HBcAg antibody and application thereof in treatment of hepatitis B related hepatocellular carcinoma

Abstract

The invention discloses a fully humanized antibody aiming at HBcAg protein expressed by hepatitis B related liver cancer cells. The antibody is proved to be capable of killing tumor cells of hepatitis B related liver cancer in in-vivo and in-vitro experiments, and the treatment effect of the PD-1 monoclonal antibody can be improved. The antibody provided by the invention has the characteristic of high-specificity recognition of hepatitis B related liver cancer tumor cells, can kill few para-carcinoma hepatocytes, and has better safety. The antibody provided by the invention can be used as a therapeutic drug for patients with advanced hepatitis B related liver cancer and a combined drug for immune checkpoint inhibitor treatment, can also be combined with an immune checkpoint inhibitor to be used as a new adjuvant and / or adjuvant therapy drug for patients with hepatitis B related liver cancer in the perioperative period, and has important value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an HBcAg antibody and its use in treating hepatocellular carcinoma. BACKGROUND

[0002] Hepatocellular carcinoma (HCC, hereinafter referred to as liver cancer) is the third most common cause of cancer-related death worldwide. Currently, the two major clinical problems of liver cancer are: 1. More than half of liver cancer patients are in the middle and advanced stages at the time of diagnosis. For patients with advanced liver cancer, systemic therapy (including targeted therapy, immunotherapy or a combination of the two) is the main treatment method. However, the effect of systemic therapy for advanced liver cancer is poor, and the response rate of single drug therapy is less than 30%; 2. For patients who can accept radical treatment such as surgery / ablation, the recurrence rate after radical treatment is high, and the 5-year recurrence rate is as high as 70%.

[0003] For patients with advanced liver cancer, current systemic therapy includes molecular targeted drugs and immune checkpoint inhibitors. Among them, immune checkpoint blockade drugs such as PD-1 monoclonal antibodies have been proven to be able to kill tumor cells by inducing systemic immunity and tumor-specific T cell expansion in a variety of malignancies such as melanoma, breast cancer, lung cancer and colon cancer. However, in patients with advanced liver cancer, the response rate of single drug immunotherapy is only about 20%. Among the multiple multi-center phase III clinical studies currently conducted worldwide, only IMBRAVE150 has been successful. This study compared atezolizumab + bevacizumab (i.e. PD-L1 monoclonal antibody combined with VEGF monoclonal antibody) with sorafenib monotherapy, and the results suggested that this combination therapy could improve the prognosis of patients with advanced liver cancer, but the tumor response rate was only 30%, and most patients had tumor progression, and the available drugs after progression were still very limited. At the same time, the targeted drugs currently used are mostly multi-target tyrosine kinase inhibitors or VEGF inhibitors, and the targets are not specific enough in liver cancer patients, often causing adverse reactions such as hypertension, hand-foot syndrome, and skin capillary proliferation, affecting the long-term regular use of drugs by patients. Therefore, finding a specific treatment target for liver cancer patients, alone or in combination with other treatments, may be able to reduce adverse reactions while improving treatment effectiveness, improving the quality of life and prognosis of patients with advanced liver cancer.

[0004] On the other hand, for patients receiving radical treatment, the current expert consensus advocates that patients with high risk of postoperative recurrence and metastasis of liver cancer should first undergo neoadjuvant local treatment or systemic anti-tumor treatment before surgery, in order to eliminate micro lesions, reduce the rate of postoperative recurrence and metastasis, and prolong survival. The results of several clinical studies published recently suggest that the overall response rate of neoadjuvant systemic therapy is about 28%, and the major pathological response rate (MPR) is about 32%. Among them, the recurrence rate of patients who can achieve MPR after surgery will be significantly reduced (Lancet Oncol 2024; 25: 1465-75). However, in the neoadjuvant therapy mode, the preventive effect of immune combined targeted therapy or double immune therapy on tumor recurrence is not significantly improved compared with immune therapy alone. This indicates that in the short-term neoadjuvant therapy, the current drug selection of combination therapy still has a lot of room for exploration. The neoadjuvant therapy mode has an advantage, that is, researchers can obtain tumor samples before and after treatment, so as to explore the changes of tumor microenvironment after neoadjuvant therapy, which is helpful to seek new treatment targets or combination therapy methods.

