant region is shown in SEQ ID No: 38, so The amino acid sequence of the constant region of the IgG2 type is shown in SEQ ID No: 39, the amino acid sequence of the constant region of the IgG4 type is shown in SEQ ID No: 40, and the amino acid sequence of the humanized antibody light chain constant region is The constant region of the human Ck type as shown in SEQ ID No: 41.
8. The anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to claim 5, wherein the humanized antibody molecule is a full-length antibody or an antibody fragment, and the humanized antibody molecule includes Fab, F (ab) One or more combinations of ₂ , Fv or ScFv.
9. A protein, characterized in that it contains the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6.
10. A polynucleotide molecule, characterized in that the polynucleotide molecule encodes the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6.
11. A recombinant DNA expression vector, characterized in that the recombinant DNA expression vector contains the polynucleotide molecule of claim 10.
12. A host cell transfected with the recombinant DNA expression vector according to claim 11, wherein the host cell includes a prokaryotic cell, yeast cell, insect cell or mammalian cell;

    Preferably, the host cell is a mammalian cell, and the mammalian cell is HEK293 cell, CHO cell or NSO cell.
13. A medicine, characterized in that the medicine contains the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6.
14. The use of the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in the preparation of drugs for the treatment of immune diseases or cancer;

    Preferably, the cancer includes glioma, melanoma, colorectal cancer, kidney cancer, lung cancer, lymphoma or leukemia;

    The immune diseases include psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis or autoimmune hepatitis.
15. The anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 is used in combination with an anti-PD-1 monoclonal antibody for the treatment of cancer or immune diseases.
16. The anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 for treating immune diseases or cancer;

    Preferably, the cancer includes glioma, melanoma, colorectal cancer, kidney cancer, lung cancer, lymphoma or leukemia;

    The immune diseases include psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis or autoimmune hepatitis.
17. A method for treating immune diseases or cancer, characterized by comprising:

    Administering an effective amount of the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 to a subject, wherein the subject suffers from an immune disease or cancer;

    Preferably, the cancer includes glioma, melanoma, colorectal cancer, kidney cancer, lung cancer, lymphoma or leukemia;

    The immune diseases include psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, ulcerative colitis or autoimmune hepatitis.
18. The method according to claim 17, characterized in that: administering to the subject an effective amount of the anti-Siglec-15 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6 and anti-PD-1 Monoclonal antibodies.