Antagonist targeting Galectin-8 and application thereof

Abstract

The invention belongs to the technical field of tumor marker detection, and particularly relates to a Galcitn-8 targeting antagonist and application thereof. The invention aims to provide an antagonist which can be combined with Galectin-8, has a function of blocking the combination of Galectin-8 and LILRB4 and comprises an antibody or an antigen binding fragment, an amino acid sequence of a complementary determining region of the antibody, nucleic acid and amino acid sequences of a variable region of the antibody, an amino acid sequence of a whole molecule of the antibody, a method for preparing the anti-Galectin-8 antibody, and a method for preparing the anti-Galectin-8 antibody. The invention relates to an anti-Galectin-8 antibody and application thereof in inhibiting the binding reaction of LILRB4 and Galectin-8 protein and regulating the immunosuppression function of MDSC, and shows the treatment value of the anti-Galectin-8 antibody in treating tumors, autoimmune diseases, transplant rejection, anaphylaxis, infection and other diseases, and an ELISA kit based on the anti-Galectin-8 antibody and used for detecting human Galectin-8.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor marker detection, and specifically relates to an antagonist targeting Galecitn-8 and an application thereof, as well as an ELISA kit for detecting human Galectin-8. Background Art

[0002] Galectins are a class of proteins that bind to specific glycans or glycosylated molecules. Previous studies have revealed that they play an important role in anti-infection immunity and in mediating cell autophagy when the endothelium is damaged (Jia, Abudu et al. 2018, Weng, Chen et al. 2018). Some of them have been found to be associated with tumor growth and tumor microenvironment regulation (Limagne, Richard et al. 2019, Yang, Sun et al. 2021). For example, Galectin 9 has been reported to promote an increase in the proportion of MDSCs in transplant reactions, thereby suppressing immunity (Yin, Li et al. 2020). Studies have shown that galectins produced by tumors are one of the main molecular mechanisms for tumors to evade immune control and affect different steps in establishing anti-tumor immune responses. The role of Galectin-8 in regulating immune function, including inflammatory response, is controversial (Tribulatti, Carabelli et al. 2020). Current studies have shown that Galectin-8 has anti-inflammatory effects in Toxoplasma infection (Bertelli, Sanmarco et al. 2020), and in tumors, it reduces cell stickiness and promotes tumor metastasis by promoting CD166 endocytosis (Renard, Tyckaert et al. 2020).

[0003] Galectin-8 was found to bind to the monocyte immune checkpoint protein LILRB4, promoting the expansion of myeloid-derived suppressor cells (MDSC) through downstream signaling pathways, thereby inducing a tumor immunosuppressive microenvironment and promoting tumor growth. Galectin-8 was also found to be highly expressed in a variety of tumors, such as breast cancer and colorectal cancer. Analysis of clinical samples showed that Galectin-8 expression was associated with poor prognosis such as lymph node spread and shortened disease-related survival.

[0004] However, the functional discovery of Galectin-8 is still in the early stages of research. Currently, no studies have shown the regulatory function of Galectin-8 on MDSC and the tumor immune microenvironment, and there is no drug that treats tumors by targeting Galectin-8. The limited understanding of its function and its characteristic that it is generally considered to be a free protein have limited its discovery as a therapeutic target. At the same time, as the functional research of Galectin-8 in tumor immune regulation has gradually deepened, there is currently a lack of an effective method for detecting human Galectin-8 protein in biological samples. Other members of the Galectin family, such as Galectin-3, Galectin-9 and Galectin-10, have commercial ELISA detection kits. The lack of Galectin-8 detection kits has become one of the factors currently limiting Galectin-8 research. Summary of the invention

[0005] The purpose of the present invention is to provide an antagonist for regulating the binding of Galectin8 to LILRB4 and its application, as well as an ELISA kit for detecting human Galectin-8, so as to meet the needs of practical applications.

[0006] The present invention innovatively targets Galectin-8 and provides a compound antagonist that can block the binding of LILRB4 to Galectin-8, thereby blocking the induction of MDSC and the display of the inhibitory phenotype, and then regulating the immunosuppressive function to achieve the purpose of activating anti-tumor immunity.

[0007] The antagonist for blocking the binding of Galectin-8 and LILRB4 provided by the present invention comprises an antibody or an antigen-binding fragment;

[0008] The CDRs of the antibody or antigen-binding fragment comprise three light chain CDRs and / or three heavy chain CDRs, wherein:

[0009] The light chain CDR is selected from the polypeptides whose amino acid sequences are as follows: SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20;

[0010] The heavy chain CDR is selected from the polypeptides whose amino acid sequences are as follows: SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23;

[0011] The antagonist binds to Galectin-8 and is able to block the binding of Galectin-8 to LILRB4.

[0012] The present invention also provides an amino acid sequence of the compound antagonist, wherein the amino acid sequence comprises an amino acid sequence constituting a light chain variable region and / or an amino acid sequence constituting a heavy chain variable region; wherein:

[0013] The amino acid sequences of the light chain variable regions are shown in SEQ ID NO: 1 and SEQ ID NO: 5;

[0014] The amino acid sequences of the heavy chain variable region are shown in SEQ ID NO:2 and SEQ ID NO:6.

[0015] The present invention also provides nucleotides for expressing the antagonist, wherein the nucleotides encode the antibody or antigen-binding fragment, and the nucleotide sequence of the antibody or antigen-binding fragment comprises a nucleotide sequence encoding a light chain variable region and / or a nucleotide sequence encoding a heavy chain variable region; wherein:

[0016] The nucleotide sequences of the genes encoding the light chain variable regions are shown in SEQ ID NO:3 and SEQ ID NO:7;

[0017] The nucleotide sequences of the genes encoding the heavy chain variable regions are shown in SEQ ID NO:4 and SEQ ID NO:8.

