Anti-trop2 protein monoclonal antibodies, methods of making and uses thereof

Abstract

The application relates to a monoclonal antibody capable of identifying a human TROP2 antigen, a preparation method thereof and the use thereof in immunodetection. The application provides an anti-TROP2 protein rabbit monoclonal antibody, the amino acid sequence of a heavy chain variable region of the monoclonal antibody is an amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence of a light chain variable region of the monoclonal antibody is an amino acid sequence shown in SEQ ID NO. 2. The antibody has high specificity and sensitivity, can specifically identify cells expressing TROP2 protein, and is suitable for immunological detection, in particular, immunohistochemical detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, in particular to an anti-TROP2 protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] TROP2 is a member of the co-stimulatory molecule B7 family, one of the immune checkpoint molecules of the B7-CD28 family. It plays an important role in T cell-mediated immune response. TROP2 protein is not expressed or very low expressed in normal tissues and cells, but is abnormally expressed in various malignant tumors, and is closely related to the progression of tumor and prognosis of patients. TROP2 protein molecule is a type I transmembrane protein encoding 316 amino acids, containing 7 exons and 6 introns. In addition, there are two main splice bodies of TROP2 protein, TROP2a (2Ig-TROP2) with extracellular segment composed of Ig-V-IgC and B7-H3b (4Ig-TROP2) with extracellular segment composed of Ig-V-IgC-Ig-V-IgC, and humans mainly express 4Ig-TROP2.

[0003] The role of TROP2 in tumor immunity is controversial. On the one hand, it can play a co-stimulatory role, stimulate the proliferation of CD4 and CD8 T cells and the secretion of cytokines such as IFN-γ, IL-8 and TNF-α, and improve the immune response of the body to tumors. On the other hand, it can inhibit the activation of CD4+ T cells and the production of cytokines such as IFN-γ and IL-4, and play a co-inhibitory role.

[0004] Studies have confirmed that the expression level of TROP2 is closely related to the occurrence, proliferation, metastasis and invasion, metabolism and tumor angiogenesis of malignant tumors. Immunohistochemical results have shown that B7-H3 is highly expressed in liver cancer, colorectal cancer, endometrial cancer, breast cancer, bile duct cancer, colorectal cancer and other cancer tissues, and is significantly related to the degree of tumor infiltration, distant metastasis and differentiation. SUMMARY

[0005] The present application provides an anti-TROP2 protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2.

[0006] Further, the monoclonal antibody specifically recognizes TROP2 protein.

[0007] Further, the monoclonal antibody is a rabbit monoclonal antibody.

[0008] Further, the clone number of the rabbit monoclonal antibody is T1815162.

[0009] The present application also provides a method for preparing the anti-TROP2 protein monoclonal antibody, wherein the antigen for immunizing the rabbit is a recombinant protein expressed by E. coli.

[0010] Further, the recombinant protein comprises a TROP2 protein fragment and a HIS protein tag.

[0011] Further, the TROP2 protein fragment is the amino acid sequence shown in SEQ ID NO. 3.

[0012] Further, the plasmid vector selected in the recombinant process is PCMV3.

[0013] The present application also provides a TROP2 protein immunodetection reagent, wherein the immunodetection reagent contains the above-mentioned anti-TROP2 protein monoclonal antibody as an effective component.

[0014] Compared with the prior art, the present application has the following beneficial technical effects: the above-mentioned technical solution provides an anti-TROP2 protein rabbit monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2. The antibody has high specificity and sensitivity, can specifically recognize cells expressing TROP2 protein, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Figure 1 is a comparison chart of immunohistochemical staining results of breast ductal invasive carcinoma (the right is the rabbit monoclonal antibody TROP2 of the present application, and the left is the commercially available TROP2).