[0005] Globally, nearly 50% of patients with liver cancer are associated with chronic hepatitis B virus (HBV, also known as hepatitis B) infection, and this proportion is higher in China, reaching 85% (J Hepatocell Carcinoma 2014; 1: 115). The HBV viral genome is an incomplete double-stranded circular DNA, and its replication is a reverse transcription replication with mRNA as an intermediate. This unique replication mode provides an opportunity for HBV-DNA to integrate into the host genome, thereby expressing the HBV viral gene fragments in host cells without forming complete viral particles (Nat Rev Immunol. 2022 Jan; 22(1): 19-32). As hepatitis progresses, the probability and complexity of HBV integration increase, and HBV gene integration-induced liver cell genome variation has been confirmed as an important cause of hepatitis B-related liver cancer. It is reported that about 85-90% of hepatitis B-related liver cancer cells have HBV genome integration, so hepatitis B-related liver cancer has a high probability of expressing HBV viral gene fragments; however, in HBV-infected liver cells (i.e. liver cells adjacent to liver cancer), only 0.1-1% of cells have HBV gene integration (Cell Mol Gastroenterol Hepatol 2023; 15: 921-929). Currently, some clinical studies of CAR-T cell therapy targeting HBV antigens are underway, but only small sample case reports (Clinical and Molecular Hepatology 2024; 30: 735-755), and no positive clinical research results have been announced. However, successful cases of CAR-T cell therapy targeting HBV antigens have shown that HBV antigens expressed by hepatitis B liver cancer cells can activate the patient's anti-tumor T cell immunity, and previous studies have found that the proportion of T cells specifically recognizing HBV antigens is negatively correlated with the patient's postoperative recurrence rate (Immunity. 2021. 54, 1825-1840). However, humoral immunity, an important part of antiviral and antitumor immunity, has not been explored in hepatitis B liver cancer. Hepatitis B liver cancer expresses a variety of hepatitis B virus-related antigens, and it is not known which hepatitis B virus antigen can activate the patient's humoral immunity and participate in the process of killing cancer cells. It is also not known whether the level of activation of humoral immunity is related to the prognosis of patients and the effectiveness of immunotherapy.

[0006] In recent years, T cell activation and expansion have been shown to be significantly associated with response to immune checkpoint molecule inhibitors, but studies have also found that not all patients with effective anti-PD-1 monoclonal antibody treatment have significant T cell expansion, suggesting that other intratumoral immune mechanisms play an anti-tumor function. Studies have reported that the infiltration level of intratumoral B cells and tertiary lymphoid structures is related to the prognosis and immunotherapy efficacy of tumor patients. Tertiary lymphoid structures are ectopic lymphocyte aggregates composed of T and B cells in non-lymphoid organs such as tumors, and play an important role in specific anti-tumor immunity. However, in hepatitis B-related liver cancer, how B cell-dominated humoral immunity participates in anti-tumor, and whether it is related to the HBV-related antigens expressed by the tumor, still needs further study.

[0007] In 2023, the research team of the Crick Institute published an article entitled Antibodies against endogenous retroviruses promote lung cancer immunotherapy in Nature. In this article, they found that there are antibodies against endogenous KPAR-related retroviruses (KARV) in the tumor of mouse lung adenocarcinoma, and after treatment with PD-L1 antibody, the affinity of the antibody in the tumor to KPAR cells increases, and the growth rate of the tumor slows down. Based on the findings in the mouse model, they further screened human patient samples of lung adenocarcinoma and found that nearly half of the lung adenocarcinoma patients could detect antibodies reactive to the envelope protein of human endogenous retrovirus HERV-K (HML-2). At the same time, after treatment with immune checkpoint inhibitors, the titers of HERV-K (HML-2) reactive antibodies in all patients increased. Therefore, they proposed that the intratumoral HERV-K (HML-2) envelope-targeting antibody response in lung adenocarcinoma patients is related to anti-tumor immunity and can increase the efficacy of immune checkpoint inhibitors through anti-tumor immunity.

[0008] Their research findings are similar to the present application in that antibodies against a certain virus in the tumor were found in solid tumors, which can exert anti-tumor function and enhance the efficacy of immune checkpoint inhibitors. However, in their study, the virus is an endogenous retrovirus, which is ubiquitous in tissues and lacks tumor tissue specificity. Therefore, in terms of specificity of distribution in tumors, KPAR retroviruses are not as high as HBV for liver cancer. Therefore, in subsequent use, it may not be possible to ensure the accuracy of the treatment target, and the safety is lower.

[0009] In addition, their findings are from mouse models, only subsequent verification in human samples, can not provide the whole humanized antibody sequence. Antibody subsequent drugability and safety are uncertain. The antibody of the application is completely based on the prospective clinical research cohort analysis, the antibody variable region sequence is a whole humanized antibody, the subsequent modification and processing of the antibody sequence are difficult, and the drug safety and reliability are high.

