Anti-human LY6D protein monoclonal antibody and application thereof

Abstract

The invention relates to the technical field of antibodies, in particular to an anti-human LY6D protein monoclonal antibody and application thereof, and the anti-human LY6D protein monoclonal antibody is 16F11, 3G10, 3B9, 6D12 and 7C10 and can specifically recognize 21-98aa epitopes of the LY6D protein. The anti-human LY6D protein monoclonal antibody can be respectively expressed by a recombinant 293F cell containing a recombinant plasmid for coding an anti-human LY6D protein monoclonal antibody 16F11, 3G10, 3B9, 6D12 or 7C10 gene. The anti-human LY6D protein monoclonal antibody can be applied to preparation of drugs such as an antibody-drug conjugate (ADC) and a protein degradation targeting chimera (PROTAC) for treating pancreatic cancer, an ELISA kit constructed based on the anti-human LY6D protein monoclonal antibody can quantitatively or qualitatively detect LY6D protein in human serum and tissue samples, and the lowest detection limit can reach 1 ng / ml.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a specific and high affinity anti-human LY6D protein monoclonal antibody and its application in preparing an antibody-drug conjugate or a protein degradation targeting chimera drug for treating pancreatic cancer, and an ELISA kit for detecting LY6D protein in human serum or tissue homogenate. BACKGROUND

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with a 5-year survival rate of less than 10%. Although surgical resection is the only means to cure the disease, 80% of patients are in the advanced stage at the time of diagnosis, and about 50% have distant metastasis, missing the best opportunity for surgery. Therefore, early diagnosis is crucial for improving treatment outcomes and prognosis.

[0003] Existing diagnostic methods mainly rely on serum carbohydrate antigen (CA19-9) and carcinoembryonic antigen (CEA). However, the elevation of CA19-9 can also occur in patients with benign diseases (such as acute and chronic pancreatitis, obstructive jaundice, cirrhosis) or other malignant tumors (such as gastrointestinal tumors), thus limiting its application in early diagnosis of pancreatic cancer. Although the research of liquid biopsy, exosome miR-21, miR-191, and miR-451a biomarkers provides a new direction for early diagnosis, these methods have not yet been widely applied.

[0004] LY6D (Lymphocyte Antigen 6D) is a glycosylphosphatidylinositol (GPI) anchored protein that plays a key role in immune system regulation and cell signaling. It can exist as a GPI-anchored protein on the membrane surface and also freely in blood and other body fluids, making it a potential biomarker and therapeutic target. Studies have shown that LY6D is abnormally expressed in various malignant tumors, it can promote the occurrence and development of pancreatic cancer, and is highly related to the prognosis of pancreatic cancer patients. However, the detection value of LY6D in the diagnosis and treatment of tumor patients has not been fully studied. Existing antibody preparation techniques such as hybridoma technology have low production and difficulty in humanization, while recombinant DNA technology can efficiently and stably produce antibodies with high affinity and low immunogenicity. In addition, the development of LY6D-targeted ADC and PROTAC drugs is still in the early stages, and specific antibodies are urgently needed as basic tools. SUMMARY

[0005] The application aims to solve the above technical problems, and provides an anti-human LY6D protein monoclonal antibody and application thereof, the anti-human LY6D protein monoclonal antibody has high specificity and strong affinity, can be stably expressed in mammalian cells CHO, and can be applied to preparation of an antibody-drug conjugate or a protein degradation targeting chimera drug for treating pancreatic cancer, and an ELISA kit for detecting LY6D protein in human serum or tissue homogenate, so that the early diagnosis rate of pancreatic cancer is improved, and the treatment strategy is expanded.

[0006] Technical scheme of the application

[0007] In a first aspect, the application provides an anti-human LY6D protein monoclonal antibody, which adopts the following technical scheme:

[0008] The anti-human LY6D protein monoclonal antibody is 16F11, 3G10, 3B9, 6D12 or 7C10, and specifically recognizes the 21-98aa epitope of the LY6D protein;

[0009] The base sequence of the amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 16F11 is shown in SEQ ID NO. 1, and the base sequence of the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO. 2;

[0010]

[0011] The amino acid sequence corresponding to SEQ ID NO. 1 is shown below as SEQ ID NO. 11:

[0012] METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGFSLSSYYMTWVRQAPGKGLEWIGIIDAGGSAYSASWAKGRFTISRTSTTVDLKMTSLTTEDTATYFCVRDSGQNYLDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK* (SEQ ID NO. 11).

[0013] atggacacgagggcccccactcagctgctggggctcctgctgctctggctcccaggtgccagatgtgcccttgtgatgacccagactccagcctcggtgtctgaaccagtgggaggcacagtcaccatcaaatgccaggctagtgagattattggtagcggcttatcctggtatcagcagaaaccagggcagcctcccaaactcctgatctatgatgcatccgatctggcatctggggtcccaaagcggttcagtggcagcagatctgggatacaatacactctcaccatcagcggcgtgcagtgtgacgatgctgccacttactactgtctaggtgtttacgcttataatagtgatgatggttttgttttcggcggagggaccgaggtggtggtcaaaggtgatccagttgcacctactgtcctcatcttcccaccagctgctgatcaggtggcaactggaacagtcaccatcgtgtgtgtggcgaataaatactttcccgatgtcaccgtcacctgggaggtggatggcaccacccaaacaactggcatcgagaacagtaaaacaccgcagaattctgcagattgtacctacaacctcagcagcactctgacactgaccagcacacagtacaacagccacaaagagtacacctgcaaggtgacccagggcacgacctcagtcgtccagagcttcaataggggtgactgttag (SEQ ID NO. 2).

[0014] The amino acid sequence corresponding to SEQ ID NO. 2 is shown below as SEQ ID NO. 12:

[0015] MDTRAPTQLLGLLLLWLPGARCALVMTQTPASVSEPVGGTVTIKCQASEIIGSGLSWYQQKPGQPPKLLIYDASDLASGVPKRFSGSRSGIQYTLTISGVQCDDAATYYCLGVYAYNSDDGFVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC* (SEQ ID NO. 12).

[0016] The base sequence of the amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 3G10 is shown in SEQ ID NO. 3, and the base sequence of the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO. 4.

[0017]

[0018] The amino acid sequence corresponding to SEQ ID NO. 3 is shown below as SEQ ID NO. 13:

[0019] METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSSYYIMWVRQAPGKGLEGVGFIDTGGSAYYASWARGRFTISRTSSTTMDLKVTSPTTEDTATYFCARNDGGSHGMDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK* (SEQ ID NO. 13).

[0020] atggacacgagggcccccactcagctgctggggctcctgctgctctggctcccaggtgccagatgtgcccttgtgatgacccagactccagcctcggtgtctgcacctgtgggaggcacagtcaccatcaagtgccaggccagtgagactattagtagcgacttatcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatgatgcatccgatctggcatctggggtcccatcgcggttcagtggcagcagatctgggacacagttcactctcaccatcagcggcgtgcagtgtgacgatgctgccacctactactgtctaggtgtttatgcttataatagtgatgatggtattgttttcgccggagggaccgaggtggtggtcaaaggtgatccagttgcacctactgtcctcatcttcccaccagctgctgatcaggtggcaactggaacagtcaccatcgtgtgtgtggcgaataaatactttcccgatgtcaccgtcacctgggaggtggatggcaccacccaaacaactggcatcgagaacagtaaaacaccgcagaattctgcagattgtacctacaacctcagcagcactctgacactgaccagcacacagtacaacagccacaaagagtacacctgcaaggtgacccagggcacgacctcagtcgtccagagcttcaataggggtgactgttag (SEQ ID NO. 4).

[0021] The amino acid sequence corresponding to SEQ ID NO. 4 is shown below as SEQ ID NO. 14:

[0022] MDTRAPTQLLGLLLLWLPGARCALVMTQTPASVSAPVGGTVTIKCQASETISSDLSWYQQKPGQPPKLLIYDASDLASGVPSRFSGSRSGTQFTLTISGVQCDDAATYYCLGVYAYNSDDGIVFAGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC* (SEQ ID NO. 14).

[0023] The base sequence of the amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 3B9 is shown in SEQ ID NO. 5, and the base sequence of the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO. 6.

[0024]

[0025] The amino acid sequence corresponding to SEQ ID NO. 5 is shown below as SEQ ID NO. 15:

[0026] METGLRWLLLVAVLKGVQCQSVEESGGGLVTPGGTLTLTCTASGFSLNSYNMGWVRQAPGKGLEWIGIMNIDGSASYASWANGRFTISKNSGTVDLKMTGLTAADTAMYFCVREGGYGYDSAMDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK* (SEQ ID NO. 15).

[0027] atggacacgagggcccccactcagctgctggggctcctgctgctctggctcccaggtgccagatgtgcccttgtgatgacccagactccagcctccgtggaggcagctgtgggaggcacagtcaccatcaagtgccaggccagtcagaacatttacagcaatttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctataggtcatccaatctggaatctggggtcccatcgcggttcaaaggcagtggatctggggcagactacactctcaccatcagcggcgtgcagtgtgccgatgctgccacttactactgtcaaggcggttactttactgctaatactgataatgatttcggcggagggaccgaggtggtggtcaaaggtgatccagttgcacctactgtcctcatcttcccaccagctgctgatcaggtggcaactggaacagtcaccatcgtgtgtgtggcgaataaatactttcccgatgtcaccgtcacctgggaggtggatggcaccacccaaacaactggcatcgagaacagtaaaacaccgcagaattctgcagattgtacctacaacctcagcagcactctgacactgaccagcacacagtacaacagccacaaagagtacacctgcaaggtgacccagggcacgacctcagtcgtccagagcttcaataggggtgactgttag (SEQ ID NO. 6).

[0028] The amino acid sequence corresponding to SEQ ID NO. 6 is shown below as SEQ ID NO. 16:

[0029] MDTRAPTQLLGLLLLWLPGARCALVMTQTPASVEAAVGGTVTIKCQASQNIYSNLAWYQQKPGQPPKLLIYRSSNLESGVPSRFKGSGSGADYTLTISGVQCADAATYYCQGGYFTANTDNDFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC* (SEQ ID NO. 16);

[0030] The base sequence of the amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 6D12 is shown in SEQ ID NO. 7, and the base sequence of the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO. 8.