[0016] Figure 2 Figure 2 is a comparison chart of immunohistochemical staining results of gallbladder tissue (the right is the rabbit monoclonal antibody TROP2 of the present application, and the left is the commercially available TROP2). DETAILED DESCRIPTION

[0017] In order to explain the possible application scenarios, technical principles, specific implementable schemes, and the purposes and effects achieved by the present application in detail, the following will be described in detail in combination with the specific embodiments listed and the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0018] The term "embodiment" is mentioned in this document means that the specific features, structures or properties described in connection with the embodiment can be included in at least one embodiment of the present application. The term "embodiment" appearing in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit the independence or association between other embodiments. In principle, in this application, as long as there is no technical contradiction or conflict, the technical features mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

[0019] Unless otherwise defined, the meanings of the technical terms used in this document are the same as those commonly understood by those skilled in the art to which the present application belongs; the use of related terms in this document is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0020] In the description of the present application, the phrase "and / or" is a description of the logical relationship between the objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this document generally represents that the associated objects before and after are a "or" logical relationship.

[0021] In this application, such as "first" and "second" language is only used to distinguish one entity or operation from another entity or operation, and does not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0022] In this application, without more limitation, the "includes", "contains", "has" or other similar expressions used in the sentence are intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of other elements in the process, method or product including the described elements, so that the process, method or product including a series of elements can not only include those defined elements, but also include other elements not explicitly listed, or also include the elements inherent in such process, method or product.

[0023] In this application, "greater than", "less than", "exceed" and other expressions are understood as not including the number; "above", "below", "within" and other expressions are understood as including the number. In addition, the meaning of "multiple" in the description of the embodiments of the present application is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly limited.

[0024] Preparation of recombinant TROP2 protein fragment of embodiment 1

[0025] I. Gene optimization and synthesis

[0026] According to the protein sequence of TROP2 with accession number NP_002344.2 in the NCBI database, protein fragments at sites 27-274 and 299-323 were selected, directly optimized into gene fragments suitable for expression in E. coli Rosetta (DE3), and cloned into the pCMV3 vector.

[0027] Transform competent E. coli Rosetta (DE3) cells with the expression vector, pick colonies from the plate and inoculate them for bacterial PCR identification. Select colonies with positive PCR results for sequencing analysis, and use the colonies with completely correct sequences.

[0028] Choosing different antigens for immunization may produce antibodies with different binding properties. This molecule also has multiple variants caused by alternative splicing, ultimately resulting in different antibodies' ability and patterns of recognition of antigen-expressing cells. The TROP2 molecule was analyzed based on the published sequence. Based on its structure, antigenicity, the hydrophilicity of its constituent amino acids, and secondary structure, regions suitable for soluble expression and good immunogenicity were selected for recombinant expression. Codon optimization was performed on the gene sequence at positions 27-274 and 299-323 of TROP2, resulting in a molecular weight of approximately 36 kDa. Through sequence optimization, the TROP2 protein was obtained using the prokaryotic expression gene sequence. The recombinant immunogen consists of an antigenic TROP2 protein fragment and a protein tag (HIS) for purification of the recombinant protein.

[0029] 2. Protein Expression and Purification

[0030] Overnight cultures from a single colony were transferred to 100 mL of LB medium at a ratio of 1:100. Ampicillin was added at a final concentration of 100 μg / mL and cultured at 37°C with shaking until the OD600 reached 0.6-0.8. 0.8 mmol / L IPTG was added and cultured at 37°C with shaking for 4 hours. The cells were harvested and then sonicated. The recombinant protein was histidine-tagged and affinity purified using a nickel column. Elution was performed with 150 mmol / L imidazole and detection was performed by SDS-PAGE. The purified protein (27-274 and 299-323 aa) expressed in the supernatant reached a protein concentration of 1 mg / mL, with a purity of 85%, meeting the requirements for animal immunization and antibody screening and identification.

[0031] Example 2 Isolation of Single B Cells Producing Rabbit Anti-Human TROP2 Monoclonal Antibodies

[0032] 1. Immunity and ELISA testing

[0033] The recombinant TROP2 protein in Example 1 was emulsified with Freund's complete adjuvant, and 6 rabbits were selected for immunization, with a dose of 300 μg per rabbit. One-time booster immunization was performed on the 21st, 28th and 49th days, respectively, and the antigen was emulsified with Freund's incomplete adjuvant, with a dose of 150 μg per rabbit. After the third immunization, serum ELISA titer detection was performed, and the A rabbit had an immune titer greater than 1:2.56K, and the immune effect met the requirements. The serum of 4 rabbits after the fourth immunization was subjected to IHC detection, and according to the results (i.e., using positive control photos corresponding to the antibody for detection, and immunohistochemical staining can be observed at the corresponding detection site in the positive control photos), one rabbit (number: A) was selected for subsequent monoclonal antibody screening.