[0010] The treatment targets and drugs for liver cancer patients in the prior art are still limited, and the treatment effect is not satisfactory. Therefore, there is an urgent need to provide new treatment targets and drugs for hepatitis B-related liver cancer patients. SUMMARY

[0011] The purpose of the application is to solve the problems of insufficient treatment targets and drugs for liver cancer patients and limited treatment effect in the prior art, and to provide a drug that can be used for the treatment of advanced hepatitis B-related liver cancer patients, or as a combined drug for immune checkpoint inhibitor therapy. It can also be used as a drug for neoadjuvant and / or adjuvant therapy for hepatitis B-related liver cancer patients during the perioperative period in combination with immune checkpoint inhibitors.

[0012] The inventors conducted a neoadjuvant immunotherapy study (NCT04615143) for hepatitis B-related liver cancer patients to explore whether neoadjuvant immunotherapy can reduce the postoperative recurrence rate of hepatitis B-related liver cancer patients. At the same time, using paired tumor specimens before and after treatment, the effect of immunotherapy on intratumoral humoral immunity was analyzed, the effective mechanism of immunotherapy was explored, and the effective target for combined immunotherapy was found.

[0013] To solve the above technical problems, the technical scheme adopted by the application is as follows:

[0014] The application discloses an isolated monoclonal antibody, characterized in that the monoclonal antibody binds to an HBcAg antigen, and the monoclonal antibody binds to at least one amino acid residue selected from E77, D78 and P79 of the A / B chain of the HBcAg dimer.

[0015] Preferably, the sequence of the A chain and the sequence of the B chain are respectively as shown in SEQ ID NO. 98.

[0016] Preferably, the monoclonal antibody is a humanized antibody.

[0017] Preferably, the monoclonal antibody is a neutralizing antibody.

[0018] Preferably, the monoclonal antibody binds to the K D value is less than or equal to 1.593E-9M.

[0019] Preferably, the monoclonal antibody binds to the K DPreferably, the monoclonal antibody binds to the K

[0020] Preferably, the monoclonal antibody binds to at least the residues E77 of the A chain. D Preferably, the monoclonal antibody binds to at least the residues D78 of the A chain.

[0021] Preferably, the monoclonal antibody binds to at least the residues E77 of the B chain.

[0022] Preferably, the monoclonal antibody binds to at least the residues D78 of the B chain.

[0023] Preferably, the monoclonal antibody binds to at least the residues E77 of the B chain.

[0024] Preferably, the monoclonal antibody binds to at least the residues D78 of the B chain.

[0025] Preferably, the monoclonal antibody binds to at least the residues P79 of the B chain.

[0026] Preferably, the monoclonal antibody binds to the amino acid residues E77 and D78 of the A chain of the HBcAg dimer, and E77, D78 and P79 of the B chain of the HBcAg dimer.

[0027] Preferably, the monoclonal antibody is mAb40, the amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain of the mAb40 comprise the sequences as shown in SEQ ID NO. 67, SEQ ID NO. 71 and SEQ ID NO. 76 respectively, and the amino acid sequences of the CDR1, CDR2 and CDR3 of the light chain of the mAb40 comprise the sequences as shown in SEQ ID NO. 83, SEQ ID NO. 87 and SEQ ID NO. 91 respectively.

[0028] Preferably, the monoclonal antibody is mAb40, the amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain of the mAb40 comprise the sequences as shown in SEQ ID NO. 67, SEQ ID NO. 71 and SEQ ID NO. 76 respectively, and the amino acid sequences of the CDR1, CDR2 and CDR3 of the light chain of the mAb40 comprise the sequences as shown in SEQ ID NO. 83, SEQ ID NO. 87 and SEQ ID NO. 91 respectively.

[0029] Preferably, the monoclonal antibody is mAb40, the amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain of the mAb40 comprise the sequences as shown in SEQ ID NO. 67, SEQ ID NO. 71 and SEQ ID NO. 76 respectively, and the amino acid sequences of the CDR1, CDR2 and CDR3 of the light chain of the mAb40 comprise the sequences as shown in SEQ ID NO. 83, SEQ ID NO. 87 and SEQ ID NO. 91 respectively.

[0030] Preferably, the coding sequence of the heavy chain variable region of the monoclonal mAb40 comprises the sequence as shown in SEQ ID NO. 80, and the coding sequence of the light chain variable region of the mAb40 comprises the sequence as shown in SEQ ID NO. 96.