[0031]

[0032] The amino acid sequence corresponding to SEQ ID NO. 7 is shown below as SEQ ID NO. 17:

[0033] METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGIDLNNYNMGWVRQAPGEGLEWIGI INFDDSVSYANWAKGRFTISRTSTTVDLKMTSLTAADTATYFCAREGGYGYDSAMDLWGPGTLVTVSS GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVT SSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQ DDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKAR GQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSV P TSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK* (SEQ ID NO. 17).

[0034] atggacacgagggcccccactcagctgctggggctcctgctgctctggctcccaggtgccagatgtgcccttgtgatgacccagactccagcctccgtgtctgaagctgtgggaggcacagtcaccatcaagtgccaggccagtcaggacatttacaataatttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctacagggcatccaatctggaatctggggtcccatcgcggttcaaaggcagtggatctgggacagagtatattctcaccatcaccgacctcgagtgtgacgatgctgccacttactactgtcaaggcggctattttactctttatacggataatagtttcggcggagggaccgaggtggtggtcaaaggtgatccagttgcacctactgtcctcatcttcccaccagctgctgatcaggtggcaactggaacagtcaccatcgtgtgtgtggcgaataaatactttcccgatgtcaccgtcacctgggaggtggatggcaccacccaaacaactggcatcgagaacagtaaaacaccgcagaattctgcagattgtacctacaacctcagcagcactctgacactgaccagcacacagtacaacagccacaaagagtacacctgcaaggtgacccagggcacgacctcagtcgtccagagcttcaataggggtgactgttag (SEQ ID NO. 8).

[0035] The amino acid sequence corresponding to SEQ ID NO. 8 is shown below as SEQ ID NO. 18:

[0036] MDTRAPTQLLGLLLLWLPGARCALVMTQTPASVSEAVGGTVTIKCQASQDIYNNLAWYQQKPGQPPKLLIYRASNLESGVPSRFKGSGSGTEYILTITDLECDDAATYYCQGGYFTLYTDNSFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC* (SEQ ID NO. 18).

[0037] The base sequence of the amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 7C10 is shown in SEQ ID NO. 9, and the base sequence of the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO. 10.

[0038]

[0039] The amino acid sequence corresponding to SEQ ID NO. 9 is shown below as SEQ ID NO. 19:

[0040] METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTVSGFSLSSDGMTWVRQAPGKGLEWIGYIYVRGSSPVYANWAKGRFTISKTSTTVDLTMTGLTTEDTATYFCARDASSSGYYGGFDPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK* (SEQ ID NO. 19).

[0041] atggacacgagggcccccactcagctgctggggctcctgctgctctggctcccaggtgccacatttgccctggtgatgacccagactccatcctccacgtctgaaccagtgggaggcacagtcaccatcaaatgccaggccagtgagagcatttatagcggcttatcctggtatcagcagaaaccagggcagcctcccaaactcctgatcgctgatgcatccactctggcatctggggtcccatcgcggttcagtgccagcagatctgggacagaatacagtctcaccgtcagcggcgtgcaatgtgacgatgctgccacttactactgtctaggtgtttatgcttataatgttgatgatgggcttgctttcggcggagggaccgaggtggtggtcaagggtgatccagttgcacctactgtcctcatcttcccaccagctgctgatcaggtggcaactggaacagtcaccatcgtgtgtgtggcgaataaatactttcccgatgtcaccgtcacctgggaggtggatggcaccacccaaacaactggcatcgagaacagtaaaacaccgcagaattctgcagattgtacctacaacctcagcagcactctgacactgaccagcacacagtacaacagccacaaagagtacacctgcaaggtgacccagggcacgacctcagtcgtccagagcttcaataggggtgactgttag (SEQ ID NO. 10).

[0042] The amino acid sequence corresponding to SEQ ID NO. 10 is shown below as SEQ ID NO. 20:

[0043] MDTRAPTQLLGLLLLWLPGATFALVMTQTPSSTSEPVGGTVTIKCQASESIYSGLSWYQQKPGQPPKLLIADASTLASGVPSRFSASRSGTEYSLTVSGVQCDDAATYYCLGVYAYNVDDGLAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC* (SEQ ID NO. 20).

[0044] The anti-human LY6D protein monoclonal antibody is prepared by the steps of antigen preparation, animal immunization, B cell screening and purification, and comprises the following steps:

[0045] Antigen preparation: the gene encoding human LY6D protein is cloned into pcDNA3.4 eukaryotic expression vector by genetic engineering technology, transformed into 293F cells for expression, and then the expressed LY6D protein is purified by affinity chromatography and the like to obtain high-purity human LY6D protein antigen;

[0046] The immunogen mFc-6his-Ly6d (1-97aa) / detection 1 mFc-6his-Ly6d (1-97aa) is expressed by 293F cells 30mL, cultured at 37℃, 125rpm, 8% CO2, and the supernatant is purified by His affinity chromatography, and the protein concentration reaches 1.04mg / mL;

[0047] The amino acid sequences of the immunogen mFc-6his-Ly6d (1-97aa) / detection 1 mFc-6his-Ly6d (1-97aa) are the same, and are shown as SEQ ID NO. 21. In the sequence, the 1st-97th amino acid corresponds to the 1st-97th amino acid sequence of Ly6d, the 98th-104th amino acid corresponds to the enterokinase sequence, the 105th-331th amino acid corresponds to the mFC sequence, and the 332nd-337th amino acid corresponds to the 6 His sequence, which is specifically as follows:

[0048] MRTALLLLAALAVATGPALTLRCHVCTSSSNCKHSVVCPASSRFCKTTNTVEPLRGNLVKKDCAESCTPSYTLQGQVSSGTSSTQCCQEDLCNEKLHADDDDKAVPRDSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKHHHHHH* (SEQ ID NO. 21).

[0049] The detection antigen 2 hFc-6His-Ly6d (21-98 aa) was also expressed by 293F cells in 30 mL, and the supernatant was purified by His affinity chromatography under the same culture conditions, and the protein concentration was 3.55 mg / mL; the amino acid sequence corresponding to the detection antigen 2 hFc-6His-Ly6d (21-98 aa) is shown in SEQ ID NO. 22, in which the 1st-78th amino acid corresponds to the amino acid sequence of the 21st-98th position of Ly6d, the 79th-85th amino acid corresponds to the TEV site, the 86th-87th amino acid MD is the natural N-terminal sequence of Ly6d protein, the 88th-318th amino acid corresponds to the hFC sequence, and the 319th-324th amino acid corresponds to the 6 histidine sequence, which is specifically as follows: LRCHVCTSSSNCKHSVVCPASSRFCKTTNTVEPLRGNLVKKDCAESCTPSYTLQGQVSSGTSSTQCCQEDLCNEKLHNENLYFQGMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH* (SEQ ID NO. 22).

[0050] Detection of 3 Ly6d (21-97aa) + 3*G4S + thrombin cleavage site + 6His tag 30 mL was expressed by 293F cells, and the supernatant was purified by His affinity chromatography, and the protein concentration was 0.4 mg / mL;

[0051] The amino acid sequence corresponding to the detection of 3 Ly6d (21-97aa) + 3*G4S + thrombin cleavage site + 6His tag is shown as SEQ ID NO. 23. In the sequence, the 1st-77th amino acid corresponds to the amino acid sequence of Ly6d at positions 21-97, the 78th-92nd amino acid corresponds to 3*G4S, the 93rd-98th amino acid corresponds to the thrombin cleavage site, and the 99th-104th amino acid corresponds to the 6 histidine sequence, as follows: LRCHVCTSSSNCKHSVVCPASSRFCKTTNTVEPLRGNLVKKDCAESCTPSYTLQGQVSSGTSSTQCCQEDLCNEKLHGGGGSGGGGSGGGGSLVPRGSHHHHHH* (SEQ ID NO. 23).

[0052] Detection of 4 Ly6d (21-97aa) + 3*G4S + thrombin cleavage site + 6His tag-mFC 30 mL was expressed by 293F cells, and the supernatant was purified by His affinity chromatography, and the protein concentration was 0.9 mg / mL.

[0053] The amino acid sequence of the detection agent 4 Ly6d (21-97aa) + 3*G4S + thrombin cleavage site + 6His tag-mFC corresponds to SEQ ID NO. 24. In the sequence, the 1st-77th amino acids correspond to the amino acid sequence at positions 21-97 of Ly6d, the 78th-92nd amino acids correspond to 3*G4S, the 93rd-98th amino acids correspond to the thrombin cleavage site, the 99th-325th amino acids correspond to the mFC sequence, and the 326th-331st correspond to 6 histidine sequences, specifically as follows: LRCHVCTSSSNCKHSVVCPASSRFCKTTNTVEPLRGNLVKKDCAESCTPSYTLQGQVSSGTSSTQCCQEDLCNEKLHGGGGSGGGGSGGGGSLVPRGSVPRDSGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKHHHHHH* (SEQ ID NO. 24).

[0054] The detection agent 5 Ly6d (21-97aa) + 3*G4S + thrombin cleavage site + 6His tag-hFC was expressed in 293F cells for 30 mL, and the supernatant was subjected to His affinity chromatography purification, and the protein concentration was 0.7 mg / mL.

[0055] The amino acid sequence of the detection agent 5 Ly6d (21-97aa) + 3*G4S + thrombin cleavage site + 6His tag-hFC corresponds to SEQ ID NO. 25. In the sequence, the 1st-77th amino acids correspond to the amino acid sequence at positions 21-97 of Ly6d, the 78th-92nd amino acids correspond to 3*G4S, the 93rd-98th amino acids correspond to the thrombin cleavage site, the 99th-329th amino acids correspond to the mFC sequence, and the 330th-335th correspond to 6 histidine sequences, specifically as follows:

[0056] LRCHVCTSSSNCKHSVVCPASSRFCKTTNTVEPLRGNLVKKDCAESCTPSYTLQGQVSSGTSSTQCCQEDLCNEKLHGGGGSGGGGSGGGGSLVPRGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH* (SEQ ID NO. 25).