[0018] The present invention also provides a method for expressing an antibody or antigen-binding fragment protein containing the compound antagonist in a eukaryotic cell, the specific steps of which are:

[0019] (1) optimizing the light chain and heavy chain sequences of the protein by eukaryotic expression to obtain a nucleotide sequence;

[0020] (2) inserting the light chain nucleotide sequence obtained in step (1) into a eukaryotic expression vector or a viral packaging vector, and inserting the heavy chain nucleotide sequence into a eukaryotic expression vector or a viral packaging vector. After transfection, collect the supernatant and perform protein purification.

[0021] The present invention also provides a method for preparing a Galectin-8 targeted compound antagonist, the specific steps of which are as follows:

[0022] (1) Immunizing mice with Galectin-8 antigen protein emulsified with CFA (complete Freund's adjuvant);

[0023] (2) harvesting mouse spleen cells, identifying and sequencing the heavy and light chains, and using sequencing data to analyze CDRs;

[0024] (3) using immune libraries to screen antigenic proteins through phage expression display;

[0025] (4) After optimizing the light chain and heavy chain sequences of the screened antibody clones for eukaryotic expression, the light chain nucleotide sequence was inserted into pcDNA3.1-IgK, and the heavy chain nucleotide sequence was inserted into pcDNA3.1-IgG1Fc to construct an expression vector;

[0026] (5) The above expression vector was transfected into 293 cells, and the supernatant was collected after 7-072 hours; the monoclonal antibody was purified by a standard purification method.

[0027] The present invention also provides an expression vector of the above-mentioned compound antagonist, which is constructed in the following manner:

[0028] (1) Prokaryotic codon optimization based on the nucleic acid sequence of Galectin-8;

[0029] (2) constructing into a prokaryotic expression vector by fusion method, the vector includes PGEX-6p-1, expression vectors PET28 and PET30 of PET system, but not limited to these vectors;

[0030] (3) The recombinant plasmid was identified by double enzyme digestion method, and the digestion products were detected by 1.0% agarose gel electrophoresis.

[0031] The present invention also provides the use of the above-mentioned compound antagonist and its expression vector in the preparation of drugs for inhibiting tumors or regulating immune responses, as well as the use of the above-mentioned compound antagonist and its expression vector in the preparation of companion diagnostic reagents or kits for related drugs.

[0032] The present invention also provides a method for using the above-mentioned compound antagonist to detect Galectin-8 content by ELISA, the specific steps of which are:

[0033] An ELISA plate is coated with a targeted Galectin-8 antagonist as described above, and another compound antagonist as described above is labeled with biotin as a primary antibody. The two compound antagonists specifically bind to free Galectin-8 protein in a sandwich format, and the signal intensity is displayed using a biotin-bound secondary antibody.

[0034] The present invention also provides an ELISA kit that can conveniently and accurately detect Galectin-8, and the detection sensitivity and specificity are very high. The kit comprises the aforementioned compound antagonist and also comprises a biotin-labeled compound antagonist, and the biotin-labeled compound antagonist is prepared by the following method:

[0035] (1) concentrating the antibody protein to a specified concentration by ultrafiltration;

[0036] (2) The protein obtained in step (1) is co-incubated with a biotin marker under appropriate conditions, and then excess biotin is washed away to obtain a biotin-labeled antibody protein.

[0037] The ELISA kit also includes a standard Galectin-8 protein, whose amino acid sequence is shown in SEQ ID NO:9 and whose encoding nucleotide sequence is shown in SEQ ID NO:10.

[0038] The ELISA kit can detect the following:

[0039] (1) Detect the concentration of human galectin-8 in liquid samples such as serum, cell culture supernatant, and cell lysate;

[0040] (2) Detect the concentration of Galectin-8 protein samples with unknown concentration.

[0041] The present invention aims to propose Galectin-8 as a factor that promotes immunosuppressive function and has the function of being a therapeutic target, and provides a targeted Galectin-8 compound antagonist that blocks the binding of Galectin-8 and LILRB4, wherein the compound antagonist comprises an antibody or an antigen binding fragment, wherein the antibody or antigen binding fragment comprises an amino acid sequence of an antibody CDR region, a variable region nucleic acid and an amino acid sequence; the present invention provides a method for preparing a targeted Galectin-8 compound antagonist, and its use in inhibiting the binding reaction of LILRB4 and Galectin-8 protein and regulating the immunosuppressive function of MDSC, showing the target value of anti-Galectin-8 antibodies in the treatment of tumors, autoimmune diseases, transplant rejection reactions, allergic reactions, infections and other diseases.

[0042] The present invention also provides a method for preparing Galectin-8 protein, the specific steps of which are:

[0043] (1) Prokaryotic codon optimization of Galectin-8 gene sequence;

[0044] (2) constructing a recombinant vector containing the Galectin-8 gene sequence;

[0045] (3) expressing the Galectin-8 protein;

[0046] (4) Isolation, purification and identification.

[0047] Wherein: the expression system can be a bacterial, yeast, insect or mammalian cell expression system.

[0048] The production method includes conventional microbial fermentation production, and expression and production in bacteria, yeast, insect, and mammalian cell expression systems using bioengineering technology.

[0049] The present invention provides two compound antagonists targeting Galectin-8, the compound antagonists include antibodies or antigen-binding fragments, the amino acid sequences of the light chains of the antibodies or antigen-binding fragments are shown in SEQ ID NO.1 and SEQ ID NO.5, and the amino acid sequences of the heavy chains are shown in SEQ ID NO.2 and SEQ ID NO.6. Correspondingly, the nucleic acid sequences of the light chains are shown in SEQ ID NO.3 and SEQ ID NO.7, and the nucleic acid sequences of the heavy chains are shown in SEQ ID NO.4 and SEQ ID NO.8, and the compound antagonists competitively bind to Galectin-8 with the above antibodies or antigen-binding fragments, and can block the binding of Galectin-8 to LILRB4.

[0050] In Example 4, the present invention discloses the binning relationship of the binding epitopes of the compound antagonists targeting Galectin-8, and in Example 6, discloses the compound antagonists having blocking function and similar binding epitopes.