[0031] Preferably, the monoclonal antibody is mAb40, the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of the mAb40 are respectively as shown in SEQ ID NO. 67, SEQ ID NO. 71 and SEQ ID NO. 76, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of the mAb40 are respectively as shown in SEQ ID NO. 83, SEQ ID NO. 87 and SEQ ID NO. 91.

[0032] Preferably, the monoclonal antibody is mAb40, the amino acid coding sequences of the heavy chain CDR1, CDR2 and CDR3 of the mAb40 are respectively as shown in SEQ ID NO. 68, SEQ ID NO. 72 and SEQ ID NO. 77, and the amino acid coding sequences of the light chain CDR1, CDR2 and CDR3 of the mAb40 are respectively as shown in SEQ ID NO. 84, SEQ ID NO. 88 and SEQ ID NO. 92.

[0033] Preferably, the sequence of the heavy chain variable region of the monoclonal mAb40 is as shown in SEQ ID NO. 79, and the sequence of the light chain variable region of the mAb40 is as shown in SEQ ID NO. 95.

[0034] Preferably, the coding sequence of the heavy chain variable region of the monoclonal mAb40 is as shown in SEQ ID NO. 80, and the coding sequence of the light chain variable region of the mAb40 is as shown in SEQ ID NO. 96.

[0035] Preferably, the monoclonal antibody is mAb32, the amino acid coding sequences of the heavy chain CDR1, CDR2 and CDR3 of the mAb32 comprise the sequences as shown in SEQ ID NO. 3, SEQ ID NO. 7 and SEQ ID NO. 11 respectively, and the amino acid coding sequences of the light chain CDR1, CDR2 and CDR3 of the mAb32 comprise the sequences as shown in SEQ ID NO. 19, SEQ ID NO. 23 and SEQ ID NO. 27 respectively.

[0036] Preferably, the sequence of the heavy chain variable region of the monoclonal mAb32 comprises the sequence as shown in SEQ ID NO. 15, and the sequence of the light chain variable region of the mAb32 comprises the sequence as shown in SEQ ID NO. 31.

[0037] Preferably, the monoclonal antibody is mAb41, the amino acid coding sequences of the heavy chain CDR1, CDR2 and CDR3 of the mAb41 comprise sequences as shown in SEQ ID NO. 35, SEQ ID NO. 39 and SEQ ID NO. 43 respectively, and the amino acid coding sequences of the light chain CDR1, CDR2 and CDR3 of the mAb41 comprise sequences as shown in SEQ ID NO. 51, SEQ ID NO. 55 and SEQ ID NO. 59 respectively.

[0038] Preferably, the monoclonal mAb41 has a heavy chain variable region sequence comprising a sequence as shown in SEQ ID NO. 47, and a light chain variable region sequence comprising a sequence as shown in SEQ ID NO. 63.

[0039] The present application discloses a nucleic acid molecule encoding the monoclonal antibody.

[0040] The present application discloses a vector or expression cassette comprising the nucleic acid molecule.

[0041] The present application discloses a cell comprising the nucleic acid molecule and / or the vector or expression cassette.

[0042] The present application discloses a monoclonal antibody expressed by the vector or expression cassette.

[0043] The present application discloses a monoclonal antibody expressed by the cell.

[0044] Preferably, the monoclonal antibody is used for treating liver cancer.

[0045] Preferably, the liver cancer is hepatocellular carcinoma.

[0046] Preferably, the hepatocellular carcinoma is caused by hepatitis B virus.

[0047] The present application discloses a composition comprising the monoclonal antibody and / or the nucleic acid and / or the vector or expression cassette and / or the cell.

[0048] Preferably, the composition is a pharmaceutical composition.

[0049] Preferably, the composition further comprises other anti-tumor components.

[0050] Preferably, the anti-tumor component is PD-1.

[0051] Preferably, the composition comprises monoclonal antibody mAb40 and PD-1.

[0052] Preferably, the composition comprises the monoclonal antibody mAb32 and PD-1.

[0053] Preferably, the composition comprises the monoclonal antibody mAb41 and PD-1.

[0054] The application discloses the use of the monoclonal antibody, the nucleic acid, the vector or the expression frame, the cell and the composition in the preparation of an antitumor drug.

[0055] Preferably, the tumor is liver cancer.