[0057] The gene encoding the human LY6D protein is cloned into a pcDNA3.4 eukaryotic expression vector by genetic engineering technology, transformed into 293F cells for expression, and then the expressed LY6D protein is purified by affinity chromatography and the like, to obtain a high-purity human LY6D protein antigen;

[0058] Animal immunization: healthy rabbits are selected, and the immunogen is used for multiple immunization, and the serum is collected and the rabbit with the best immune effect is selected;

[0059] Spleen cell separation: the spleen cells of the immunized rabbit are separated, and B cell sorting and culture are performed;

[0060] Monoclonal antibody screening and preparation: the B cells are sorted by flow cytometry, a linear expression module (LEM) is constructed, the supernatant is collected after transfecting the host cells, and a monoclonal antibody clone (such as 3B9, 3G10, 7C10, 16F11, 6D12, etc.) with high binding capacity to the 21-98aa epitope of the LY6D protein is screened out.

[0061] In a second aspect, the application provides a recombinant plasmid pcDNA3.4 containing a base sequence encoding the heavy chain variable region VH and the light chain variable region VL of the 16F11, 3G10, 3B9, 6D12 or 7C10 gene of the anti-human LY6D protein monoclonal antibody.

[0062] In a third aspect, the application provides a recombinant 293F cell expressing an anti-human LY6D protein monoclonal antibody, which contains the above-mentioned recombinant plasmid pcDNA3.4.

[0063] In a fourth aspect, the application provides a use of the anti-human LY6D protein monoclonal antibody described above in the preparation of an antibody-drug conjugate (ADC) or a protein degradation targeting chimera drug (PROTAC) for treating pancreatic cancer.

[0064] By using the technical solution described above, the antibody-drug conjugate (ADC), i.e., the anti-human LY6D protein monoclonal antibody, is coupled with a cytotoxic drug (such as a maytansine derivative DM1 or a camptothecin analogue) through a cleavable or non-cleavable linker to form a targeted delivery system, which realizes targeted delivery of the drug to LY6D highly-expressed tumor cells, precisely kills the tumor cells, and reduces the toxicity to normal tissues.

[0065] Further, the protein degradation targeting chimera drug of the application recruits the PROTAC to the surface of the tumor cells by targeting LY6D to induce ubiquitination and degradation of the LY6D protein and block the cancer-promoting signaling pathway, which can overcome the drug resistance problem of traditional small molecule inhibitors and provide a new strategy for treating pancreatic cancer.

[0066] In a fifth aspect, the application provides an ELISA kit for detecting LY6D protein in human serum or tissue homogenate, which comprises an enzyme-labeled plate, a capture antibody, a detection antibody, a Neutravidin-HRP secondary antibody, and a standard.

[0067] The capture antibody is 3B9, 3G10, 7C10, 16F11, or 6D12.

[0068] The detection antibody is biotin-labeled 3B9, 3G10, 7C10, 16F11, or 6D12.

[0069] The standard is a purified Ly6d (21-97aa)+3*G4S+6His tag protein with a concentration range of 0-10 ng / mL.

[0070] The ELISA kit described above for detecting LY6D protein in human serum or tissue homogenate further comprises a diluent, a washing solution, a blocking solution, a coloring solution, and an ELISA termination solution.

[0071] By using the technical solution described above, the ELISA kit for detecting LY6D protein in human serum or tissue homogenate of the application can quantitatively or qualitatively detect the LY6D protein level in the serum or tissue sample, the minimum detection limit of the kit is 1 ng / ml, and the kit is suitable for early diagnosis of pancreatic cancer.

[0072] Further, through immunohistochemistry (IHC) and immunofluorescence (IF), the expression and distribution of LY6D protein in pancreatic cancer tissues can be visually detected by tissue section staining, which can provide auxiliary basis for pathological diagnosis. Through Western Blot, the molecular weight of LY6D protein in the sample can be specifically recognized, and the relative expression level can be quantitatively analyzed, which can provide accurate and reliable experimental data support for the expression verification of LY6D protein. Through flow cytometry (Flow Cytometry), the expression level of LY6D protein on the surface of tumor cells can be quantitatively analyzed, and based on the difference in protein expression, a reference basis for the development of individualized treatment plan can be provided.

[0073] The beneficial technical effects of the present application

[0074] The anti-human LY6D protein monoclonal antibody of the present application can obtain high-purity anti-human LY6D protein monoclonal antibody by recombinant DNA technology and recombinant 293F cell expression system, combined with GS screening system and affinity chromatography purification. The preparation process is stable and efficient, and is suitable for large-scale production.

[0075] The ELISA kit for detecting LY6D protein in human serum or tissue homogenate of the present application contains capture antibody (3B9, 3G10, 7C10, 16F11 or 6D12) and biotin-labeled (3B9, 3G10, 7C10, 16F11 or 6D12) detection antibody, which is widely used in pancreatic cancer sample detection and can be used in various detection methods such as ELISA, IHC, IF and flow cytometry. The minimum detection limit of the ELISA kit is 1 ng / ml, and the detection sensitivity is high.

[0076] The anti-human LY6D protein monoclonal antibody of the present application can be used in the preparation of antibody-drug conjugate (ADC) or protein degradation targeting chimera drug (PROTAC) for treating pancreatic cancer, which can accurately target LY6D positive tumor cells, improve efficacy and reduce side effects, providing a new effective means for the treatment of pancreatic cancer, and having good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0077] Figure 1 , SDS-PAGE diagram of anti-human LY6D monoclonal antibody (3B9, 3G10, 7C10, 16F11, 6D12);

[0078] Figure 2a 、 Figure 2b 、 Figure 2c, respectively, are the response curve graphs of sandwich ELISA of anti-human LY6D monoclonal antibodies (3B9, 3G10, 7C10, 16F11, 6D12) in different detection antigens 3 Ly6d (21-97aa) + 3*G4S + 6His tag, detection antigen 4 Ly6d (21-97aa) + 3*G4S + 6His tag-mFC and detection antigen 5 Ly6d (21-97aa) + 3*G4S + 6His tag-hFC;

[0079] Figure 3 , the scatter plot of LY6D content detection in serum of human pancreatic ductal adenocarcinoma (PDAC);

[0080] Figure 4, optical microscope images of immunohistochemical (IHC) staining of different antibodies (3B9, 3G10, 7C10, 16F11, 6D12, IgG as control);

[0081] Figure 5 , Western Blot of different antibodies (3B9, 3G10, 7C10, 16F11, 6D12, IgG as control, actin protein as internal reference) in PANC-1 cells;

[0082] Figure 6 , scatter plot of flow cytometry detection of different antibodies (3B9, 3G10, 7C10, 16F11, 6D12, IgG as control) in PANC-1 cells. DETAILED DESCRIPTION

[0083] The present application will be described in detail below through specific examples or application examples and in conjunction with the accompanying drawings, but the present application is not limited thereto.

[0084] In each embodiment of the present application, the PBS buffer used is a diluent obtained by diluting 10 times 10×PBS buffer (Shanghai Yezheng Biomedicine Technology Co., Ltd., Catalog No. PS110S) with deionized water;

[0085] The TBST buffer is TBS-T instant granules (1 L of 1×) (MCE, Catalog No. HY-K1025);

[0086] The name, model number and manufacturer information of the detection equipment used in each embodiment of the present application are as follows:

[0087] The microplate reader used for OD450nm\630nm detection is Varioskan LUX multifunctional microplate reader, Thermofisher

[0088] Flow cytometer, model BD LSRFortessa X20 (12F), produced by BD.

[0089] The various reagents, raw materials used in the embodiments of the present application are all commercially available, except for the special annotations in the embodiments.

[0090] The heritage resources used in the embodiments of the present application are as follows:

[0091] Plasmid pcDNA3.4, item number HG-VPH1386, HonorGene;

[0092] Trelief 5α competent cells, item number DZC101-96B100, GenScript Biotech Corporation;

[0093] 293T cells: SNL-015, Wuhan Shengene Biotech Co., Ltd;

[0094] 293F cells: A14528, Thermo Fisher Scientific.

[0095] Example 1

[0096] Preparation of an anti-human LY6D protein monoclonal antibody, specifically comprising the following steps

[0097] 1. Antigen preparation

[0098] · Immunogen mFc-6his-Ly6d (1-97aa) / Detection Original 1 mFc-6his-Ly6d (1-97aa) (persons skilled in the art can prepare the recombinant antigen according to existing genetic engineering technology) is expressed by 293F cells 30mL, cultured at 37℃, 125rpm, 8% CO2, and the supernatant is purified by His affinity chromatography, and the protein concentration reaches 1.04mg / mL;

[0099] · Detection Original 2 hFc-6His-Ly6d (21-98 aa) (persons skilled in the art can prepare the recombinant antigen according to existing genetic engineering technology) is also expressed by 293F cells 30mL, cultured under the same conditions, and the supernatant is purified by His affinity chromatography, and the protein concentration is 3.55mg / mL;

[0100] • In the detection of LY6D protein in human serum and tissue samples, the large tag (hFc or mFc) is relatively large, and there is steric hindrance. The 98th aa in the sequence is N (asparagine), and when G follows N, the presence of G will have some impact on N (such as the stability of N, such as deamidation, etc.), so N is deleted and G4S linker is added when redesigning the paired protein. Therefore, 21-97aa-3G4S linker is expressed, as follows:

[0101] • Detection antigen 3 Ly6d (21-97aa)+3*G4S+thrombin cleavage site+6His tag (a person skilled in the art can prepare a recombinant antigen according to existing genetic engineering technology) is expressed in 293F cells 30mL, and the supernatant is purified by His affinity chromatography, and the protein concentration is 0.4mg / mL.

[0102] • Detection antigen 4 Ly6d (21-97aa)+3*G4S+thrombin cleavage site+6His tag-mFC (a person skilled in the art can prepare a recombinant antigen according to existing genetic engineering technology) is expressed in 293F cells 30mL, and the supernatant is purified by His affinity chromatography, and the protein concentration is 0.9mg / mL.

[0103] • Detection antigen 5 Ly6d (21-97aa)+3*G4S+thrombin cleavage site+6His tag-hFC (a person skilled in the art can prepare a recombinant antigen according to existing genetic engineering technology) is expressed in 293F cells 30mL, and the supernatant is purified by His affinity chromatography, and the protein concentration is 0.7mg / mL.

[0104] 2、Animal immunization (rabbit)

[0105] • Select healthy rabbits with a body weight of 2.5-3.0 kg and an age of 6 months for immunization, and use immunogen mFc-6his-Ly6d (1-97aa) for multiple immunizations. The first immunization dose is 400μg / one, mixed with an equal volume of complete Freund's adjuvant (C.F.A) (Sigma-Aldrich, catalog number: F5881) to prepare an emulsifying agent, and injected subcutaneously (S.Q.) on the abdomen and back.