[0051] The present invention provides a drug for inhibiting the biological function produced by the combination of LILRB4 and ligand Galectin-8, wherein the drug contains the above-mentioned Galectin-8-targeted compound antagonist.

[0052] In Example 9, the present invention discloses that the compound antagonist has the function of blocking MDSC induction in vitro.

[0053] In Example 11 and Example 12, the present invention discloses that the compound antagonist has an inhibitory effect on tumor growth in mice when used alone or in combination with PD-L1 antibody.

[0054] The present invention provides the use of a compound antagonist in the preparation of a tumor diagnostic reagent or therapeutic drug for inhibiting the function of Galectin-8. The compound antagonist comprises the above-mentioned antibody or antigen-binding fragment.

[0055] In Example 13, the present invention discloses a method for preparing an ELISA kit comprising the compound antagonist, a biotin-labeled compound antagonist and a standard Galectin-8 protein and its application.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] Currently, the development of tumor immunity research and treatment methods faces many challenges, among which the focus on MDSC is a new hot issue in this field. There are no mature drugs for the MDSC-induced targets that have been discovered so far. Galectin-8 has been found to bind to the monocyte immune checkpoint protein LILRB4 and promote the expansion of MDSC through downstream signaling pathways. However, due to the lack of knowledge about the function of Galectin-8 and its characteristics of free protein, the application of related antibody research using Galectin-8 as a target in detection and drug development remains to be explored.

[0058] At present, there are no reports on related blocking functions and clear blocking of LILRB4 ligand Galectin-8. The targeted Galectin-8 antagonist disclosed in the present invention has the effect of combining and blocking MDSC induction, thus having high specificity and functionality.

[0059] Compared with the existing technology, the technology related to this application has the outstanding advantage that the value of Galectin-8 as a therapeutic target is demonstrated through the development of targeted Galectin-8 antagonists. At the same time, the disclosed ELISA detection kit has the potential value of companion diagnosis, which can quickly and accurately detect the Galectin-8 content in biological samples, screen tumors with high expression of Galectin-8, screen out potential effective populations, and provide a basis for the subsequent clinical development of drugs.

[0060] Although the specific antibody sequences provided by the present invention cannot represent all antagonist structures that target Galectin-8 to block LILRB4, the present application clearly defines a class of antagonists that are novel, creative, and practical by targeting the specific functions of the target. Inspired by the content of the present application, the antagonists developed with Galectin-8 as the target and having the function of blocking immunosuppression are essentially the same class as the antagonists disclosed in the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The enzyme-linked immunosorbent assay is used to perform preliminary affinity comparison analysis and screening of multiple antibody clones. Each point represents an antibody clone, and the horizontal and vertical axes are the binding signal intensities of all antibody clones with human and monkey Galectin-8 proteins, respectively. The black bold data points in the upper right quadrant are the screened antibody clones with high affinity for both antigens and sequence differences.

[0062] Figure 2Gator and ELISA were used to analyze the epitopes, blocking functions and affinity of multiple antibody clones. Figure A is a network diagram of epitope similarity between antibodies with different numbers after processing the epitope binning detection data using Cytoscape; Figure B is an ELISA test of the ability of representative antibodies to block Galectin-8 and LILRB4 binding; Figures C, D, E, and F are the affinity test curves and affinity constant values ​​of two antibodies A237 and A269 with strong blocking ability with human / monkey Galectin-8 protein, respectively.

[0063] Figure 3 The expression of Galectin-8 in human tumors and the in vitro function of A269 are detected. Figures A and B are flow cytometric analysis and statistical analysis of human tumor cell lines with different Galectin-8 expression levels stained with A269 antibody; Figure C is a flow cytometric cell clustering diagram, showing the effect of A269 antibody on the phenotypic changes of M-MDSC induced by Galectin-8 in PBMC; Figures D and E are representative images and case statistics of the intensity classification evaluation of Galectin-8 expression in human melanoma tissues; Figures F and G are representative images and case statistics of the intensity classification evaluation of Galectin-8 expression in human colorectal cancer tissues; Figures H and I are representative images and case statistics of the intensity classification evaluation of Galectin-8 expression in human breast cancer tissues.

[0064] Figure 4 The results of using A269 antibody alone to treat mixed transplants of various human tumor cells and human PBMC in immunodeficient mice. Figures A and B are the tumor growth curves and in vitro tumor sizes of transplants formed by mixing human melanoma cells A375 and human PBMC after A269 antibody treatment; Figures C and D are the tumor growth curves and in vitro tumor sizes of transplants formed by mixing human colorectal cancer cells SW480 and human PBMC after A269 antibody treatment; Figures E and F are the tumor growth curves and in vitro tumor sizes of transplants formed by mixing human colorectal cancer cells HCT116 and human PBMC after A269 antibody treatment.

[0065] Figure 5 The results of using A269 antibody alone or in combination with PD-L1 antibody to treat transplanted tumors in wild-type mice. Figure A is a tumor growth curve; Figure B is an in vitro tumor image and weight statistics; Figures C, D, and E are immunohistochemical staining of tumor tissue sections, showing the levels of infiltrating CD8-positive cells and FOXP3-positive cells in the tumor microenvironment; Figure F is a mouse weight change curve, showing the weight changes of mice in different groups.

[0066] Figure 6The present invention is an ELISA kit for detecting the level of human Galectin-8 protein in biological samples such as cell supernatants. Figure A shows the schematic diagram of the ELISA kit; Figure B shows the standard curve and fitting parameters made with recombinant human Galectin-8 protein standard; Figure C shows the results of using the kit to analyze the expression level of Galectin-8 protein in the supernatants of various human tumor cell cultures. DETAILED DESCRIPTION

[0067] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0068] The experimental methods without specific conditions in the examples were carried out according to conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning-A Laboratory Manual) (3rd Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight, and the materials and reagents used are all commercially available products.