[0056] The application provides a fully humanized antibody sequence against HBcAg protein expressed by hepatitis B related hepatocarcinoma cells, and the antibody has an antitumor effect. The antibody is derived from a BCR sequence that is significantly amplified after PD1 monoclonal antibody treatment and undergoes an affinity maturation process, and BLI shows that the antibody has high affinity for HBcAg and can efficiently bind to HBcAg protein. Meanwhile, the antibody has been proved to be able to kill tumor cells of hepatitis B related hepatocarcinoma and increase the treatment effect of the PD1 monoclonal antibody in an in-vivo experiment. Therefore, the antibody has the opportunity to play an antitumor effect as a treatment antibody for hepatitis B related hepatocarcinoma, or to be used as an auxiliary drug of an immune checkpoint inhibitor such as the PD-1 monoclonal antibody to enhance the response rate.

[0057] The results of flow cytometry, immunoelectron microscopy and the like provided in the application show that HBcAg is significantly highly expressed in tumors and is lowly expressed in paracancerous hepatocytes. Therefore, the antibody provided in the application should have the characteristics of high specific recognition of hepatitis B related hepatocarcinoma tumor cells in subsequent clinical use, and rarely kill paracancerous hepatocytes, and has good safety. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 Patients in the response group mainly characterized by plasma cell expansion have strong functions of secreting IgG1 subtype antibodies. Among them, (a) composition of tumor microenvironment of patients in different groups before and after neoadjuvant therapy, wherein the number of plasma cells of the B-responder group is significantly increased after treatment; (b) distribution of BCR subtypes of plasma cells of patients in different groups before and after neoadjuvant therapy.

[0059] Figure 2 Results of affinity detection of mAb32, mAb40 and mAb41 antibodies to HBcAg.

[0060] Figure 3 Multiple immunofluorescence staining of tumor sections of hepatitis B related hepatocarcinoma patients co-stained by HBcAg, hepatocarcinoma tumor marker GPC3 and antibodies of the application.

[0061] Figure 4The antibody (mAb40) of the present application binds to HBV+ liver cancer cells and increases cell apoptosis.

[0062] Figure 5 The antibody provided by the present application is determined for treatment efficacy in a HBcAg+ liver cancer mouse subcutaneous tumor model.

[0063] Figure 6 Expression and binding of HBcAg on tumor and paracancerous tissue of hepatitis B related liver cancer, wherein (a, b) are immunofluorescence staining for detecting expression and binding of HBcAg on tumor and paracancerous tissue of fresh hepatitis B related liver cancer; (c) is flow cytometry for detecting binding of commercial HBcAb on tumor and paracancerous tissue of fresh hepatitis B related liver cancer.

[0064] Figure 7 Immunoelectron microscopy images of tumor tissue and paracancerous tissue of hepatitis B related liver cancer, wherein red triangles in the figure represent HBcAg protein labeled by intracellular colloidal gold particles, and green triangles represent HBcAg protein labeled by colloidal gold particles in the intercellular space.

[0065] Figure 8 Structure analysis of HBcAg and mAb40 Fab complex. (a) CryoEM density map of HBcAg and mAb40 Fab complex; (b) density map focusing on the local refinement of one HBcAg dimer and one Fab complex, the densities of mAb40 light chain, heavy chain, phage dimer chain A and chain B are represented by yellow-green, pink, blue and cyan, respectively; (c) the overall structure of the complex is shown in the form of a schematic diagram; (d) details of the interaction between HBcAg dimer and mAb40 Fab; the residues involved in forming hydrogen bonds (represented by red dashed lines) and salt bridges (represented by blue dashed lines) are represented by sticks, and the colors are the same as their chains. DETAILED DESCRIPTION

[0066] The specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0067] Subject main conditions of Example 1

[0068] The main inclusion criteria of the subjects include:

[0069] 1) 18-75 years old;

[0070] 2) Patients with resectable hepatocellular carcinoma who have experienced first recurrence after radical treatment;

[0071] 3) At least one RECIST 1.1 standard measurable lesion: tumor ≥1 cm evaluated by imaging (MR / CT);

[0072] 4) Patients receive specimen collection;

[0073] 5) ECOG score 0-1, good physical condition;

[0074] 6) Liver function Child-Pugh classification A.

[0075] The main exclusion criteria for subjects include:

[0076] 1) Extrahepatic metastasis;

[0077] 2) History of tumor targeting, immunotherapy;

[0078] 3) Recurrent lesions have a history of local embolization perfusion therapy, radiotherapy;

[0079] 4) There is an active autoimmune disease that needs systemic treatment;

[0080] 5) Accompanied by other malignant tumors or other malignant tumors within 5 years before enrollment;

[0081] 6) Severe heart, kidney and other organ dysfunction;

[0082] 7) Participated in other drug clinical trials within 12 months before enrollment.