[0106] • The subsequent multiple immunization dose is 200μg / one, mixed with an equal volume of incomplete Freund's adjuvant (I.F.A) (Sigma-Aldrich, catalog number: F5506) to prepare an emulsifying agent, and also injected by S.Q.

[0107] • During the immunization process, collect serum at predetermined times for subsequent detection.

[0108] • According to the serum test results, the rabbit with the best immune effect was sacrificed and the spleen was collected for subsequent cell separation and screening.

[0109] 3. Serum ELISA detection

[0110] Conventional ELISA experiments were carried out on serum (serum after rabbit immunization) using the detection antigen 1 mFc-6his-Ly6d (1-97aa).

[0111] • The detection antigen 1 mFc-6his-Ly6d (1-97aa) was diluted to 1 μg / mL with 1x PBS buffer, coated a 384-well plate at 25 μl / well, and incubated at 4°C overnight;

[0112] • Wash 5 times with PBST (1*PBS + 0.1% Tween 20) 75 μl / well;

[0113] 50 μl / well 5% skim milk + 1% BSA in PBST (1*PBS + 0.05% Tween 20) to block non-specific binding sites, incubate at room temperature for 1 h, then wash as before;

[0114] • The post-immune serum was first diluted 1:1000, then 3-fold diluted into 8 gradients (0.001, 0.0003333, 0.0001111, 0.000037, 0.0000123, 0.0000041, 0.0000014, 0.0000005); add 25 μl / well, incubate at room temperature for 1 h, then wash as before;

[0115] • Secondary antibody (HRP Goat Anti-Rabbit IgG (H+L), Jackson ImmunoResearch, 111-035-045) was diluted 1:5000 with 1x PBS buffer, added to the 384-well plate at 25 μl / well, and incubated at room temperature for 1 h in the dark, then washed as before;

[0116] • Color developing solution (TMB, Thermo Fisher, CAT: 34029) was diluted 1:5 with 1x PBS buffer, added to the 384-well plate at 25 μl / well, and developed at room temperature for 3 min in the dark;

[0117] • Add 10 μl of stop solution (2M H2SO4) to each well of the 384-well plate to stop the reaction;

[0118] • Read the plate at 450nm\630nm, use 450nm minus 630nm background, read the plate results (OD 450-630 values) as shown in Table 1:

[0119] Table 1. Rabbit six immunization serum routine ELISA test results

[0120]

[0121] Table 1, "negative" refers to the serum without immunization; "five immunization" refers to the serum collected after 5 times of immunization; "six immunization" refers to the serum collected after 6 times of immunization; NC represents blank control;

[0122] In Table 1, the detection value of the "negative" column as a negative control has obvious difference with the values of the "five immunization" and "six immunization" columns, indicating that the experiment can effectively distinguish negative samples and positive samples (five immunization and six immunization serum samples) after immunization, reflecting the specificity of the experiment, that is, the ELISA detection method can specifically detect the target substance in the serum after immunization, and will not produce non-specific reaction to the negative sample.

[0123] As can be seen from Table 1, with the decrease of serum dilution, the OD 450-630 value shows a gradient downward trend, and its change completely conforms to the gradient law of concentration dilution. In this experiment, 2 rabbits were included, and the sixth immunization serum (six immunization serum) of rabbit No. E20756 showed strong reactivity to the detection original 1 (mFc-6his-Ly6d (1-97aa)) protein, which met the screening standard of monoclonal antibody.

[0124] 4. Preparation of polyclonal antibody

[0125] Protein A purification, the steps are as follows:

[0126] 1) Sample incubation: including matrix washing, equilibration, incubation. That is, take the matrix (Biolab, 1024-1822) out of the 4°C refrigerator, use a pipette to suck the appropriate volume of matrix into the purification column tube. After the matrix storage solution is completely drained, wash with pure water for 4-6 column volumes; equilibrate the filler with 1*PBS for 6 column volumes;

[0127] Mix the equilibrated matrix with the E20756 four immunization serum sample, label it, seal it, and incubate it in a rotary incubator, with the incubation conditions being 20 rpm, 4°C, and 40-60 min;

[0128] 2) Flow through: after the above incubation is completed, centrifuge and collect the supernatant, i.e. the flow-through. Reserve about 5 mL of supernatant for suspending the matrix, and transfer the matrix to the purification column tube. After the flow-through in the column tube is completely drained, collect all the flow-through serum;

[0129] • 3) Wash and elution: elute the column with 1*PBS for 2-3 column volumes, collect the first column volume of wash (white, Biosharp BL601A) and combine with flow-through serum; sequentially use 0.1M Gly (pH 3.0) and 0.1M Gly (pH 2.2) for elution, after elution, adjust the pH of the antibody solution to 8.0-8.5, use Nanodrop one antibody detector to determine the antibody concentration, and then perform antigen affinity purification;

[0130] • Antigen affinity purification

[0131] Similar to the above Protein A purification, including sample incubation, flow-through, wash and elution, etc. Finally, perform polyclonal antibody routine ELISA experiment, method same as above serum ELISA detection, plate reading results (OD 450-630 values) are shown in Table 2:

[0132] Table 2. Purified polyclonal antibody routine ELISA detection results of rabbit four immune serum

[0133]

[0134] 1:1k, 1:3k, 1:9k, 1:27k, 1:81k, 1:243k, 1:729k, 1:2187k in Table 2 are corresponding to different dilutions, and Blank is a blank control.

[0135] As can be seen from the above Table 2, the polyclonal antibody obtained after the above purification of the rabbit four immune serum E20756 has strong immunoreaction to the detection antigen 1 (mFc-6his-Ly6d (1-97aa)), which indicates that the purified polyclonal antibody has good specificity and binding ability to the target detection antigen 1 (mFc-6his-Ly6d (1-97aa)).

[0136] 5. Spleen cell separation

[0137] • Transfer the spleen of E20756 after rabbit six immunization into a sterile culture dish, and pre- place RPMI 1640 medium (Gibco, CAT: C11875500BT) containing 1% Pen Strep (gibco, CAT: 15140-122) in the culture dish. Use sterile tissue forceps and dissecting scissors to cut the spleen, and place it in a sterile cell filter. Then, gently grind the spleen tissue with a plastic grinding rod, so that the spleen cells pass through the cell filter net into the culture medium.

[0138] • Transfer the spleen cell suspension from the culture dish to a 50 mL conical tube (Jetex, CFT010500), and add 1% Pen / Strep RPMI 1640 medium to adjust the total volume to approximately 50 mL. Centrifuge at 400 g for 5 min and discard the supernatant. Resuspend the cells in 13 mL of 1×ACK erythrocyte lysis buffer (BioGems, 64010-00-100) and incubate at room temperature for 1 min. Then, add 1% Pen / Strep RPMI 1640 medium to bring the volume to 50 mL, and gently invert the conical tube to mix the cell suspension. Centrifuge again at 400 g for 5 min, discard the supernatant, and resuspend the cells in 50 mL of RPMI 1640 medium containing 1% Pen / Strep.

[0139] • After resuspending the cells in B cell culture medium (ABClonal), filter the cell suspension through a 70μm cell filter (Jet, CSS013070) to remove any cell clumps, and take samples for cell counting to ensure the number of cells required for subsequent experiments.

[0140] Take 1×10 8 Up to 1.5×10 8 Spleen cells (in this example, E20756 rabbit cultured at 1.38 × 10⁻⁶) 8 Cells were used for culture, and the cell density was adjusted to 2-3 × 10⁻⁶. 6 cells / mL;

[0141] The remaining spleen cells were 9 × 10 7 / pipe, 5×10 7 Cells were resuspended at a density of 1 / mL in cryopreservation solution (90% serum + 10% DMSO, Sigma, D4540-1L), aliquoted into cryovials, and placed in a programmed cooling box (Corning, 432008) at a cooling rate of -1℃ / min overnight at -80℃. The next day, the frozen cells were transferred to a liquid nitrogen tank for long-term storage.

[0142] 6. B cell sorting and routine ELISA detection

[0143] • B cell sorting experiment: two B cell sorting experiments were performed. First, the E20756 rabbit six-immune spleen cells were incubated with 3 μg of sorting immunogen protein biotin-hFc-6His-Ly6d (21-98 aa) at room temperature for 20 min; then, the first centrifugal washing operation was performed, followed by the addition of 1 ul of fluorescent secondary antibody, and after the second centrifugal washing, the sorting was performed using a flow cytometer, and the collected cells were cultured at 37°C for 12 days to obtain single B cell clone samples for subsequent RNA extraction;

[0144] The collected supernatant, i.e. B cell supernatant (collected after B cells were sorted and cultured for 12 days), was used for ELISA detection;

[0145] • B cell supernatant routine ELISA detection: the detection protein 2 hFc-6His-Ly6d (21-98 aa) screened was used to coat the 384-well plate, then 10 μL of B cell supernatant and 15 μL of diluent (1% BSA and 0.05% Tween-20 in PBS solution) were added to each well, and a secondary antibody (HRP Goat Anti-Rabbit IgG (H+L), Jackson ImmunoResearch, 111-035-045) was used.

[0146] 7. Preparation of LEM vector: total RNA was extracted from the single B cell clone samples screened above, reverse transcription was performed using a reverse transcription kit (ABScript II RT, ABclonal, RK21400) to synthesize cDNA, VH and VL genes were amplified, and a linear expression module (LEM) was constructed. The specific steps are as follows:

[0147] (1) RNA extraction: total RNA was isolated and extracted from the single B cell clone samples obtained from the previous screening, which was the initial template for obtaining antibody genes (VH and VL);

[0148] (2) cDNA synthesis: a specific reverse transcription kit (ABScript II RT, ABclonal, Catalog No. RK21400) was used to convert the extracted total RNA into complementary DNA (cDNA) through a reverse transcription reaction, solving the problem of RNA degradation, and providing a stable template for subsequent gene amplification;

[0149] (3) VH / VL gene amplification: using synthetic cDNA as a template, the heavy chain variable region gene (VH) and the light chain variable region gene (VL) of the antibody are specifically amplified by PCR technology, and these two genes are the key fragments that determine the specificity of the antibody;

[0150] (4) LEM construction: the amplified VH and VL genes are reacted with 2xVazyme LAmp Master Mix (Novazyme, CAT: P311-02) to form a linear expression module LEM (Linear Expression Module, LEM) that can be directly used for expression, laying a foundation for subsequent in vitro expression or vector integration of the antibody;

[0151] The reaction system of the above reaction is as follows:

[0152] LEM-H reaction system

[0153]

[0154] The running program of the above LEM construction process is as follows:

[0155] 1) pre-denaturation at 94℃ for 30 min;

[0156] 2) denaturation at 94℃ for 20 s;

[0157] 3) annealing at 55℃ for 30 s;

[0158] 4) extension at 68℃ for 4 min;

[0159] 5) post-extension at 68℃ for 5 min;

[0160] 6) 30 cycles;

[0161] After the above program is run, 1% agarose gel is used for PCR product detection, and the detection results show that the main band (purity > 85%) is recovered using a PCR product purification kit (Tiangen, DP204-3).

