Rabbit monoclonal antibody against prostate stem cell antigen (PSCA) and its application

Abstract

This invention relates to the field of biotechnology and discloses a rabbit monoclonal antibody OTIR4B1 against human prostate stem cell antigen (PSCA). The antibody is produced by sorting and culturing specific B cells and then performing molecular clonal recombination. The immunogen of the rabbit monoclonal antibody OTIR4B1 is a synthetic polypeptide ranging from 50 to 90 amino acids. The amino acid sequence of the OTIR4B1 antibody light chain (VL) is shown in SEQ ID NO. 1; the amino acid sequence of the OTIR4B1 antibody heavy chain (VH) is shown in SEQ ID NO. 2. The VL region of the OTIR4B1 antibody includes three antigenic determinants: CDR1, CDR2, and CDR3, whose amino acid sequences are shown in SEQ ID NO. 3-5, respectively. The VH region of the OTIR4B1 antibody also includes three antigenic determinants: CDR1, CDR2, and CDR3, whose amino acid sequences are shown in SEQ ID NO. 6-8, respectively. This invention also relates to the application of rabbit monoclonal antibodies against human prostate stem cell antigen (PSCA) in the preparation of immunoassay tools for detecting PSCA protein, including but not limited to the application in immunohistochemical kits and kits for labeling tumors, providing an auxiliary means for the diagnosis of PSCA-related diseases and laying the foundation for the preparation of engineered antibodies in the next step.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to rabbit monoclonal antibodies against human prostate stem cell antigen (PSCA) and their application in immune detection. Background Technology

[0002] Prostate cancer is an epithelial malignant tumor that occurs in the prostate gland. It is the most common non-dermatological malignancy and one of the most common cancers in men worldwide. PSCA, or prostate stem cell antigen, is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein belonging to the Thy-1 / Ly-6 family. PSCA is considered a key biomarker for several primary cancers, including bladder cancer, prostate cancer, and pancreatic cancer.

[0003] PSCA was initially designated as a "stem cell antigen," but it is now known to be primarily expressed in differentiated cells rather than stem cells. PSCA is expressed in small amounts in normal prostate tissue and benign lesions, with expression localized to the basal cell layer of the prostate. It is highly specific in prostate cancer, especially androgen-independent prostate cancer and bone metastases, with expression localized to prostate cancer cells; it is not expressed in the intercellular matrix or muscle. In normal tissues, PSCA is mainly expressed in the prostate epithelium. It is also expressed in non-prostate tissues such as the kidney, small intestine, bladder transitional epithelium, placental trophoblast, and neuroendocrine cells of the colon, but at levels less than 1% of those in normal prostate tissue, indicating that PSCA expression has relatively high prostate tissue specificity.

[0004] PSCA expression is low in normal prostate tissue. Increased PSCA expression is associated with increased tumor stage, grade, and the formation of androgen-independent and metastatic cancers, and it does not decrease with cancer progression, making it an ideal target antigen for prostate cancer diagnosis and treatment. Currently, immunohistochemistry (IHC) is commonly used in clinical practice to detect protein expression in tumor cells. However, the core of IHC experiments is the use of monoclonal antibodies that specifically bind to the protein, and their performance directly determines the sensitivity and specificity of the entire detection. Therefore, finding highly specific and sensitive anti-PSCA antibodies is crucial for establishing a method for detecting PSCA in cells. A search of patent websites in China, the United States, EPO, and WIPO, using PSCA or PSCA+monoclonal antibody or antibody as keywords, yielded no patents related to monoclonal antibodies with high affinity and specific binding to PSCA. No immunoassay kits using the anti-PSCA rabbit monoclonal antibody OTIR4B1 were also found. Using PSCA or PSCA+monoclonal antibody or antibody as keywords, no similar literature related to the content of this invention was found on PubMed. A search of the National Medical Products Administration website revealed that no testing reagents related to PSCA have been registered. Summary of the Invention

[0005] The purpose of this invention is to provide the highly specific and affinity-rich rabbit monoclonal antibody OTIR4B1 against PSCA and its application in the preparation of an immunoassay tool for detecting PSCA protein, thereby providing auxiliary diagnosis and monitoring for benign prostatic hyperplasia and prostate cancer, and also laying the foundation for the preparation of engineered antibodies in the next step.

[0006] The rabbit monoclonal antibody is rabbit monoclonal antibody OTIR4B1.

[0007] The rabbit monoclonal antibody OTIR4B1 uses a synthetic PSCA polypeptide as an immunogen. It is obtained by immunizing New Zealand white rabbits, collecting peripheral blood mononuclear cells (PMBCs) from the immunized rabbits, sorting and culturing specific B cells, screening, and using molecular clonal recombination technology.