[0034] II. Spleen cell separation and B lymphocyte sorting

[0035] Monoclonal antibody preparation was performed on the selected rabbit. The spleen was removed 3 days after the final immunization, and the rabbit spleen was placed in RMPI basic medium containing 100 U / mL penicillin and 100 μg / mL streptomycin, cut into pieces with a surgical blade, and then transferred to a 100 μm cell screen for grinding. The obtained cell suspension was filtered to remove large cell clumps and tissue envelopes, and after centrifugation at 400 g for 5 minutes, the supernatant was removed and the spleen cell clumps were retained. The spleen cell clumps were resuspended with a hypotonic solution and lysed red blood cells, and then centrifuged at 400 g for 5 minutes, and the spleen cells were retained. The spleen cells were resuspended with RMPI basic medium containing 100 U / mL penicillin and 100 μg / mL streptomycin, centrifuged at 400 g for 5 minutes, and the obtained spleen cells were resuspended with complete medium (RMPI basic medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin) for standby.

[0036] The specific steps of B lymphocyte sorting are described in Chinese Patent 201910125091.4 "Method for efficiently separating single antigen-specific B lymphocytes from spleen cells".

[0037] About 2000 single B cell clones were sorted and cultured, and positive clones specifically recognizing the recombinant TROP2 protein in Example 1 were screened out by ELISA. According to the ELISA data, 50 supernatants from high to low were selected for IHC verification.

[0038] III. Detection of positive clones in B cell culture

[0039] Single B cell culture supernatant was subjected to IHC verification using a multi-tumor tissue chip and a normal tissue chip containing TROP2 positive and negative, and the IHC results of the supernatant of 8 clones were better. LEM supernatant was prepared for IHC verification to determine the clone (T1815162) with excellent sensitivity and specificity.

[0040] The screening criteria for sensitivity and specificity are: immunohistochemical staining is observed at the corresponding test site in the positive control photo, while no immunohistochemical staining is observed at the non-test site. The staining intensity is equal to or even higher than that of the control antibody.

[0041] IV. Cloning of the Rabbit Monoclonal Antibody Gene (T1815162) and Construction of Rabbit Monoclonal Antibody Expression Plasmid

[0042] Positive clones were harvested, lysed, and RNA was extracted and reverse-transcribed into cDNA. Using PCR, naturally paired rabbit monoclonal antibody light and heavy chain variable region genes were amplified from the cDNA of the corresponding positive clones and sequenced. Rabbit monoclonal antibody expression vector plasmids were constructed using the naturally paired rabbit monoclonal antibody light and heavy chain variable region gene sequences.

[0043] Example 2 was completed by Fuzhou Maixin Biotechnology Development Co., Ltd. on behalf of Dima Biotechnology (Wuhan) Co., Ltd., and Fuzhou Maixin Biotechnology Development Co., Ltd. performed the immunohistochemical screening.

[0044] Example 3 TROP2 rabbit monoclonal antibody expression

[0045] 1. Plasmid Amplification and Extraction

[0046] Take out a tube (100 μL) of competent bacteria (DH5α), insert it into ice, and ice bath for 5-10 minutes; add 5 μL of plasmid and shake gently, then place on ice for 30 minutes; shake gently and place in a 42°C water bath for 90 seconds for heat shock, then quickly return to ice and let it stand for 5 minutes; add 800 μL of LB culture medium (Note: does not contain antibiotics) to the above in the clean bench, mix gently, fix on a shaker and shake at 37°C for 1 hour; take 50-100 μL of the above transformation mixture in the clean bench, add it dropwise to the marked solid LB flat plate culture dish containing Amp, and spread it evenly with a glass coating rod (sterilized); first place it upright in a 37°C incubator for 30 minutes to allow the bacterial liquid on the surface to completely penetrate into the culture medium, and then invert it and place it in a 37°C incubator for overnight culture. Use a pipette to pick up a single colony and place it into 4 mL of LB medium (containing 2 uL of 200 mg / mL Amp) and culture it in a shaking incubator at 37°C for 16 h at 37°C and 220 rpm.