[0162] (5) LEM vector expression: 293T cells are transferred into a culture well plate, and LEM vector transfection is performed according to the virus transfection method (this method is a routine operation method for those skilled in the art) (LEM vector 1.2 μg, 293T cell number 3.6X10 6 ), and the temperature is controlled at 37℃ for 48 h after transfection of the LEM vector to collect the cell supernatant;

[0163] The 293T cell is a mammalian cell, and the optimal temperature for its in vitro culture is usually 37℃ (simulating the physiological environment in vivo), which can ensure normal cell metabolism, efficient expression of vectors, and secretion of target proteins.

[0164] The cell supernatant was subjected to routine ELISA detection and capture ELISA detection using the detection antigen 2hFc-6His-Ly6d (21-98 aa). The detection results are shown in Tables 3 and 4.

[0165] Table 3: Routine ELISA detection results of cell supernatant

[0166]

[0167] In Table 3 above, NC represents unimmunized serum, and positive represents positive serum.

[0168] As can be seen from Table 3 above, except for 13H9 and 17A2, 3A10, 3B9, 3G10, 6D12, 7A7, 7C10, 8B3, 10B4, 14D7, 14E1, 16F11, 1D7, 2G6, 3G12, 6C11, 8H3, 9B2, and the like, 17 clone samples have good binding capacity with the detection antigen 2hFc-6His-Ly6d (21-98 aa), and the preferred ones are 3B9, 3G10, 7C10, 16F11, or 6D12.

[0169] Table 4: Capture ELISA detection results of cell supernatant

[0170]

[0171] In Table 4 above, NC represents unimmunized serum, and Blank represents a blank control.

[0172] As can be seen from Table 4, 3A10, 3B9, 3G10, 6D12, 7A7, 7C10, 8B3, 10B4, 14D7, 14E1, 16F11, 1D7, 2G6, 3G12, 6C11, 8H3, 9B2, 13H9, 17A2, and the like, 19 clone samples have good capture antibody function, and the more preferred ones are 3B9, 3G10, 7C10, 16F11, or 6D12.

[0173] 8. Construction of monoclonal antibody vector

[0174] The vector pcDNA3.4_OSLIC_Hc and pcDNA3.4_OSLIC_Lc were respectively single-enzymed (Invitrogen™, V79020) using NheI (ABclonal, RK21111) and XbaI (ABclonal, RK21112) to obtain linearized pcDNA3.4 plasmids;

[0175] The VH and VL genes corresponding to the 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D12 in the LEM preparation process were respectively recombined with the linearized pcDNA3.4 plasmid by homologous recombinase (ABclonal, RK21020) to form the connection products corresponding to the 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D1.

[0176] The Trelief 5α competent cells (Qingke Biology, DZC101-96B100) were ice-bathed for 30 min, then heat-shocked at 42°C for 1 min, and then ice-bathed for 2 min. 5 μl of the connection products corresponding to the 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D1 were respectively added to 50 μl of Trelief 5α competent cells, and then sequentially treated by ice-bathing for 30 min, heat-shocking at 42°C for 1 min, and ice-bathing for 2 min to complete the transformation, thereby obtaining the transformation systems corresponding to the 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D1.

[0177] 400 μl of preheated LB medium was added to the transformation systems corresponding to the 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D1, and then cultured at 37°C for 1 h to obtain bacterial liquid corresponding to the 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D1. 100 μl of the bacterial liquid was respectively inoculated on Amp-resistant plates (Biofroxx, 1146GR005).

[0178] After the single colonies were respectively inoculated in LB medium for expansion culture, the recombinant plasmid pcDNA3.4 was extracted using a plasmid extraction kit (Tiangen Biology, DP103), and then sent to Shanghai Qingke Biology Technology Co., Ltd. for sequencing verification by Sanger method. The results showed that the recombinant plasmid pcDNA3.4 containing the VH and VL genes corresponding to the 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D1 was constructed correctly.

[0179] 9. Monoclonal antibody expression

[0180] The correct recombinant plasmid pcDNA3.4 verified by sequencing was transfected into host cells 293F cells, and after culture, the supernatant containing the target antibody was collected, and the core was to realize the in vitro scale expression of the antibody. The specific steps are as follows:

[0181] Cell preparation: 1 day before transfection, take 20 ml of 293F cell suspension with a concentration of 1.2×10 6 ~1.6×10 6 cells / ml, add to the reaction tube (NEST, 788211) for standby;

[0182] Transfection mixture preparation: take 13ug (heavy chain: light chain = 1:2) of the above obtained recombinant plasmid of the corresponding 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D12 respectively, dilute with Opti-MEM medium (Gibco™, item number 31985070), respectively add 39ul of PEI-MAX (Polysciences 24765-1) (PEI volume is 3:1 ratio of PEI (ul): total DNA (ug)) transfection reagent with a concentration of 1 mg / ml (pH value 7.1), the transfection volume is 30ml, gently mix, incubate at room temperature for 15 min, to form a stable plasmid-transfection reagent complex of recombinant plasmid pcDNA3.4 and transfection reagent;

[0183] Transfection and culture: add 10ml of the above plasmid-transfection reagent complex to the above reaction tube (NEST, 788211) containing 20ml of 293F cell suspension with a concentration of 1.2×10 6 ~1.6×10 6 cells / ml, incubate at 37℃, 5% CO2 for 4-6h, then dilute the transfection reagent toxicity with fresh complete medium (DMEM high glucose medium, Gibco™, 11965092), that is, the recombinant 293F cells containing the corresponding 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D1, control the temperature at 37℃ for 72~96h, then collect the cell culture supernatant, that is, the cell culture supernatant containing the anti-human LY6D monoclonal antibody (corresponding to the 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D1).

[0184] 10. Monoclonal antibody purification

[0185] This step uses the specific binding of Protein A to the Fc fragment of the antibody to separate and purify the target anti-human LY6D monoclonal antibody from the above anti-human LY6D monoclonal antibody-containing cell culture supernatant (corresponding to 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D1), and ensure the quality of the antibody by dialysis and concentration determination.

[0186] Preparation of cell culture supernatant and Protein A Agarose:

[0187] Centrifuge the cell culture supernatant at 400xg to remove cell debris and other impurities;

[0188] Take 300 μl Protein A Agarose suspension (Nanobiology, 17013-070100) and add it to a 1.5 ml EP tube (NEST, 61540). Wash with PBS buffer for 2 times to obtain the washed Protein A Agarose;

[0189] Antibody binding: Mix the washed Protein A Agarose with the centrifuged cell supernatant and incubate for 3-4 h with shaking, then centrifuge at 400xg;

[0190] Transfer the Protein A Agarose to an empty column (Pierce™ Centrifuge Columns MAX (Thermo 89868)), wash with PBS buffer for 2 times to remove non-specifically bound impurities, then add elution buffer and collect the eluate containing anti-human LY6D monoclonal antibody.

[0191] Dialysis and concentration determination:

[0192] Determine the concentration of anti-human LY6D monoclonal antibody protein in the eluate using a Nanodrop (Ossen, Nano-300);

[0193] Place the eluate at 4°C and dialyze against 1*PBS buffer for at least 5 h to remove the elution buffer components;

[0194] After dialysis, determine the concentration of anti-human LY6D monoclonal antibody protein again using a Nanodrop, and store the sample after confirming the concentration.

[0195] The antibody concentrations corresponding to the 5 antibodies 3B9, 3G10, 7C10, 16F11 and 6D1 are as follows in Table 5;

[0196] Table 5, Nanodrop determination of anti-human LY6D monoclonal antibody protein concentration

[0197]

[0198] The base sequences of the obtained five antibodies 3B9, 3G10, 7C10, 16F11 and 6D1 were verified, and the results showed that the base sequence of the heavy chain variable region VH encoding the antibody 16F11 was consistent with SEQ ID NO. 1, and the base sequence of the light chain variable region VL was consistent with SEQ ID NO. 2; the base sequence of the heavy chain variable region VH encoding the antibody 3G10 was consistent with SEQ ID NO. 3, and the base sequence of the light chain variable region VL was consistent with SEQ ID NO. 4; the base sequence of the heavy chain variable region VH encoding the antibody 3B9 was consistent with SEQ ID NO. 5, and the base sequence of the light chain variable region VL was consistent with SEQ ID NO. 6; the base sequence of the heavy chain variable region VH encoding the antibody 6D12 was consistent with SEQ ID NO. 7, and the base sequence of the light chain variable region VL was consistent with SEQ ID NO. 8; the base sequence of the heavy chain variable region VH encoding the antibody 7C10 was consistent with SEQ ID NO. 9, and the base sequence of the light chain variable region VL was consistent with SEQ ID NO. 10.