[0083] Example 2 Preparation of HBcAg antibody

[0084] The specific treatment plan for the subjects is as follows: after signing the informed consent, the subjects receive a total of 2 courses of neoadjuvant therapy, one course every three weeks, intravenous infusion of tiragulimab (anti-PD-1 single card, trade name of Bazi'an) 200 mg. After 1 week of 2 courses of treatment, the subjects will complete imaging examination and surgical evaluation, and complete surgical treatment within 6 weeks after enrollment. After 4-6 weeks of surgery, the subjects start postoperative tiragulimab adjuvant therapy, one course every three weeks, intravenous infusion of tiragulimab 200 mg. Adjuvant therapy lasts for 1 year, or until the subject develops unacceptable toxicity or tumor recurrence. The primary endpoint of the study is 1-year disease-free survival.

[0085] During the treatment of the subjects, the inventors collect the tumor specimens before and after neoadjuvant therapy for single cell suspension dissociation, and perform single cell level transcriptome sequencing and B cell receptor (BCR) sequencing. According to the study endpoint, i.e. whether the patient has tumor progression / recurrence within 1 year, the subjects are divided into treatment response group and non-response group, and the changes of immune microenvironment of the two groups of patients are compared.

[0086] The present application uses the 17 subjects in this clinical study for relevant analysis. The analysis results suggest that there is a group of patients with response group patients characterized by plasma cell expansion, who have strong IgG1 subtype antibody secretion function Figure 1 Further, by detecting the BCR repertoire before and after immunotherapy, 557 BCR antibody sequences significantly expanded after treatment, with somatic hypermutation and IgG1 subtype were identified, from which 126 with the highest expansion amplitude and somatic hypermutation rate were screened for in vitro synthesis. The synthesis process is as follows: according to the sequences of the 126 antibody complete heavy chain and complete light chain variable region obtained by BCR sequencing, clone into pcDNA3.4 vector expressing mouse IgG2a and IgK. Then the recombinant plasmid encoding the target protein is transiently transfected into suspended CHO LITE cells, and then the successfully transformed colonies are expanded. The cell culture fluid is centrifuged, filtered and purified. Finally, the molecular weight and purity of the purified protein are determined by SDS-PAGE and HPLC analysis, obtaining antibodies with human variable domains and mouse constant domains.

[0087] Example 3 Screening and affinity determination of HBcAg antibodies

[0088] Further, the 126 antibodies were further subjected to binding affinity detection of HBcAg. First, ELISA kit was used to qualitatively detect whether the antibodies could bind to HBcAg, and 42 antibodies that could bind to recombinant HBcAg antibody protein were screened out. At the same time, BLI technology was used for qualitative analysis of whether the antibodies could bind to HBcAg, and 30 antibodies that could bind to HBcAg were screened out. Further, BLI technology was used to quantitatively detect the affinity of the 30 antibodies to HBcAg. The specific steps are as follows: 3 molar concentration ratio (MCR) of biotin and antibody were incubated at room temperature for 1 hour, and excess biotin reagent was removed using a gravity desalting column, then 1 nM of biotin-antibody conjugate was loaded onto a SA sensor, and different concentration gradients of HBcAg were used to analyze the binding kinetics. The corresponding equilibrium dissociation constant KD was determined by the global fitting 1:1 binding algorithm of Octet analysis studio software, and 3 antibodies with higher affinity (mAb32, mAb40, mAb41) Figure 2 ) were selected for in vivo and in vitro experimental detection of their binding capacity to HBcAg and killing capacity to hepatitis B related hepatocellular carcinoma. The related sequences of mAb32, mAb41 and mAb40 antibodies obtained are as follows:

[0089] mAb32 antibody:

[0090] Heavy chain FWR1: QLLLLQSGGGLVKPGGHMRLSCEGS (SEQ ID NO. 1).

[0091] Heavy chain FWR1 coding sequence:

[0092] CAACTACTACTACTGCAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGCACATGAGACTCTCCTGTGAAGGCTCT (SEQ ID NO. 2).

[0093] Heavy chain CDR1 : GFTFGDYY (SEQ ID NO. 3).

[0094] Heavy chain CDR1 coding sequence: GGATTCACATTTGGTGACTATTAC (SEQ ID NO. 4).

[0095] Heavy chain FWR2: MNWVRQAPGKGLEWIAS (SEQ ID NO. 5).

[0096] Heavy chain FWR2 coding sequence:

[0097] ATGAATTGGGTCCGCCAGGCTCCAGGAAAGGGACTCGAATGGATTGCATCC (SEQ ID NO. 6).