[0069] Example 1: Production process of Galectin-8 protein

[0070] Galectin-8 sequence from Uniprot:O00214

[0071] By genetic engineering, the target gene is connected to the prokaryotic expression vector by universal biological gene synthesis after prokaryotic codon optimization, and then transformed into Escherichia coli for expression. The purified Galectin-8 protein is obtained by affinity chromatography, ion exchange chromatography, molecular sieve chromatography and ultrafiltration; the methods include:

[0072] 1.1 Production methods and processes

[0073] 1.1.1 Construction strategy of Galectin-8 gene recombinant plasmid

[0074] According to the gene sequence, after codon optimization and gene synthesis, it is directionally cloned into the prokaryotic expression vector commonly used in the industry, such as PGEX-6p-1; the expression vectors PET28 and PET30 of the PET system, but not limited to these vectors. The commonly used multiple cloning restriction sites are selected to insert the target gene into the expression vector to construct a recombinant plasmid.

[0075] 1.1.2 Directional cloning and identification of recombinant plasmids of target genes

[0076] The nucleic acid sequence of Galectin-8 was synthesized after prokaryotic codon optimization, constructed into PGEX-6p-1 vector by fusion method, and the recombinant plasmid was identified by double restriction method, and the restriction products were detected by 1.0% agarose gel electrophoresis.

[0077] 1.2.1 Prokaryotic expression of Galectin-8

[0078] 1.2.1.1 Recombinant plasmid transformation

[0079] The expression host bacteria selected in this experiment is E. coli BL21 (DE3), which is a protein expression host for highly efficient expression of exogenous genes mediated by T7 RNA polymerase. The Galectin-8 recombinant plasmid verified by sequencing was transformed into BL21 (DE3) competent cells. The steps are as follows:

[0080] (1) Take 100 μl of competent cells from a -80°C refrigerator, thaw in an ice bath, add 1 μl of recombinant plasmid, flick to mix, and place in an ice bath for 30 min;

[0081] (2) Heat shock at 42°C in a water bath for 90 seconds, then quickly transfer to ice and place for 3–5 minutes;

[0082] (3) Add 500 μl of sterile LB liquid medium (without antibiotics) and culture at 37°C, 200 rpm, with shaking for 30 min;

[0083] (4) Take 200 μl of the bacterial solution and spread it on LB agar medium containing 100 μg / mL ampicillin antibiotics, invert the plate, and culture it at 37°C overnight.

[0084] 1.2.1.2 Protein induction expression

[0085] BL21 (DE3) containing Galectin-8 recombinant plasmid and empty vector were selected as controls, single colonies were picked and inoculated into 5 mL LB medium (containing 10-200 μg / mL ampicillin), respectively, and cultured at 37°C, 200 rpm with shaking until the OD value was 0.6-0.8, IPTG (final concentration 0.1-1 mM) was added to the culture solution, and cultured at 16°C, 200 rpm with shaking for 12 h to induce the expression of the target gene Galectin-8.

[0086] 1.2.1.3 Purification of recombinant protein Galectin-8

[0087] The induced bacteria were collected by centrifugation, crushed by high pressure and then centrifuged at high speed to collect the supernatant. The recombinant protein Galectin-8 was purified by three steps of chromatography and one step of ultrafiltration. All purification operations were carried out at 4°C.

[0088] A. Nickel column purification

[0089] Use Ni2+-NTA affinity chromatography column to purify the target protein. First, add 3 column volumes of 0.5M NaOH to wash the column (column regeneration); then wash the column with 3-5 column volumes of Lysis Buffer (column balancing); after balancing, add the supernatant of the previous step to the column. When the liquid is almost gone, elute with Wash Buffer (containing 25mM imidazole) and Elution Buffer (containing 300mM imidazole), respectively, collect the eluate from the Elution Buffer, and prepare the collected target protein for ion exchange chromatography purification;

[0090] B. Ion exchange chromatography

[0091] The elution sample was diluted and loaded onto a cationic chromatography column Sourse S10 (cytiva), and gradient eluted with 0.01-1M NaCl. After elution, the sample was identified by SDS-page gel, collected, ultrafiltered, and then subjected to molecular sieve chromatography;

[0092] C. Molecular sieve chromatography

[0093] After cation exchange chromatography, the samples were loaded onto Superdex 200 (cytiva), and samples were collected according to the sample collection peak and SDS-page gel identification results; the target protein was ultrafiltered and purified using a 10kD ultrafiltration tube; the concentration of the target protein was detected using Nano2000, and the purified samples were stored at -80°C.

[0094] Example 2: Preparation of Galectin-8 Antibody

[0095] 2.1 Animal immunization

[0096] Mice were immunized with Galectin-8 antigen protein emulsified with CFA (complete Freund's adjuvant). The mice were stimulated with the same immunogen once a week. The serum collected from tail bleeding was diluted 1000 times, 100,000 times, and 100,000,000 times in an ELISA plate coated with Galectin-8 protein to evaluate the antiserum titer. After 7 weeks of immunization, blood was collected again in the eighth week, and the dose was increased in the ninth week.

[0097] 2.2 Sequencing library construction

[0098] Mouse spleen cells were harvested for library construction and sequencing, and RACE (rapid amplification of cDNA ends) identification of heavy and light chains was performed according to the following protocol: (1) mRNA denaturation, (2) cDNA synthesis, (3) 5' RACE reaction, (4) analysis of PCR results (observation of amplified DNA fragments on agarose gel - the size of the correct antibody variable region DNA fragment should be between 500-700 base pairs, (5) TOPO cloning PCR positive bands; (6) PCR amplification of TOPO clones, followed by gel electrophoresis and recovery from agarose gel, (7) a total of 218 clones were measured, and (8) CDR analysis was performed using sequencing data (VBASE2 available through abysisvbase2.org was used to define the CDR region). The sequencing results of antibodies A269 and A371 are as follows.