[0199] 11. Monoclonal antibody SDS-PAGE detection

[0200] By distinguishing the antibody subunit under reducing / non-reducing conditions, combined with specific electrophoresis and imaging parameters to obtain protein band information, the specific steps are as follows:

[0201] Gel preparation: 4-20% precast gel (KingsRy, item number M00928) was selected, and the electrophoresis device was assembled according to the precast gel instruction;

[0202] Sample preparation:

[0203] Reducing sample: 5ul of 4x reducing loading buffer (6*loading buffer, ABClona, containing reducing agent DTT) was added to 5ug of antibody sample (anti-human LY6D monoclonal antibody 3B9, 3G10, 7C10, 16F11, 6D12 in Table 5 above), and after mixing well, heating at 95℃ for 10 min;

[0204] Non-reducing sample: 5ul of 4x non-reducing loading buffer (6*loading buffer, ABClona, without reducing agent DTT) was directly added to 5ug of antibody sample (anti-human LY6D monoclonal antibody 3B9, 3G10, 7C10, 16F11, 6D12 in Table 5 above), and mixed well (without heating);

[0205] Sample loading: For both reducing and non-reducing samples, load 4 μg of anti-human LY6D monoclonal antibody protein (3B9, 3G10, 7C10, 16F11, 6D12) per well, along with 5 μl of protein maker (TransGen Biotech, DM131-01) for molecular weight reference.

[0206] Electrophoresis conditions: 180V constant voltage electrophoresis for 45 minutes;

[0207] Staining and destaining: After electrophoresis, remove the gel and stain it thoroughly in Coomassie Brilliant Blue staining solution. After staining, treat it with destaining solution until the background is clear and the protein bands are obvious.

[0208] The destaining gel was scanned using a Clinx ChemiScope 6000 imaging system to acquire and save protein band images for subsequent analysis. The SDS-PAGE image of the obtained anti-human LY6D monoclonal antibody is shown below. Figure 1 As shown in the figure, 1, 2, 3, 4, and 5 correspond to reducing samples of 3B9, 3G10, 7C10, 16F11, and 6D1, respectively; 6, 7, 8, 9, and 10 correspond to non-reducing samples of 3B9, 3G10, 7C10, 16F11, and 6D1, respectively. Figure 1 The images show complete antibody bands and heavy and light chain bands for monoclonal antibodies 3B9, 3G10, 7C10, 16F11 and 6D1. The purity of each monoclonal antibody 3B9, 3G10, 7C10, 16F11 and 6D1 is ≥90%.

[0209] 12. Routine ELISA detection of monoclonal antibodies

[0210] An ELISA assay procedure for detecting the binding activity of monoclonal antibodies (5 strains including 3B9) against the original 2 hFc-6His-Ly6d (21-98 aa) was developed. This procedure involves antigen coating, antibody incubation, enzyme-labeled secondary antibody binding, and colorimetric reaction to quantitatively analyze the specific binding ability of the antibody to the antigen. Based on the antigen coating method, the target antigen (hFc-6His-Ly6d (21-98 aa)) was immobilized on a 384-well plate. Then, a cascade reaction of "monoclonal antibody to be tested → HRP-labeled secondary antibody → TMB colorimetric reaction" was used to convert the antibody binding signal into a quantifiable result. The operational steps are as follows:

[0211] Antigen coating: Dilute the original 2 protein hFc-6His-Ly6d (21-98 aa) to 1 μg / mL with PBS, coat 25 μl per well of a 384-well plate, and incubate overnight at 4°C to immobilize the antigen on the surface of the plate, providing a target for subsequent antibody binding.

[0212] Washing: Add 75 μl washing solution to each well, wash 5 times; wash away unbound free antigen;

[0213] Blocking: Add 50 μl blocking solution to each well, incubate at room temperature for 1 h; repeat the above washing process; block the blank sites of the well plate to reduce non-specific binding;

[0214] Incubation of the antibody to be tested: The monoclonal antibody to be tested (3B9, 3G10, 7C10, 16F11, 6D12) is diluted by 3 times in gradient, 25 μl per well, and incubated at room temperature for 1 h, and then washed as above, so that the different concentrations of the antibody to be tested can specifically bind to the fixed antigen (the detected protein), and the subsequent gradient concentration can be used to determine the affinity of the antibody;

[0215] Incubation of the secondary antibody: HRP (horseradish peroxidase) labeled secondary antibody (Jackson ImmunoResearch, 111-035-045, 1:5000) is added, 25 μl per well, and incubated at room temperature for 1 h in the dark, and then washed, 75 μl of washing solution is added to each well, and washed 5 times; the HRP labeled secondary antibody binds to the monoclonal antibody to be tested, providing enzyme molecules for subsequent color development;

[0216] Color development: TMB color development solution (Thermo 34029, 1:5 dilution) is added, 25 μl per well, and color developed at room temperature in the dark for 3 min;

[0217] Termination: 10 μl of termination solution (2M H2SO4) is added to terminate the reaction;

[0218] Result reading: The microplate reader reads the absorbance values at 450 nm (main wavelength, detects color development signal) and 630 nm (reference wavelength, corrects background), and the microplate reader reads the absorbance values at 450 nm / 630 nm as shown in Table 6:

[0219] Table 6, ELISA detection results of monoclonal antibodies

[0220]

[0221] The Blank in Table 6 above is the blank control, and the microplate reader data corresponding to each antibody is shown on the left at 450 nm and on the right at 630 nm;

[0222] As can be seen from Table 6, the five antibodies 3B9, 3G10, 7C10, 6D12 and 16F11 have good binding ability with the specific antigen (hFc-6His-Ly6d (21-98 aa).

[0223] 13. Monoclonal antibody capture ELISA detection, specifically comprising the following steps:

[0224] Coating antibody: Dilute the recombinant monoclonal antibody (3B9, 3G10, 7C10, 16F11, 6D12) to 1 μg / mL with PBS, add 25 μl of the dilution to coat the 384-well plate, and incubate at 4°C overnight;

[0225] Washing and blocking: follow the washing and blocking steps in the above-mentioned monoclonal antibody regular ELISA detection;

[0226] Antigen dilution and incubation: dilute the biotinylated antigen hFc-6His-Ly6d (21-98 aa) by 1:3 from 1 μg / ml, add 25 μl of the diluted biotinylated antigen to each well, and incubate at room temperature for 1 h;

[0227] Secondary antibody incubation: add NeutrAvidin-HRP secondary antibody (item number: Thermo 31001), dilute by 1:20000, add 25 μl of the diluted NeutrAvidin-HRP secondary antibody to each well, and incubate at room temperature in the dark for 1 h, then wash, add 75 μl of washing solution to each well, and wash 5 times;

[0228] Color development, termination, and result reading: the same as the monoclonal antibody regular ELISA detection, and the plate reading results are shown in Table 7:

[0229] Table 7. Monoclonal antibody capture ELISA detection results

[0230]

[0231] As can be seen from Table 7, the five antibodies 3B9, 3G10, 7C10, 6D12, and 16F11 all have capture functions.

[0232] 14. Monoclonal antibody sandwich ELISA detection of antigen

[0233] Dilute the recombinant monoclonal antibody (3B9, 3G10, 7C10, 16F11, 6D12) to 1 μg / mL with PBS, add 25 μl to coat the 384-well plate, and incubate at 4°C overnight;

[0234] Washing and blocking: follow the washing and blocking steps in the above-mentioned monoclonal antibody regular ELISA detection;

[0235] The detection of Ly6d (21-97aa) + 3*G4S + 6His tag -mFc, Ly6d (21-97aa) + 3*G4S + 6His tag -hFc, Ly6d (21-97aa) + 3*G4S + 6His tag respectively with 1 ug / ml starting, 1:3 gradient dilution, 25ul per well into the plate, incubated at room temperature for 1h;

[0236] Add biotinylated detection antibody (E20756 pAb-Biotin) (1 ug / ml, prepared according to existing genetic engineering technology), 25ul per well, incubated at room temperature for 1h, then washed, the washing was added with 75ul per well, washed 5 times;

[0237] Add NeutrAvidin-HRP secondary antibody (Thermo 31001, 1:20000 dilution), 25ul per well, incubated at room temperature for 1h, then washed, the washing was added with 75ul per well, washed 5 times;

[0238] Color development, termination and reading of OD 450-600 The results are the same as the conventional ELISA detection of monoclonal antibody;

[0239] The results of sandwich ELISA detection of antigen by monoclonal antibody (3B9, 3G10, 7C10, 16F11, 6D12) are shown in Figure 2a 、 Figure 2b 、 2c The horizontal coordinate in each figure is the concentration of the detection antigen, and the vertical coordinate is OD 450-600 , Figure 2a -c corresponds to the detection antibody E20756 pAb-Biotin, and the antigen is Ly6d (21-97aa) + 3*G4S + 6His tag -mFc, Ly6d (21-97aa) + 3*G4S + 6His tag -hFc, Ly6d (21-97aa) + 3*G4S + 6His tag; from Figure 2a -c can be seen that with the increase of Protein concentration (antigen protein concentration), OD450-600 (absorbance value, reflecting the strength of antigen-antibody reaction) shows an upward trend, that is, it conforms to the rule that the antigen concentration is positively correlated with the signal in ELISA detection, indicating that the detection system is basically effective, and the antibody can specifically bind to the corresponding antigen and produce a detectable signal. It is shown that the anti-human LY6D monoclonal antibody of the application can effectively bind to the antigen, the detection system is reliable, and can be used for preliminary antigen detection analysis, which also indicates the specificity of the antigen and antibody interaction.

[0240] 15. Monoclonal antibody sandwich ELISA to detect cell supernatant

[0241] Dilute the recombinant monoclonal antibodies (3B9, 3G10, 7C10, 16F11, 6D12) to 1 μg / mL with PBS, 25 μl per well to coat 384-well plates, 4°C overnight;

[0242] Wash and block, as described in the wash and block steps of the monoclonal antibody regular ELISA assay above;

[0243] Dilute the cell supernatant with a 3-fold gradient starting from 1:2, 25 μl per well, incubate at room temperature for 1 h;

[0244] Add biotinylated detection antibody (1 ug / ml), 25 μl per well, incubate at room temperature for 1 h;

[0245] Add NeutrAvidin-HRP secondary antibody (Thermo 31001, 1:20000 dilution), 25 μl per well, incubate at room temperature in the dark for 1 h;

[0246] Develop, stop and read results as in the monoclonal antibody regular ELISA assay, the plate reading results for different monoclonal antibodies (3B9, 3G10, 7C10, 16F11, 6D12) are shown in Tables 8.1, 8.2, 8.3, 8.4, 8.5, respectively;

[0247] Table 8.1. Monoclonal antibody 3B9 sandwich ELISA to detect cell supernatant results

[0248]

[0249] 8.2 Monoclonal antibody 3G10 sandwich ELISA to detect cell supernatant results

[0250]

[0251] 8.3 Monoclonal antibody 7C10 sandwich ELISA to detect cell supernatant results

[0252]

[0253] 8.4 Monoclonal antibody 16F11 sandwich ELISA to detect cell supernatant results

[0254]

[0255] 8.5 Monoclonal antibody 6D12 sandwich ELISA to detect cell supernatant results

[0256]

[0257] From Table 8.1, 8.2, 8.3, 8.4, 8.5, it can be seen that 16 pairs of antibody pairs can be paired to recognize cell supernatant: 3B9-3G10 (biotin), 3B9-7C10 (biotin), 3B9-6D12 (biotin), 3G10-3B9 (biotin), 3G10-7C10 (biotin), 3G10-6D12 (biotin), 7C10-3B9 (biotin), 7C10-3G10 (biotin), 7C10-6D12 (biotin), 6D12-3B9 (biotin), 6D12-3G10 (biotin), 6D12-7C10 (biotin), 16F11-3B9 (biotin), 16F11-3G10 (biotin), 16F11-7C10 (biotin), 16F11-6D12 (biotin).