[0098] Heavy chain CDR2: MSPRETAT (SEQ ID NO. 7).

[0099] Heavy chain CDR2 coding sequence: ATGAGTCCTCGGGAAACCGCCACA (SEQ ID NO. 8).

[0100] Heavy chain FWR3: YYADVVRGRFIISRDNAQQSTYLQMDILRGDDSAVYY (SEQ ID NO. 9).

[0101] Heavy chain FWR3 coding sequence:

[0102] TACTACGCAGACGTTGTGAGGGGTCGATTCATTATCTCTAGGGACAACGCCCAGCAGTCAACTTATCTGCAAATGGACATCCTGCGCGGCGACGATTCGGCCGTCTATTAC (SEQ ID NO. 10).

[0103] Heavy chain CDR3: CARGVNSRSSNYVYGFEVW (SEQ ID NO. 11).

[0104] Heavy chain CDR3 coding sequence:

[0105] TGTGCGCGAGGGGTTAACAGCCGCTCAAGTAACTACGTCTACGGTTTTGAAGTCTGG (SEQ ID NO. 12).

[0106] Heavy chain FWR4: GQGTTVTVSS (SEQ ID NO. 13).

[0107] Heavy chain FWR4 coding sequence: GGCCAGGGGACCACGGTCACCGTCTCTTCA (SEQ ID NO. 14).

[0108] Heavy chain variable region sequence:

[0109] QLLLLQSGGGLVKPGGHMRLSCEGSGFTFGDYYMNWVRQAPGKGLEWIASMSPRETATYYADVVRGRFIISRDNAQQSTYLQMDILRGDDSAVYYCARGVNSRSSNYVYGFEVWGQGTTVTVSS (SEQ ID NO. 15).

[0110] Heavy chain variable region coding sequence:

[0111] CAACTACTACTACTGCAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGCACATGAGACTCTCCTGTGAAGGCTCTGGATTCACATTTGGTGACTATTACATGAATTGGGTCCGCCAGGCTCCAGGAAAGGGACTCGAATGGATTGCATCCATGAGTCCTCGGGAAACCGCCACATACTACGCAGACGTTGTGAGGGGTCGATTCATTATCTCTAGGGACAACGCCCAGCAGTCAACTTATCTGCAAATGGACATCCTGCGCGGCGACGATTCGGCCGTCTATTACTGTGCGCGAGGGGTTAACAGCCGCTCAAGTAACTACGTCTACGGTTTTGAAGTCTGGGGCCAGGGGACCACGGTCACCGTCTCTTCA (SEQ ID NO. 16).

[0112] Light chain FWR1 : EVVLTQSPGTLSLFPGDRATLSC (SEQ ID NO. 17).

[0113] Light chain FWR1 coding sequence:

[0114] GAAGTTGTGTTGACGCAGTCTCCGGGCACCCTGTCTTTGTTTCCGGGGGATAGGGCCACCCTCTCCTGC (SEQ ID NO. 18).

[0115] Light chain CDR1: RASRTIDNTYLA (SEQ ID NO. 19).

[0116] Light chain CDR1 encoding sequence:

[0117] AGGGCCAGTCGGACGATTGACAACACCTACCTAGCC (SEQ ID NO. 20).

[0118] Light chain FWR2: WYQQKPGQAPRLLIY (SEQ ID NO. 21).

[0119] Light chain FWR2 encoding sequence: TGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAC (SEQ ID NO. 22).

[0120] Light chain CDR2: GASSRAT (SEQ ID NO. 23).

[0121] Light chain CDR2 encoding sequence: GGAGCATCCAGCAGGGCCACC (SEQ ID NO. 24).

[0122] Light chain FWR3: GVPDRFSGGGSGTDFTLTIRRLEPEDYAVYY (SEQ ID NO. 25).

[0123] Light chain FWR3 encoding sequence:

[0124] GGCGTCCCAGATAGGTTCAGTGGCGGTGGGTCTGGGACCGACTTCACTCTCACCATCCGCAGACTGGAGCCTGAAGATTATGCAGTGTATTAC (SEQ ID NO. 26).

[0125] Light chain CDR3: CQQYASPPYTF (SEQ ID NO. 27).

[0126] Light chain CDR3 encoding sequence: TGTCAACAGTACGCCTCACCTCCGTACACTTTC (SEQ ID NO. 28).

[0127] Light chain FWR4:GRGTQLEIK (SEQ ID NO.29).