[0099] A269 light chain variable region nucleic acid sequence: SEQ ID No.7

[0100] A269 light chain variable region amino acid sequence: SEQ ID No.5

[0101] A269 light chain CDR1: RASESVDSYGNSFMH (SEQ ID NO.12)

[0102] A269 light chain CDR2: RASNLES (SEQ ID NO.13)

[0103] A269 light chain CDR3: QQSNEDPYT (SEQ ID NO. 14)

[0104] A269 heavy chain variable region nucleic acid sequence: SEQ ID No. 8;

[0105] A269 heavy chain variable region amino acid sequence: SEQ ID No.6,

[0106] A269 heavy chain CDR1: GYTFTDY (SEQ ID NO. 15)

[0107] A269 heavy chain CDR2: FPGGDY (SEQ ID NO. 16)

[0108] A269 heavy chain CDR3: RYSHWNFDV (SEQ ID NO. 17)

[0109] A371 light chain variable region nucleic acid sequence: SEQ ID No.3

[0110] A371 light chain variable region amino acid sequence: SEQ ID No.1

[0111] A371 light chain CDR1: KASQNVGTNVA (SEQ ID NO. 18)

[0112] A371 light chain CDR2: SASYRYS (SEQ ID NO. 19)

[0113] A371 light chain CDR3: QQYNSYPYT (SEQ ID NO. 20)

[0114] A371 heavy chain variable region nucleic acid sequence: SEQ ID No.4

[0115] A371 heavy chain variable region amino acid sequence: SEQ ID No.2

[0116] A371 heavy chain CDR1: GYTFTTY (SEQ ID NO. 21)

[0117] A371 heavy chain CDR2: NPRNGR (SEQ ID NO. 22)

[0118] A371 heavy chain CDR3: DYRFGYALDY (SEQ ID NO. 23).

[0119] 2.3 Affinity screening

[0120] Later, the immune library is used to screen the antigen protein through phage expression display. Figure 1 As shown, each data point represents the affinity of an antibody clone to human and cynomolgus macaque Galectin-8 antigens. The antibody clones in the upper right quadrant have strong affinity to both antigens. Among the antibody sequences with higher affinity, the sequence similarity is analyzed and differentiated molecules are selected as much as possible, which are marked with black and bold points.

[0121] Example 3: Expression and purification of Galectin-8 antibody

[0122] 3.1 Vector construction

[0123] After the light chain and heavy chain sequences of A269 and A371 were optimized for eukaryotic expression, the light chain nucleotide sequence was inserted into pcDNA3.1-IgK, and the heavy chain nucleotide sequence was inserted into pcDNA3.1-IgG1Fc to construct expression vectors.

[0124] 3.2 Cell transfection

[0125] Transfect into 293 cells and collect the supernatant after 72 hours.

[0126] 3.3 Protein purification

[0127] Monoclonal antibodies were purified by standard purification methods (Tian Di Ren He, Jiangsu, China).

[0128] Example 4: Gator adsorption assay for antibody clustering

[0129] Prepare the Gator detection instrument, place the hFc probe in the max plate with 256μl equilibration solution added to each well, dilute the anti-Galectin-8 antibody to 5μg / mL as the loading protein, dilute the recombinant Galectin-8 protein to 5μg / mL as the binding antigen, and then dilute the detection antibodies to 5μg / mL and add them in sequence. Set up the program, first balance and regenerate the probe, then determine the first baseline, balance the baseline again after loading the protein, and then bind the antigen protein. After the antigen protein is obviously bound, place the probe in different antibodies in turn and observe the binding of the second antibody. If there is still obvious binding, the antibody epitope does not have a competitive relationship. If the binding is affected, the antibody and the loading antibody have an epitope competition relationship. According to the offset value of the second binding curve, draw a network diagram of the epitope similarity between different antibodies, such as Figure 2 As shown in A. Among them, the antibodies with similar epitopes to antibody A269 (No. 34) and competitive relationships are A264 (No. 33), A281 (No. 37), A291 (No. 38), A294 (No. 40), and A53 (No. 47). Their CDR sequences are as follows:

[0130]

[0131]

[0132] Example 5: Biotin labeling of proteins

[0133] Galectin-8 recombinant protein or A269 antibody protein was concentrated to a concentration of 2 mg / mL using a 10 kDa ultrafiltration tube (Millipore, MA, USA), and then the protein was labeled using a biotin labeling kit (Elarerite, Wuhan, China) according to the instructions. The labeled protein was added with glycerol (final concentration 50%) and stored at −20 °C.

[0134] Example 6: ELISA showed that antibodies can block the binding of Galectin-8 and LILRB4

[0135] First, dilute the LILRB4-his protein to 0.5 μg / mL with ELISA coating solution (Solarbio, Beijing, China), take 100 μL from each well and add it to the ELISA special plate (costar, ME, USA) for coating. The negative control is coated with 100 μL of protein-free coating solution. Coat overnight at four degrees. After washing 3 times with PBST, block with 100 μL of 5% BSA (Sangon, Wuhan, China) dissolved in PBS and block in an incubator at 37 degrees for 90 minutes. Pre-incubate the recombinant Galectin-8 protein labeled with 1.5 μg / mL biotin with different purified antibodies and incubate at 37°C for 30 minutes. After washing the blocked ELISA plate twice with PBST, add the mixture of antibody and LILRB4 protein to each well and incubate at 37°C for 60 minutes. After washing with PBST for 5 times, the cells were incubated with horseradish peroxidase-conjugated streptavidin (GenScript, Nanjing, China) diluted in PBS for binding in an incubator at 37°C for 30 min. After washing with PBST for 5 times, 100 μL of colorimetric solution (Solabo, Beijing, China) was added to each well and the cells were placed in an incubator for 10 min. 50 μL of stop solution (Sangon, Shanghai, China) was then added and the absorbance at 450 nm was read using an ELISA reader (Thermo Fisher, MA, USA). Figure 2 As shown in B, this experiment shows that the binding of the full-length Galectin-8 protein to the purified protein of the extracellular region of LILRB4 can be blocked by antibodies 26 (A237) and 34 (A269).