[0258] Example 2 Preparation and detection of enzyme-linked immunoassay kit

[0259] The content of LY6D protein in pancreatic cancer samples was detected by double antibody sandwich ELISA method. The ELISA kit contains capture antibody (take monoclonal antibody 3G10), biotin-labeled detection antibody (biotin-labeled monoclonal antibody 3B9), Neutravidin-HRP secondary antibody and standard (purified Ly6d (21-97aa)+3*G4S+6His tag protein). The specific operation steps are as follows:

[0260] Coating: add capture antibody (2 μg / mL) to 96-well enzyme-labeled plate, 100 μL per well, 4°C incubate overnight;

[0261] Washing: discard the liquid in the hole, add 300 μL of washing liquid (20X ELISA washing liquid (Shengong, E661005) to each hole, wash 5 times, 1 min each time.

[0262] Blocking: add 250 μl of blocking solution (containing 5% skimmed milk, 1% BSA and 0.05% Tween-20 in PBS solution) to each well, 4°C incubate overnight;

[0263] Washing: same as above;

[0264] Loading: Add standard sample (dilute standard (purified Ly6d (21-97aa) + 3*G4S + 6His tag) with 1*PBS buffer) with concentration gradient (0, 0.01, 0.04, 0.156, 0.625, 2.5, 5, 10 ng / mL series concentration) and pancreatic cancer patient serum sample (dilute 3 times with 1*PBS buffer for pancreatic cancer patient serum), 100 μL per well, set PBS blank control well, incubate at room temperature for 1 h, set 2 replicate wells for each sample;

[0265] Washing: Same as above;

[0266] Detection antibody: Add biotin-labeled monoclonal antibody (1 ug / ml), 100 μL per well, incubate at room temperature for 1 h;

[0267] Washing: Same as above

[0268] Secondary antibody: Add Neutravidin-HRP (0.1 ug / ml), 100 μL per well, incubate at room temperature for 1 h;

[0269] Washing: Same as above;

[0270] Color development: Add 100 μL color developing solution (1-Step™ Ultra TMB-ELISA substrate solution (Thermofisher, 34028)) to each well, incubate at room temperature in the dark for 15-30 min. (Adjust according to color development, color development time in this example is 3 min);

[0271] Termination: Add 100 μL termination solution to each well to terminate the color development reaction.

[0272] Detection: Read the absorbance value at 450 nm (read 630 nm as the reference wavelength) on the microplate reader.

[0273] Preparation of standard curve: Take the calibrated OD450 value of the standard sample (calibrated OD450 value = OD450 average value of each sample minus OD450 average value of the blank control well (0 point)) as the abscissa, and the concentration (ng / ml) of the standard sample as the ordinate, linearly regress the standard curve, and obtain the curve equation: y = 0.47508x, R2= 0.9824.

[0274] Take different batches of the above ELISA kit respectively, and perform linear verification, the result is that when the linear range is 0.01-10 ng / mL series concentration, R 2 ≥0.9800.

[0275] The storage stability of the above-mentioned ELISA kit was verified. The results showed that after storage at 4°C for 1 month, 3 months, 6 months, 9 months, and 12 months, the average OD of the ELISA kit remained stable. 450 The rate of change (%) of the values ​​was 0.4%-4.3%, which meets the requirement of a rate of change (%) ≤5%. This indicates that the above-mentioned ELISA kit has good storage stability.

[0276]

[0277] The calibrated OD450 values ​​of each serum sample from pancreatic cancer patients (calibrated OD450 value = average OD450 value of each sample minus the average OD450 value of the blank control well (0 point)) are shown in Table 9.

[0278] Table 9. Detection of LY6D content in serum of human pancreatic cancer patients

[0279] Based on the calibrated OD450 value of PDAC (pancreatic cancer patient serum samples) and the above-mentioned standard curve equation, the corresponding LY6D content in the pancreatic cancer patient serum samples was calculated, and the scatter plot was obtained as follows. Figure 3 As shown (calculated based on absorbance from Table 9).

[0280] From Table 9 and Figure 3 In summary, the ELISA kit of the present invention for detecting LY6D protein in human serum or tissue homogenate can quantitatively or qualitatively detect LY6D protein in human serum samples, with a detection limit of 1 ng / ml.

[0281] Example 3 Immunohistochemical (IHC) Detection

[0282] 1. Sample preparation: Paraffin-embedded tumor tissue from surgically removed pancreatic cancer patients was continuously sliced ​​to a thickness of 4μm, attached to poly-L-lysine-treated glass slides, and baked in a 60℃ oven for 0.5-1h for later use.

[0283] 2. Dewaxing to water: The sections were sequentially immersed in xylene I for 10 min and xylene II for 10 min to dewax; then they were transferred to anhydrous ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min and 75% ethanol for 5 min to perform gradient hydration; finally, they were washed 3 times with PBS buffer (0.01 mol / L, pH 7.4) for 3 min each time.

[0284] 3. Antigen retrieval: using the hot retrieval method, the section was immersed in 0.01 mol / L citrate buffer (pH 6.0), placed in a microwave oven and heated to boiling, and kept on medium heat for 15 min. After the microwave oven was turned off, it was naturally cooled to room temperature, and washed with PBS buffer for 3 times, each for 3 min.

[0285] 4. Endogenous peroxidase blocking: 3% hydrogen peroxide solution (peroxidase blocker) was added dropwise to cover the tissue section, and incubated at room temperature for 10 min in the dark, and washed with PBS buffer for 3 times, each for 3 min.

[0286] 5. Blocking treatment: 5% bovine serum albumin (BSA) blocking solution (prepared with 0.05% Tween-20 in PBS) was added dropwise to completely cover the tissue, and incubated at room temperature for 30 min, and the blocking solution was poured off without washing.

[0287] 6. Primary antibody incubation: the anti-LY6D monoclonal antibody (3B9, 3G10, 7C10, 16F11, 6D12) obtained in the examples of the present application was diluted with an antibody diluent (1% BSA in PBS) at a ratio of 1:200, and added dropwise to the tissue section, and incubated in a wet box at 4°C overnight (16-18 h). The negative control used a same-type control antibody instead of the primary antibody, and the rest of the steps were the same.

[0288] 7. Secondary antibody incubation: the next day, the section was taken out, and warmed at room temperature for 30 min, and washed with PBS buffer for 3 times, each for 5 min. HRP-labeled goat anti-rabbit IgG secondary antibody (dilution ratio 1:500) was added dropwise, and incubated at room temperature for 1 h, and washed with PBS buffer for 3 times, each for 5 min.

[0289] 8. Color development and counterstaining: the color developing solution was prepared according to the instructions of the DAB color developing kit (Roche, 11718096001), added dropwise to the section, and the color development was observed in real time under a microscope, and after 3-5 min, the color development was terminated with PBS. The section was immersed in hematoxylin staining solution for counterstaining for 3 min, and washed with tap water for 10 min to return to blue, and after differentiation with 1% hydrochloric acid ethanol for 10 s, immediately washed with tap water to terminate the differentiation.

[0290] 9. Dehydration, transparency and mounting: the section was sequentially immersed in 75% ethanol for 3 min, 85% ethanol for 3 min, 95% ethanol for 3 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min for dehydration, and then xylene I for 5 min, xylene II for 5 min for transparency. Finally, neutral balsam was added dropwise, covered with a cover glass, and dried at room temperature before microscopic examination.

[0291] 10. Result observation: the different antibodies (3B9, 3G10, 7C10, 16F11, 6D12, IgG as control) were used for immunohistochemical (IHC) staining, and the optical microscope images were observed, and the results are shown in Figure 4a , Figure 4a LY6D protein positive expression in the cytoplasm was located in brownish yellow particles, and from Figure 4a it can be seen that when the different antibodies (3B9, 3G10, 7C10, 16F11, 6D12, IgG as control) were used to detect LY6D expression in pancreatic cancer tissues, the staining patterns of each antibody were significantly heterogeneous. This result shows that LY6D presents a heterogeneous expression characteristic in pancreatic cancer tissues - the expression level is significantly different in different regions or cell groups.

[0292] According to the above method, the mouse pancreatic cancer tissue was used for immunohistochemical (IHC) detection, and the results are shown in Figure 4b , from Figure 4b it can be seen that the different antibodies (3B9, 3G10, 7C10, 16F11, 6D12) of the application do not recognize mice.

[0293] Example 4 Western blot detection

[0294] 1. Sample preparation: collect human pancreatic cancer cells PANC-1 (Shanghai Hongshun Biotechnology Co., Ltd.) in the logarithmic growth phase, wash the cells twice with pre-cooled PBS buffer, add RIPA lysis buffer (Qiang, Biyun Tian Biotechnology) containing protease inhibitors and phosphatase inhibitors, lyse on ice for 30 min, and vortex mix every 10 min. 4℃, 12000rpm, centrifuge for 15 min, collect the supernatant, determine the protein concentration by BCA protein quantitative kit (Wuhan Sanyou Biotechnology Co., Ltd.), adjust the protein concentration of each group to be consistent, add 5x SDS loading buffer (containing β-mercaptoethanol), and boil at 100℃ for 10 min to denature the protein, and prepare for use.

[0295] 2. SDS-PAGE electrophoresis: prepare 10% separation gel and 5% concentrated gel, load 30μg total protein per well according to the predetermined order, use 1x SDS electrophoresis buffer as running buffer, and run at a constant voltage of 80V for 30 min in the concentrated gel stage and 120V for 90 min in the separation gel stage, until the bromophenol blue indicator reaches the bottom of the gel.