[0008] The rabbit monoclonal antibody OTIR4B1 has a light chain variable region (VL) containing 109 amino acids, and its amino acid sequence is shown in SEQ ID NO. 1; the heavy chain (VH) of the antibody contains 112 amino acids, and its amino acid sequence is shown in SEQ ID NO. 2.

[0009] The rabbit monoclonal antibody OTIR4B1 comprises three antigenic determinants in its VL region: CDR1, CDR2, and CDR3, with corresponding regions of 27aa-34aa, 52aa-54aa, and 91aa-99aa, respectively. Its amino acid sequences are shown in SEQ ID No. 3-5.

[0010] The rabbit monoclonal antibody OTIR4B1 comprises three antigenic determinants in its VH region: CDR1, CDR2, and CDR3, with corresponding regions of 25aa-32aa, 50aa-56aa, and 93aa-101aa, respectively. Its amino acid residue sequences are shown in SEQ ID No. 6-8.

[0011] The rabbit monoclonal antibody OTIR4B1 described above can bind with high specificity to PSCA and can be prepared into various immunoassay kits for detecting PSCA using methods known to those skilled in the art. In particular, it can be used in immunohistochemical kits and tumor labeling kits. Attached Figure Description

[0012] Figure 1 This is an electrophoresis diagram of the full-length amplification products of the heavy and light chains of the rabbit monoclonal antibody OTIR4B1. M is the DNA molecular weight marker.

[0013] Figure 2 The Western blot results of rabbit monoclonal antibody OTIR4B1 specifically recognizing the full-length PSCA (PSCA-FL) protein are shown in the figure.

[0014] Figure 3 Immunohistochemical results of a formalin-fixed, paraffin-embedded human prostatic hyperplasia sample 1 (primary antibody: PSCA monoclonal antibody OTIR4B1);

[0015] Figure 4 Immunohistochemical results of a formalin-fixed, paraffin-embedded human prostatic hyperplasia sample 2 (primary antibody: PSCA monoclonal antibody OTIR4B1);

[0016] Figure 5 Immunohistochemical results of formalin-fixed and paraffin-embedded human duodenum (primary antibody: PSCA monoclonal antibody OTIR4B1). Detailed Implementation

[0017] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer.

[0018] Example 1: Preparation of PSCA rabbit monoclonal antibody

[0019] I. Preparation of PSCA Immunogen

[0020] An immunogenic polypeptide was designed based on the PSCA 1-114aa sequence. The polypeptide sequence range is 50-90aa, and the polypeptide purity is above 90%, meeting the purity requirements for monoclonal antibody preparation. The polypeptide was synthesized by a third party, Zhongtai Biochemical Co., Ltd.

[0021] II. Animal Immunization

[0022] The synthesized PSCA peptide was emulsified with complete Freund's adjuvant and administered subcutaneously to approximately 2 kg New Zealand White rabbits at a dose of 500 μg per rabbit. A second immunization was performed two weeks later, using incomplete Freund's adjuvant emulsification at a dose of 250 μg per rabbit. Tail blood was collected after both immunizations and serum titers were determined using a serially diluted ELISA method. The OD450 at an ELISA titer of 128,000 was considered to be greater than 1.0. Based on the results, it was determined whether to collect PBMCs or continue immunization. Rabbits with the highest antibody titers were selected for PBMC collection.

[0023] III. PBMC Isolation, Specific B Cell Sorting, and Clonal Recombination: Rabbits were fixed supine on the operating table. The fur around the heart was trimmed, and the skin was disinfected with alcohol. A 50ml syringe was used to puncture the area where the heartbeat was most pronounced. Blood flowed into the syringe immediately after the needle pierced the heart. The needle was quickly withdrawn, and the whole blood was transferred to a sterile 50ml tube. This was mixed with an equal volume of PBS and slowly added dropwise onto the lymphocyte separation medium. The mixture was centrifuged at 400×g for 30 minutes at room temperature. After centrifugation, the liquid surface separated into four layers from top to bottom: a yellow plasma layer, a white thin film layer (mononuclear cell layer), a separation medium layer, and a red blood cell layer. The mononuclear cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation medium, yielding rabbit PBMCs. Antigen-specific B cells were further sorted from the rabbit PBMCs for culture. Positive clones were selected from the cultured B cell supernatant using an antigen-coated ELISA plate. Cells from positive clones were collected, lysed, and RNA was extracted and reverse transcribed into cDNA. The full-length light and heavy chain sequences of naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones. A rabbit monoclonal antibody expression vector was constructed using clonal recombination, and the sequence was confirmed by sequencing. The results of the amplified full-length PCR products are shown below. Figure 1 .