[0047] 1 mL of bacterial solution was added to 100 mL of LB medium (containing 50 μL of 200 mg / mL Amp) and cultured at 37°C in a shaking incubator for 16 h. Plasmids were extracted using the SanPrep Endotoxin-Free Plasmid Mini-Scale DNA Extraction Kit (Sanggong) according to the manufacturer's instructions.

[0048] 2. Transfection

[0049] Adjust the concentration of 293F cells to 2.5-3 × 10 cells / mL using Expi293F™ Expression Medium. 6 viable cells / mL and culture overnight.

[0050] 1) Count the viable cells using a hemocytometer; the concentration of viable cells is approximately 4.5-5.5×10 6 viable cells / mL, and the number of viable cells must meet the requirements of the expression system;

[0051] 2) Dilute the cells to a concentration of 3 × 10 cells / mL using Expi293F™ Expression Medium. 6 viable cells / mL;

[0052] 3) Add the plasmid DNA to the Opti-MENTM I induced serum medium, pipette gently, and invert to mix thoroughly;

[0053] 4) Gently invert the ExpiFectamine TM 293 Reagent 4-5 times, ExpiFectamine TM 293 Mix the Reagent and Opti-MENTM I Regulated Serum Medium, gently pipette and invert 2-3 times, and let it stand at room temperature for 5 minutes;

[0054] 5) Combine the solutions from steps 3) and 4) by gently pipetting and inverting 2-3 times to mix thoroughly;

[0055] 6) placing the solution from step 5) at room temperature for 10-20 minutes;

[0056] 7) Slowly pipette the mixture into the cell culture medium and gently shake the flask;

[0057] 8) Incubate in a shaking incubator at 8% CO2 at 37°C for 5-7 days;

[0058] 9) After 18-22 hours, add ExpiFectamine TM 293 Transfection Enhancer1 and ExpiFectamine TM 293 Add Transfection Enhancer 2 (Note: Mix in advance before use), shake gently to mix, and continue culturing.

[0059] 3. Purification of Monoclonal Antibodies

[0060] Antibodies were purified from the supernatant using HiTrap rProtein A FF affinity chromatography according to the manufacturer's instructions. Purity was assessed by SDS-PAGE gel analysis and concentration was determined by the Bradford method. Purified antibodies were stored at -20°C. Example 4. Immunohistochemical staining and identification of tissue microarrays

[0061] 1. Chip Preparation Process

[0062] Each sample is first stained with HE sections to determine the location of the tumor. Circle the tumor target site and prepare for punching. When making a blank recipient wax block, place the plastic frame on the mold, pour the melted paraffin (melting point at 55-58°C) into the mold, cool to room temperature, place the mold in a -20°C refrigerator for 6 minutes, and remove the wax block from the mold. Select a 1mm diameter sample needle on the tissue sample machine to punch a hole in the recipient wax block with a depth of 3-4mm. Use another 1mm diameter punching needle to punch a hole in the marked part of the wax block to collect a tissue core. The length is about 0.1mm shallower than the hole depth of the recipient wax block. Insert the collected tissue core directly or carefully pick it up with tweezers and insert it into the empty hole of the recipient wax block. Repeat this process until all sample points are prepared. Finally, use a glass slide to flatten all the tissue cores to make the tissue chip wax block flat and smooth. The prepared tissue microarray wax block is placed back into a wax mold and placed in a 60°C oven for 15 minutes to allow the tissue core and the recipient wax to fuse. The mold is then gently removed from the oven and the semi-melted paraffin is allowed to cool at room temperature for approximately 30 minutes. The block is then placed in a -20°C freezer for 6 minutes. The tissue microarray block is then removed from the mold and sliced ​​or stored in a 4°C refrigerator until ready for use. After trimming, serial sections are cut to a thickness of 3 μm. Serial sections are then floated in cold water and allowed to unfold naturally. Separate sections are then transferred to 45°C warm water for 30 seconds. The sections are then mounted on polylysine-treated glass slides. The prepared tissue microarray is then baked in a 65°C oven for 2 hours, removed from the oven, cooled to room temperature, and stored in a -4°C refrigerator.