[0136] Example 7: Determination of antibody affinity using Gator

[0137] Prepare the Gator detection instrument, place the hFc probe into the max plate with 256μl equilibration solution added to each well, dilute different anti-Galectin-8 antibody proteins to 5μg / mL as loading proteins, set up buffer as blank control, dilute recombinant human and monkey Galectin-8 proteins to 1.25, 2.5, 5, and 10μg / mL, respectively, and add them in sequence. Set up the program, first equilibrate and regenerate the probe, then determine the first baseline, equilibrate the baseline again after loading the protein, and then combine with antibodies of different concentrations. After the antibody is obviously bound, run the probe into the next step buffer for dissociation. End the run when obvious dissociation is observed, fit the dissociation curve, and obtain the affinity constant. Figure 2 C shows the affinity test and fitting curve of antibody A237 and human Galectin-8. The calculated affinity constant KD=1.35E-09. Figure 2 D shows the affinity test and fitting curve of antibody A237 and monkey Galectin-8. The calculated affinity constant KD=1.26E-09. Figure 2E shows the affinity test and fitting curve of antibody A269 and human Galectin-8. The calculated affinity constant KD=8.47E-11. Figure 2 F shows the affinity test and fitting curve of antibody A269 and monkey Galectin-8, and the calculated affinity constant KD=2.11E-09.

[0138] Example 8: Flow cytometry demonstrated that hybridoma supernatants can bind to Galectin-8 on the cell surface

[0139] Count the cells of various tumor cell lines in culture and adjust the cell number to 1x10 6 / mL, add 200μL to 1.5mL EP tubes (Axygen, CA, USA). Add A269 antibody or isotype control IgG at a final concentration of 10μg / mL to each EP tube. Place all EP tubes on a culture shaker in a cell culture incubator and mix for 30 minutes, then take out the EP tubes, centrifuge at 400rcf for 5 minutes and discard the supernatant. Resuspend and wash the cells with 500μL cell staining buffer (Invitrogen, CA, USA), and repeat the above steps once. Dilute Alexa Fluor 647 fluorescent specific anti-human IgG antibody (Invitrogen, CA, USA) at 1:500 with cell staining buffer, add 200μL of antibody diluent to each EP tube, and incubate on a shaker at 65rpm for 30 minutes at room temperature. Centrifuge at 400rcf for 5 minutes and discard the supernatant, resuspend and wash the cells with 1000μL cell staining buffer, and repeat the above steps once. After washing, 300 μL of cell staining buffer was added to each EP tube for resuspending, and the cells were transferred to a flow cytometry tube (Falcon, NY, USA) and analyzed by BD FACS (BD, NJ, USA). Figure 3 A shows a representative peak graph of A269 antibody binding to the surface of various tumor cells. Figure 3 B is the statistical analysis of the mean fluorescence intensity of flow cytometry results.

[0140] Example 9: Anti-Galectin-8 antibodies can block the proportion of monocytes in PBMCs that differentiate into myeloid-derived suppressor cells (MDSCs) increased by Galectin-8

[0141] Resuscitated human PBMC (Sany Hematopoietic, Fujian, China) were cultured in AIM-V medium (Gibco, CA, USA). The next day, recombinant human granulocyte-monocyte colony stimulating factor (GM-CSF) (Sangong, Wuhan, China) was added, and Galectin-8 protein and A269 antibody were added at the same time. After a total of 3 days of treatment, the cells in each well were collected, washed twice with PBS, and then stained for dead cells (ZombieDye) (Biolegend, CA, USA) at room temperature in the dark for 15 minutes, centrifuged at 400g for 5 minutes, and the cells were resuspended and washed with cell staining buffer (Invitrogen, CA, USA). The above steps were repeated once. A mixture of fluorescent antibodies diluted in cell staining buffer was added, and the mixture was mixed and incubated for 30 minutes at room temperature in the dark. The antibody mixture includes anti-CD11b-APC antibody (Invitrogen, CA, USA), anti-CD33-PE antibody (Invitrogen, CA, USA), and anti-HLA-DR-APC.Cy7 antibody (Biolegend, CA, USA). After staining, the cells were washed twice with cell staining buffer, and the cells were transferred to the wells of a 96-well plate (costar, ME, USA). MACS Quant 10 (Miltenyi Biotec, Cologne, Germany) was used for flow cytometry analysis to circle the CD11b-positive cells among the living cells. In this gate, the proportion of HLA-DR-, CD33+ cells to CD11b+ cells was analyzed. This group of cells was defined as MDSC. Figure 3 As shown in C, A269 antibody reduced the proportion of MDSCs induced by Galectin-8.

[0142] Example 10: Detection of Galectin-8 expression in human tumor tissue

[0143] Tissue microarrays (Tabos, Xi'an, China) were dewaxed in xylene multiple times, hydrated with ethanol gradients, and antigen retrieval was performed with citrate. Subsequently, endogenous enzymes were blocked with 3% hydrogen peroxide for 15 minutes and blocked with goat serum for 1 hour at room temperature. Galectin-8 antibody was diluted to a working concentration and incubated with the tissue overnight at 4 degrees Celsius. The next day, after incubation with biotin-labeled secondary antibodies for 1 hour, the DAB color development kit (Biyuntian, Shanghai, China) was used for color development and hematoxylin staining.

[0144] Example 11: Anti-Galectin-8 antibodies have therapeutic effects on transplanted tumors in mice

[0145] NCG mice of the same age (Jicui Yaokang, Shanghai, China) were selected and divided into groups of 6, with two groups for each tumor. They were subcutaneously injected with 2.1E+06, 4E+06, and 1.8E+06 / mouse of human melanoma A375 cells, human colorectal cancer SW480 cells, and human colorectal cancer HCT116 cells mixed with human PBMCs, respectively. The tumor growth was observed and the size was measured. When the tumor size was measurable and growing steadily, control PBS or A269 antibody drug was given, where the dose of A269 was 5 mg / kg, once every three days, for a total of 4 times, and the tumor growth was continued to be observed until the tumor size exceeded 1000 cubic millimeters. The results of A375 tumor growth are shown in Figure 2. Figure 4 As shown in A, Figure 4 B shows the statistical analysis of in vitro tumor size and weight; SW480 tumor growth curve and in vitro tumor results are shown in Figure 4 CD shows; HCT116 tumor growth curve and in vitro tumor results are shown in Figure 4 As shown in EF, A269 antibody showed therapeutic effects in limiting tumor growth in various tumors.