[0024] IV. Preparation and Purification of Monoclonal Antibodies To obtain rabbit monoclonal antibodies recognizing human PSCA protein, this invention loads the heavy and light chain genes of rabbit monoclonal antibodies into an expression vector, and transfects the plasmid into HEK293 cells. After 120-144 hours of transfection, the culture supernatant contains recombinant rabbit monoclonal antibodies recognizing human PSCA protein. The cell suspension is collected, the supernatant is obtained by centrifugation, and the antibody is purified by affinity chromatography. The concentration of the purified monoclonal antibody is determined by the BCA method, and then aliquoted and lyophilized.

[0025] Example 2: Identification of anti-PSCA rabbit monoclonal antibody OTIR4B1

[0026] I. Western blot identification of rabbit monoclonal antibody OTIR4B1

[0027] Western blot (WB) was used for detection. The left lane contained cell lysates transfected with the pCMV6-Entry control plasmid; the right lane contained cell lysates transfected with the full-length pCMV6-PSCA expression plasmid. 5 μg of each lysate was loaded for SDS-PAGE, and WB was performed after transfer to a membrane.

[0028] The results showed that the rabbit monoclonal antibody OTIR4B1 could specifically detect both intact and secreted PSCA protein. Antibody dilution ratio: 1:5000-8000, 1 mg / ml. See attached results. Figure 2 .

[0029] II. Titer of Rabbit Monoclonal Antibodies

[0030] The rabbit monoclonal antibody OTIR4B1 was diluted using a serial dilution method, and the antibody titer was measured by indirect ELISA. The results showed that the titer of the rabbit monoclonal antibody OTIR4B1 involved in this invention was 1.5 × 10⁻⁶. 7 .

[0031] Example 3: Analysis of the variable region gene and amino acid sequence of rabbit monoclonal antibody OTIR4B1

[0032] Using the recombinant plasmid of the OTIR4B1 antibody as a DNA template, sequencing primers for the light chain variable region and heavy chain variable region were designed based on the vector sequences at the 5' ends of the light and heavy chains on the template. Sequencing was performed using an ABI 3730 sequencer. The nucleotide sequences of the light and heavy chain variable regions of the rabbit monoclonal antibody OTIR4B1 were obtained by sequencing.

[0033] Using the internet and the IMGT / V-QUEST analysis software at http: / / www.imgt.org, the nucleotide sequences of the light and heavy chains were sequenced and analyzed. The light chain amino acid sequence of the rabbit monoclonal antibody OTIR4B1 is shown in SEQ ID NO.1, and the heavy chain amino acid sequence is shown in SEQ ID NO.2. The full length of VL is 109 amino acids. The number of amino acids in the four domains of its FR are 26, 17, 36, and 10, respectively, and the number of amino acids in the three domains of CDR are 8, 3, and 9, respectively. The regions of CDR1, CDR2, and CDR3 are 27aa-34aa, 52aa-54aa, and 91aa-99aa, respectively, and their amino acid sequences are QSVYKNNR, KAS, and QGEFDCSRG, respectively.

[0034] Analysis revealed that the rabbit monoclonal antibody OTIR4B1 VH has a full length of 112 amino acids. The number of amino acids in the four domains of its FR is 24, 17, 36 and 11, respectively. The number of amino acids in the three domains of its CDR is 8, 7 and 9, respectively. CDR1, CDR2 and CDR3 are 25aa-32aa, 50aa-56aa and 93aa-101aa, respectively. Their amino acid sequences are GFSLTSYH, VYSSSKS and VRKGDRTLY, respectively.

[0035] Example 4: Immunohistochemical detection using monoclonal antibody OTIR4B1 as the primary antibody

[0036] I. Experimental Methods:

[0037] 1. Formalin-fixed prostatic hyperplasia and duodenal tissue were obtained, paraffin-embedded, and sectioned using a Finesse tissue slicer to a thickness of 6 μm.

[0038] 2. Dewaxing and hydration: Analytical grade xylene for 3×10 min, anhydrous ethanol for 3×10 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and deionized water for 3 min × 3 times.

[0039] 3. Add antigen retrieval solution (0.01M, pH 6.0 sodium citrate buffer) and autoclave for 3 minutes. When the autoclave temperature drops to approximately 90°C, open the autoclave, remove the specimen, and allow it to cool naturally to room temperature. Immerse in deionized water for 3 minutes each time, repeating 3 times.

[0040] 4. Inactivate endogenous peroxidase in tissues with 3% hydrogen peroxide and let stand at room temperature for 10 minutes. Soak in deionized water for 5 minutes three times.

[0041] 5. Add blocking solution (PBS + 5% skim milk powder + 5% normal goat serum) and incubate at 37°C for 60 minutes.

[0042] 6. Remove the blocking buffer, do not rinse. Add PSCA monoclonal antibody (OTIR4B1) at a dilution ratio of 1:600-1:800 (1 mg / ml), diluted with the blocking buffer. Incubate in a humidified chamber at 37°C for 60 min. Wash twice with PBST (0.1% Tween-20), 5 min each time. Wash once with PBST (0.02% Tween-20), 5 min each time.