[0063] II. IHC Staining and Analysis

[0064] Routine dewaxing of xylene for 3 times, 6 minutes each time, 100%, 100%, 95%, 85% gradient ethanol hydration, 3 minutes each time, and finally tap water rinse. Antigen retrieval was performed, and then the sections were placed in a wet box and rinsed with PBS for 3 x 3 minutes. 3% H2O2 was added dropwise and incubated for 10 minutes, and then rinsed with PBS for 3 x 3 minutes. The sections were spun dry, the appropriate dilution of the primary antibody was added dropwise (the first dilution was designed according to the concentration of the antibody), incubated at room temperature (25°C) for 1 hour, rinsed with PBS for 3 x 3 minutes, the secondary antibody was added dropwise and incubated at room temperature for 15-30 minutes, rinsed with PBS for 3 x 3 minutes, the PBS was spun off, and fresh DAB developing solution was used to develop for 3-10 minutes. Hematoxylin counterstaining was performed for 25 seconds, and PBS was returned to blue for 30 seconds. Sequential dehydration was performed according to the alcohol gradient of 85% (3 minutes)-95% (3 minutes)-100% (3 minutes)-100% (3 minutes), and finally xylene clearing for 3 minutes, and neutral resin mounting.

[0065] The results of immunohistochemical staining are divided into: positive and negative. Positive expression must be at the site of cell and tissue-specific antigens to be considered positive. In the case of clear distribution of tissue staining and accurate cell localization, the staining results are further divided according to the difference in staining intensity, as follows:

[0066] 1. The sample is weakly positive; marked as "+";

[0067] 2. The sample is moderately positive; marked as "++";

[0068] 3. The sample is highly positive; marked as "+++".

[0069] 4. The sample is negative, marked as "-".

[0070] III. Data Statistics

[0071] 1. Tumor tissue chip detection results:

[0072] The antibody TROP2 (T1815162) of the application and the commercially available antibody TROP2 (rabbit polyclonal antibody) were simultaneously detected on 49 cases of breast ductal invasive carcinoma, and the detection results were compared. The immunohistochemical results of TROP2 were statistically analyzed. The entire test process adopted a double-blind design, and the statistical results are as follows:

[0073]

[0074] The results show that the rabbit monoclonal antibody TROP2 (T1815162) has accurate staining localization, clear staining and no non-specific staining, and a clean background. In the immunohistochemical detection, the positive rate is comparable to that of the commercially available antibody, and the positive intensity of 15 cases of TROP2 rabbit monoclonal antibody is higher than that of the commercially available antibody, indicating that the sensitivity of the rabbit monoclonal antibody TROP2 (T1815162) is higher than that of the commercially available antibody.

[0075] Figure 1 Figure 1 is a comparison chart of immunohistochemical staining results of breast duct invasive carcinoma (left: rabbit monoclonal antibody TROP2 of the present application, right: commercially available TROP2).

[0076] 2. Normal tissue chip test results:

[0077] The normal tissue chip includes 30 normal tissue samples, which are mainly selected from fresh and timely fixed surgical specimens; each tissue includes 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectum, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infant), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

[0078] The rabbit monoclonal antibody TROP2 (T1815162) and the commercially available TROP2 antibody were synchronously detected on the normal tissue chip, and the positive and negative detection results were consistent, indicating that the specificity of the antibody in the normal tissue was equivalent to that of the commercially available antibody. Figure 2 Figure 1 is a comparison chart of immunohistochemical staining results of breast duct invasive carcinoma (left: rabbit monoclonal antibody TROP2 of the present application, right: commercially available TROP2).

[0079] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of the present application, the patent protection scope of the present application should not be limited. Any technical solutions obtained by replacing or modifying the equivalent structure or equivalent process based on the essential concept of the present application, using the content described in the specification and drawings of the present application, and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, etc., are all included in the patent protection scope of the present application.

Claims

1. An anti-TROP2 protein monoclonal antibody, characterized in that: The heavy chain amino acid sequence of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1; the light chain amino acid sequence of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody is a rabbit monoclonal antibody with clone number T1815162.

3. A TROP2 protein immunoassay reagent, characterized in that: The immunoassay reagent contains the anti-TROP2 protein monoclonal antibody according to claim 1 as its active ingredient.

4. The immunoassay reagent according to claim 3, characterized in that The immunoassay includes immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

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