[0146] Example 12: The therapeutic effect of anti-Galectin-8 antibody combined with PD-L1 antibody on transplanted tumors in mice.

[0147] 12.1 Construction of a stable cell line of mouse colorectal cancer cell line Mc38 overexpressing human Galectin-8

[0148] The full-length human Galectin-8 expression vector was transferred to Mc38 cells (ATCC, VA, USA) by lentivirus (Jima, Shanghai, China). A blank control group without transfection was set up and cultured in an incubator for 72 hours. Then, 2 μg / mL of puromycin (InvivoGen, MA, USA) was added for screening. After 3-4 days, when all the cells in the blank control group died, the puromycin was maintained to a sufficient amount. After the cells were collected and lysed, the expression level of Galectin-8 protein was detected by gel immunoblotting. After expanded culture, Mc38-Gal-8 cells overexpressing Galectin-8 were obtained for cryopreservation and subsequent experiments.

[0149] 12.2 Therapeutic effect of anti-Galectin-8 antibody combined with PD-L1 antibody on transplanted tumors in mice

[0150] Wild-type C57BL / 6 mice of the same age (Jihui, Shanghai, China) were selected and divided into groups of 8, with two groups for each tumor. They were subcutaneously injected with 1.5E+06 mouse colorectal cancer MC38-Gal-8 cells per mouse, and the tumor growth was observed and the size was measured. When the tumor size was measurable and growing steadily, control PBS or A269 antibody drug was given, where the dose of A269 was 5 mg / kg, once every three days, for a total of 4 times, and the tumor growth was continued to be observed until the tumor size exceeded 1000 cubic millimeters. The tumor growth results are shown in Figure 2. Figure 5 As shown in A, Figure 5 B shows the statistical analysis of in vitro tumor size and weight. When used alone, the A269 antibody showed a therapeutic effect in limiting tumor growth, and when used in combination with the PD-L1 antibody, the therapeutic effect on tumors was stronger than that of PD-L1 monotherapy.

[0151] 12.3 Analysis of the infiltration of tumor microenvironment in mice by anti-Galectin-8 antibody combined with PD-L1 antibody

[0152] Mouse tissues were embedded in paraffin and sectioned, and the sections were dewaxed in xylene multiple times, hydrated with ethanol gradients, and antigen retrieval was performed with citrate. Subsequently, endogenous enzymes were blocked with 3% hydrogen peroxide for 15 minutes and blocked with goat serum for 1 hour at room temperature. FOXP3 antibody (CST, MA, USA) and CD8 antibody (Abcam, MA, USA) were diluted to working concentrations and incubated at 4 degrees Celsius overnight. The next day, after incubation with biotin-labeled secondary antibodies for 1 hour, the DAB color development kit (Biyuntian, Shanghai, China) was used for color development and stained with hematoxylin (Biyuntian, Shanghai, China).

[0153] Example 13: Preparation and use of ELISA kit

[0154] 5.1 Reagent contents

[0155] Sample diluent: PBS solution containing 3% BSA;

[0156] Primary antibody diluent: PBS solution containing 3% BSA and 0.05% Tween-20;

[0157] Secondary antibody diluent: PBS solution containing 0.05% Tween-20;

[0158] Enzyme-labeled secondary antibody: horseradish peroxidase-labeled anti-human secondary antibody;

[0159] Washing solution: PBS solution containing 0.05% Tween-20;

[0160] Substrate color development solution A: 13.6g sodium acetate, 1.6g sodium sulphate, 0.3ml 30% hydrogen peroxide, add distilled water to 500ml.

[0161] Substrate color development solution B: 0.2 g disodium EDTA, 0.95 g sodium bicarbonate, 50 mL glycerol, 0.15 g TMB, add distilled water to 500 mL.

[0162] The PBS is prepared according to the conditions of NaCl 8.0 g, KCl 0.2 g, Na2HPO4 3.58 g, KH2PO4 0.24 g, and NaN3 1.0 g, the pH value is adjusted to 7.4, and the volume is fixed to 1 L.

[0163] 5.2 Construction of a kit for detecting Galectin-8 protein

[0164] 5.2.1 Coating ELISA Plates

[0165] The A371 antibody protein was diluted to 10 μg / mL with 0.05 mol / L carbonate buffer at pH 9.5, added to the coated plate, 100 μL / well, placed at 4°C overnight, washed 3 times with PBS containing 0.05% Tween-20, added with a blocking solution containing 3% BSA, and placed at 37°C for 1 hour, then the blocking solution was discarded, and the plate was naturally dried for 48 hours at room temperature with a humidity of 40% to complete the coating; the coated ELISA plate was stored at -20°C.

[0166] 5.2.2 Antibody biotin labeling

[0167] As described in Example 5

[0168] 5.3 How to use the kit

[0169] 5.3.1 Preparation of standard curve

[0170] Galectin-8 protein standard was taken and diluted with PBS to a final concentration of 4000, 2000, 1000, 500, 250, 125, and 62.5 pg / mL. 100 μL was added to each well, and 3 replicates were made for each concentration. After incubation at 37°C for 2 hours, the plate was washed 5 times with PBST, 300 μL / well each time; biotin-labeled A269 antibody was diluted to 5 μg / mL and added to 100 μL / well. After incubation at 37°C for 1 hour, the plate was washed 5 times with PBST; then HRP-conjugated streptavidin (GenScript, Nanjing, China) was added to 100 μL / well, incubated at 37°C for 0.5 hour, and the plate was washed 5 times with PBST; finally, ELISA colorimetric solution was added for color development for 10-30 minutes, and OD450 was read with an enzyme reader.