[0043] 7. Add reagent 1 from Polink-2 kit (Catlog No. D37-15) and incubate at 37°C for 10-20 minutes. Wash three times with PBS, 5 minutes each time. Add reagent 2 from Polink-2 kit (Catlog No. D37-15) and incubate at 37°C for 10-20 minutes. Wash three times with PBS, 5 minutes each time.

[0044] 8. Develop color using DAB solution (Zhongshan Jinqiao ZLI-9019) for 3–10 minutes. Wash with distilled water.

[0045] 9. Counterstain cell nuclei with hematoxylin for 2 min, rinse with distilled water, and differentiate with 1% hydrochloric acid. Rinse 3 times with distilled water and let stand at room temperature for 1 min.

[0046] 10. Dehydration and clearing: 75% ethanol for 5 min, 100% ethanol for 5 min x 3 times, 85% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 3 x 5 min; xylene for 3 x 5 min, neutral resin for mounting.

[0047] 11. Microscopic examination, see Figures 3-5 .

[0048] II. Experimental Results:

[0049] Depend on Figures 3-5 The results showed that the antibody, at lower dilutions (higher dilution ratios), exhibited significant specific cell membrane staining in prostatic hyperplasia and duodenal tissues. These results are consistent with the intracellular localization and tissue expression specificity of PSCA, indicating that the monoclonal antibody OTIR4B1 can be used for immunohistochemical detection of PSCA protein levels. OTIR4B1 staining demonstrates its ability to effectively differentiate tissues with high PSCA expression in prostatic hyperplasia. Figure 3 ) and tissues with low PSCA expression in benign prostatic hyperplasia ( Figure 4 Furthermore, PSCA is expressed only in the neuroendocrine cells of the intestine. Figure 5OTIR4B1 staining clearly demonstrates that only neuroendocrine cells were stained. OTIR4B1 immunohistochemical staining indicates that the monoclonal antibody OTIR4B1 has high sensitivity and can be used for immunohistochemical diagnosis and differentiation of benign prostatic hyperplasia, prostate cancer, and other related diseases. sequence list <110> Wuxi Aorui Dongyuan Biotechnology Co., Ltd. <120> Rabbit monoclonal antibody against prostate stem cell antigen (PSCA) and its application <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 109 <212> PRT <213> Oryctolagus cuniculus <400> 1 Ala Gln Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Ser Ile Ser Cys Gln Ala Ser Gln Ser Val Tyr Lys Asn 20 25 30 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Lys Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Glu Phe Asp Cys 85 90 95 Ser Arg Gly Phe Gly Gly Gly Thr Glu Val Val Val Lys 100 105 <210> 2 <211> 112 <212> PRT <213> Oryctolagus cuniculus <400> 2 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr His 20 25 30 Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly 35 40 45 Ile Val Tyr Ser Ser Ser Lys Ser Ser Tyr Ala Ser Trp Ala Asn Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Arg Ile Thr 65 70 75 80 Ser Pro Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys Val Arg Lys Gly 85 90 95 Asp Arg Thr Leu Tyr Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 3 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 3 Gln Ser Val Tyr Lys Asn Asn Arg 1 5 <210> 4 <211> 3 <212> PRT <213> Oryctolagus cuniculus <400> 4 Lys Ala Ser 1 <210> 5 <211> 9 <212> PRT <213> Oryctolagus cuniculus <400> 5 Gln Gly Glu Phe Asp Cys Ser Arg Gly 1 5 <210> 6 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 6 Gly Phe Ser Leu Thr Ser Tyr His 1 5 <210> 7 <211> 7 <212> PRT <213> Oryctolagus cuniculus <400> 7 Val Tyr Ser Ser Ser Lys Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Oryctolagus cuniculus <400> 8 Val Arg Lys Gly Asp Arg Thr Leu Tyr 1 5

Claims

1. A rabbit monoclonal antibody against human prostate stem cell antigen (PSCA), characterized in that: The rabbit monoclonal antibody is rabbit monoclonal antibody OTIR4B1, which includes a light chain variable region (VL) and a heavy chain variable region (VH). The light chain variable region (VL) includes CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID No. 3-5, respectively. The heavy chain variable region (VH) includes CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID No. 6-8, respectively.

2. The rabbit monoclonal antibody according to claim 1, characterized in that: The light chain variable region (VL) contains 109 amino acids, and its amino acid sequence is shown in SEQ ID No. 1; the heavy chain variable region (VH) contains 112 amino acids, and its amino acid sequence is shown in SEQ ID No.

2.

3. The use of the rabbit monoclonal antibody against human prostate stem cell antigen (PSCA) as described in claim 1 or 2 in the preparation of a prostate stem cell antigen (PSCA) immunoassay kit.

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