[0296] 3. Membrane transfer: use wet transfer method to assemble the transfer sandwich of gel and PVDF membrane in transfer buffer, and transfer at a constant current of 300mA for 90 min at 4℃.

[0297] 4. Blocking: Place the PVDF membrane in a TBST buffer containing 5% skimmed milk powder, and block the non-specific protein binding sites on the membrane at room temperature for 1 h on a shaker to block the non-specific protein binding sites on the membrane.

[0298] 5. Primary antibody incubation: Discard the blocking solution, add anti-LY6D monoclonal antibody (3B9, 3G10, 7C10, 16F11, 6D12) diluted with 5% skimmed milk powder TBST at a dilution ratio of 1:500, and incubate overnight at 4°C on a shaker. At the same time, incubate anti-β-actin monoclonal antibody at a dilution ratio of 1:10,000 as an internal control.

[0299] 6. Washing: Recover the primary antibody solution, wash the membrane with TBST buffer at room temperature on a shaker for 3 times, 10 min each time, to remove unbound primary antibodies.

[0300] 7. Secondary antibody incubation: Add HRP-labeled goat anti-rabbit IgG secondary antibody diluted with 5% skimmed milk powder TBST at a ratio of 1:5000, and incubate at room temperature for 1 h on a shaker.

[0301] 8. Washing: Wash the membrane with TBST buffer at room temperature on a shaker for 3 times, 15 min each time, to completely remove unbound secondary antibodies.

[0302] 9. Color development and analysis: Use the ECL chemiluminescence kit (Yeasen, 36208ES60) for development, capture images through the gel imaging system (Bio-Rad Company, ChemiDoc XRS+), and analyze the gray value of the protein band using ImageJ software. The resulting Western Blot image is shown in Figure 5 , Figure 5 Ctrl is the control group, and OE is the overexpression group. As can be seen from Figure 5 , the actin protein band signal as an internal control is relatively consistent in both groups, while the LY6D total protein band signal detected using 3B9, 3G10, 7C10, 16F11, 6D12, etc. is significantly stronger than that of the control group (Ctrl group), so the LY6D expression level corresponding to different antibodies (3B9, 3G10, 7C10, 16F11, 6D12) is significantly higher than that of the control group (Ctrl group, IgG as control).

[0303] Example 5 Flow cytometry detection

[0304] Collect the single cell suspension of human pancreatic cancer cell PANC-1, after washing, add blocking solution, incubate at room temperature for 5 min. Add the monoclonal antibody of the application, avoid light incubation for 30 min. After washing, add fluorescently labeled rabbit secondary antibody, avoid light incubation for 30 min. After washing, flow cytometry detection, analyze the expression of LY6D protein, the specific steps are as follows:

[0305] 1. Single cell suspension preparation: Take human pancreatic cancer cells PANC-1 in the logarithmic growth phase, digest with 0.25% trypsin-EDTA solution at 37°C for 2 min, add DMEM medium containing 10% fetal bovine serum to terminate digestion, gently blow to prepare single cell suspension, filter through 200 mesh cell sieve to remove cell clumps, count and adjust the cell concentration to 1×10^6 / ml.

[0306] 2. Blocking treatment: Take 100 μl of single cell suspension into a flow tube, add 10 μl of 10% BSA blocking solution, mix gently, incubate at room temperature for 5 min in the dark.

[0307] 3. Primary antibody incubation: Add the monoclonal antibody prepared in the application, dilute with 1% BSA-containing PBS at a ratio of 1:100 according to the antibody instruction, mix gently, incubate at 4°C in the dark for 30 min. Set up blank control tubes, only add 1% BSA-containing PBS buffer, and the rest of the operation is consistent with the experimental group.

[0308] 4. Washing: Add 2 ml of 1% BSA-containing PBS buffer to each tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, repeat the washing for 2 times.

[0309] 5. Secondary antibody incubation: Add fluorescently labeled secondary antibody (AF488 Goat anti-Rabbit), dilute with 1% BSA-containing PBS at a ratio of 1:200 according to the antibody instruction, mix gently, incubate at 4°C in the dark for 30 min.

[0310] 6. Washing: Add 2 ml of 1% BSA-containing PBS buffer to each tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, repeat the washing for 2 times.

[0311] 7. Resuspension: The precipitate is resuspended with 300 μl of 1% BSA-containing PBS buffer, and impurities are removed through a 300 mesh cell sieve.

[0312] 8. Flow detection: upflow cytometry, excitation wavelength 488nm, collect fluorescence signal. Use FlowJo software to analyze data, set negative threshold with isotype control, calculate LY6D protein positive cell percentage and average fluorescence intensity, which represent LY6D protein expression level, corresponding to different antibodies (3B9, 3G10, 7C10, 16F11, 6D12) respectively. The scatter plot of flow cytometry detection with IgG as control is shown in FIG. 8, from which it can be seen that, in PANC-1 cells overexpressing LY6D (OE group), LY6D expression level corresponding to different antibodies (3B9, 3G10, 7C10, 16F11, 6D12) is significantly higher than that of control group (Ctrl group, IgG as control). Figure 6 Figure 6

[0313] In summary, the anti-human LY6D protein monoclonal antibody and its application of the present application provide a new tool for early diagnosis and precise treatment of pancreatic cancer, which has significant clinical value and industrialization prospect.

[0314] The above are preferred embodiments of the present application, which do not limit the protection scope of the present application, and any equivalent variations made according to the present application shall be covered within the protection scope of the present application.​​

Claims

1. An anti-human LY6D protein monoclonal antibody, characterized in that, The anti-human LY6D protein monoclonal antibody specifically recognizes the 21-98 aa epitope of the LY6D protein.

2. The anti-human LY6D protein monoclonal antibody of claim 1, wherein, The anti-human LY6D protein monoclonal antibody is 16F11, 3G10, 3B9, 6D12 or 7C10. The amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 16F11 is shown in SEQ ID NO. 11, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

12. The amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 3G10 is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

14. The amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 3B9 is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

16. The amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 6D12 is shown in SEQ ID NO. 17, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

18. The amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 7C10 is shown in SEQ ID NO. 19, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

20.

3. The anti-human LY6D protein monoclonal antibody of claim 1, wherein, The anti-human LY6D protein monoclonal antibody is 16F11, 3G10, 3B9, 6D12 or 7C10. The base sequence of the amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 16F11 is shown in SEQ ID NO. 1, and the base sequence of the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

2. The base sequence of the amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 3G10 is shown in SEQ ID NO. 3, and the base sequence of the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

4. The base sequence of the amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 3B9 is shown in SEQ ID NO. 5, and the base sequence of the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

6. The base sequence of the amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 6D12 is shown in SEQ ID NO. 7, and the base sequence of the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

8. The base sequence of the amino acid sequence of the heavy chain variable region VH of the anti-human LY6D protein monoclonal antibody 7C10 is shown in SEQ ID NO. 9, and the base sequence of the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.

10.

4. A nucleic acid, characterized in that, The nucleic acid encodes the heavy chain variable region VH amino acid sequence and the light chain variable region VL amino acid sequence of the anti-human LY6D protein monoclonal antibody 16F11, 3G10, 3B9, 6D12 or 7C10; The base sequence encoding the heavy chain variable region VH amino acid sequence of the anti-human LY6D protein monoclonal antibody 16F11 is shown in SEQ ID NO. 1, and the base sequence of the light chain variable region VL amino acid sequence is shown in SEQ ID NO. 2; The base sequence encoding the heavy chain variable region VH amino acid sequence of the anti-human LY6D protein monoclonal antibody 3G10 is shown in SEQ ID NO. 3, and the base sequence of the light chain variable region VL amino acid sequence is shown in SEQ ID NO. 4; The base sequence encoding the heavy chain variable region VH amino acid sequence of the anti-human LY6D protein monoclonal antibody 3B9 is shown in SEQ ID NO. 5, and the base sequence of the light chain variable region VL amino acid sequence is shown in SEQ ID NO. 6; The base sequence encoding the heavy chain variable region VH amino acid sequence of the anti-human LY6D protein monoclonal antibody 6D12 is shown in SEQ ID NO. 7, and the base sequence of the light chain variable region VL amino acid sequence is shown in SEQ ID NO. 8; The base sequence encoding the heavy chain variable region VH amino acid sequence of the anti-human LY6D protein monoclonal antibody 7C10 is shown in SEQ ID NO. 9, and the base sequence of the light chain variable region VL amino acid sequence is shown in SEQ ID NO.

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5. A recombinant plasmid pcDNA3.4 plasmid, characterized in that, The nucleic acid as claimed in claim 4.

6. A recombinant 293F cell expressing an anti-human LY6D protein monoclonal antibody, characterized in that, The recombinant plasmid pcDNA3.4 plasmid as claimed in claim 5.

7. The anti-human LY6D protein monoclonal antibody as claimed in any one of claims 1-3 for use in the preparation of an antibody-drug conjugate or a proteolysis targeting chimera drug for treating pancreatic cancer.

8. A pharmaceutical composition for treating pancreatic cancer, characterized by, The pharmaceutical composition is an antibody-drug conjugate, wherein the antibody-drug conjugate is the anti-human LY6D protein monoclonal antibody as claimed in any one of claims 1-3 conjugated with a cytotoxic drug through a cleavable or non-cleavable linker.

9. A proteolysis targeting chimera drug for treating pancreatic cancer, characterized by, The anti-human LY6D protein monoclonal antibody as claimed in any one of claims 1-3.

10. An ELISA kit for detecting LY6D protein in human serum or tissue homogenate, characterized by, The enzyme-labeled plate, the capture antibody, the detection antibody, the Neutravidin-HRP secondary antibody and the standard are included. The capture antibody is the anti-human LY6D protein monoclonal antibody 3B9, 3G10, 7C10, 16F11 or 6D12 as claimed in any one of claims 1-3. The detection antibody is the biotin-labeled anti-human LY6D protein monoclonal antibody 3B9, 3G10, 7C10, 16F11 or 6D12 as claimed in any one of claims 1-3. The standard is a purified Ly6d (21-97aa)+3*G4S+6His tag protein with a concentration range of 0-10 ng / mL.

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