[0171] 5.3.2 Sample testing

[0172] The cell culture supernatant was centrifuged at 1000 g for 10 minutes to remove the precipitated impurities. Then the supernatant was diluted with PBS for a certain multiple and 100 μL was added to each well. Three replicates were made for each sample and incubated at 37 °C for 2 hours. The plate was washed 5 times with PBST, 300 μL / well each time. The biotin-labeled A269 antibody was diluted to 5 μg / mL and added to 100 μL / well. After incubation at 37 °C for 1 hour, the plate was washed 5 times with PBST. Then HRP-conjugated streptavidin (GenScript, Nanjing, China) was added to 100 μL / well. After incubation at 37 °C for 0.5 hour, the plate was washed 5 times with PBST. Finally, ELISA colorimetric solution was added for color development for 10-30 minutes, and OD450 was read with an enzyme reader.

[0173] The present invention is not limited to the above embodiments. Any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. An antagonist targeting Galectin-8, It is characterized in that Used to block the binding of LILRB4 to Galectin-8, thereby blocking the induction of MDSC and the display of inhibitory phenotypes, and then regulating the immunosuppressive function to activate anti-tumor immunity; specifically comprising an antibody or antigen-binding fragment; the CDR of the antibody or antigen-binding fragment comprises three light chain CDRs and / or three heavy chain CDRs; wherein: The light chain CDR is selected from the polypeptides whose amino acid sequences are as follows: SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20; The heavy chain CDR is selected from polypeptides whose amino acid sequences are as follows: SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.21, SEQ ID NO.22, and SEQ ID NO.

23.

2. An amino acid sequence of the antagonist according to claim 1, It is characterized in that The amino acid sequence comprises an amino acid sequence constituting a light chain variable region and / or an amino acid sequence constituting a heavy chain variable region; wherein: The amino acid sequences of the light chain variable regions are shown in SEQ ID NO: 1 and SEQ ID NO: 5; The amino acid sequences of the heavy chain variable region are shown in SEQ ID NO:2 and SEQ ID NO:

6.

3. A nucleotide sequence of the antagonist according to claim 1, It is characterized in that The nucleotide encodes the antibody or antigen-binding fragment, and the antibody or antigen-binding fragment nucleotide sequence comprises a nucleotide sequence encoding a light chain variable region and / or a nucleotide sequence encoding a heavy chain variable region; wherein: The nucleotide sequences of the genes encoding the light chain variable regions are shown in SEQ ID NO:3 and SEQ ID NO:7; The nucleotide sequences of the genes encoding the heavy chain variable regions are shown in SEQ ID NO:4 and SEQ ID NO:

8.

4. A method for preparing the Galectin-8 targeting antagonist as claimed in claim 1, It is characterized in that The specific steps are as follows: (1) Immunizing mice with Galectin-8 antigen protein emulsified with CFA (complete Freund's adjuvant); (2) harvesting mouse spleen cells, identifying and sequencing the heavy and light chains, and using sequencing data to analyze CDRs; (3) using immune libraries to screen antigenic proteins through phage expression display; (4) After optimizing the light chain and heavy chain sequences of the screened antibody clones for eukaryotic expression, the light chain nucleotide sequence was inserted into pcDNA3.1-IgK, and the heavy chain nucleotide sequence was inserted into pcDNA3.1-IgG1Fc to construct an expression vector; (5) The above expression vector was transfected into 293 cells, and the supernatant was collected after 70-72 hours; the monoclonal antibody was purified by standard purification methods.

5. A method for expressing a protein containing the antibody or antigen-binding fragment of the antagonist of claim 1 in a eukaryotic cell, comprising the following steps: (1) optimizing the light chain and heavy chain sequences of the protein by eukaryotic expression to obtain a nucleotide sequence; (2) inserting the light chain nucleotide sequence obtained in step (1) into a eukaryotic expression vector or a viral packaging vector, and inserting the heavy chain nucleotide sequence into a eukaryotic expression vector or a viral packaging vector. After transfection, collect the supernatant and perform protein purification.

6. An expression vector of the antagonist according to claim 1, constructed by the following method: (1) Prokaryotic codon optimization based on the nucleic acid sequence of Galectin-8; (2) constructing into a prokaryotic expression vector by fusion method, wherein the vector is PGEX-6p-1, expression vector PET28 or PET30 of PET system; (3) The recombinant plasmid was identified by double enzyme digestion method, and the digestion products were detected by 1.0% agarose gel electrophoresis.

7. Use of the antagonist according to claim 1 and the expression vector according to claim 6 in the preparation of drugs for inhibiting tumors or regulating immune responses, and in the preparation of companion diagnostic reagents or kits for related drugs.

8. A method for using the antagonist as claimed in claim 1 to detect Galectin-8 content by ELISA, comprising the following steps: One of the targeted Galectin-8 antagonists is coated on an ELISA plate, and another compound antagonist as described above is labeled with biotin as a primary antibody. The two compound antagonists specifically bind to free Galectin-8 protein in a sandwich format, and the signal intensity is displayed using a secondary antibody conjugated to biotin.

9. An ELISA kit for detecting Galectin-8, It is characterized in that The kit comprises the antagonist as claimed in claim 1, and also comprises a biotin-labeled compound antagonist, including a standard Galectin-8 protein, whose amino acid sequence is shown in SEQ ID NO: 9, and whose encoding nucleotide sequence is shown in SEQ ID NO: 10; The biotin-labeled compound antagonist is prepared by the following method: (1) concentrating the antibody protein to a specified concentration by ultrafiltration; (2) The protein obtained in step (1) is co-incubated with a biotin marker under appropriate conditions, and then excess biotin is washed away to obtain a biotin-labeled antibody protein.

10. The ELISA kit for detecting Galectin-8 according to claim 9, It is characterized in that The testing contents include: (1) Detect the concentration of human galectin-8 in liquid samples such as serum, cell culture supernatant, and cell lysate; (2) Detect the concentration of Galectin-8 protein samples with unknown concentration.

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