[0128] Light chain FWR4 coding sequence: GGCCGGGGGACCCAGTTGGAAATCAAA (SEQ ID NO.30).

[0129] Light chain variable region sequence:

[0130] EVVLTQSPGTLSLFPGDRATLSCRASRTIDNTYLAWYQQKPGQAPRLLIYGASSRATGVPDRFSGGGSGTDFTLTIRRLEPEDYAVYYCQQYASPPYTFGRGTQLEIK (SEQ ID NO. 31).

[0131] Light chain variable region encoding sequence:

[0132] GAAGTTGTGTTGACGCAGTCTCCGGGCACCCTGTCTTTGTTTCCGGGGGATAGGGCCACCCTCTCCTGCAGGGCCAGTCGGACGATTGACAACACCTACCTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGGAGCATCCAGCAG GGCCACCGGCGTCCCAGATAGGTTCAGTGGCGGTGGGTCTGGGACCGACTTCACTCTCACCATCCGCAGACTGGAGCCTGAAGATTATGCAGTGGTATTACTGTCAACAGTACGCCTCACCTCCGTACACTTTCGGCCGGGGGACCCAGTTGGAAATCAAA(SEQ ID NO.32).

[0133] mAb41 antibody:

[0134] Heavy chain FWR1:VVQLQESGPGLVKPSQTLSLTCSVS (SEQ ID NO.33).

[0135] Heavy chain FWR1 encoded sequence:

[0136] GTGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGCAGTGTGTCT (SEQ ID NO. 34).

[0137] Heavy chain CDR1 : GGSVSSSAYY (SEQ ID NO. 35).

[0138] Heavy chain CDR1 coding sequence: GGTGGGTCCGTCAGTAGTAGTGCTTACTAC (SEQ ID NO. 36).

[0139] Heavy chain FWR2: WTWIRQVPGKGLEWIGY (SEQ ID NO. 37).

[0140] Heavy chain FWR2 coding sequence:

[0141] TGGACCTGGATCCGCCAGGTCCCAGGGAAGGGCCTGGAATGGATTGGGTAC (SEQ ID NO. 38).

[0142] Heavy chain CDR2: VYNSGIN (SEQ ID NO. 39).

[0143] Heavy chain CDR2 coding sequence: GTCTACAACAGTGGGATCAAC (SEQ ID NO. 40).

[0144] Heavy chain FWR3: YYNPSLQRRVAMSLDTSKNEFSLKLMSVTAADTAVYF (SEQ ID NO. 41).

[0145] Heavy chain FWR3 coding sequence:

[0146] TACTACAACCCGTCCCTCCAGAGGCGAGTCGCCATGTCACTTGACACGTCTAAGAATGAGTTCTCGCTGAAATTGATGTCTGTGACTGCCGCGGACACGGCCGTATATTTC (SEQ ID NO. 42).

[0147] Heavy chain CDR3: CARDVAGGFGPGGLAYW (SEQ ID NO. 43).

[0148] Heavy chain CDR3 coding sequence:

[0149] TGTGCGAGAGACGTTGCCGGCGGCTTTGGTCCGGGGGGCCTTGCTTACTGG (SEQ ID NO. 44).

[0150] Heavy chain FWR4: GPGRLVTVSS (SEQ ID NO. 45).

[0151] Heavy chain FWR4 coding sequence: GGCCCGGGACGCCTGGTCACCGTCTCCTCT (SEQ ID NO. 46).

[0152] Heavy chain variable region sequence:

[0153] VVQLQESGPGLVKPSQTLSLTCSVSGGSVSSSAYYWTWIRQVPGKGLEWIGYVYNSGIN YYNPSLQRRVAMSLDTSKNEFSLKLMSVTAADTAVYFCARDVAGGFGPGGLAYWG PGRLVTVSS (SEQ ID NO. 47).

[0154] Heavy chain variable region coding sequence:

[0155] GTGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCC CTCACCTGCAGTGTGTCTGGTGGGTCCGTCAGTAGTAGTGCTTACTACTGGACCTG GATCCGCCAGGTCCCAGGGAAGGGCCTGGAATGGATTGGGTACGTCTACAACAGT GGGATCAACTACTACAACCCGTCCCTCCAGAGGCGAGTCGCCATGTCACTTGACA CGTCTAAGAATGAGTTCTCGCTGAAATTGATGTCTGTGACTGCCGCGGACACGGC CGTATATTTCTGTGCGAGAGACGTTGCCGGCGGCTTTGGTCCGGGGGGCCTTGCTT ACTGGGGCCCGGGACGCCTGGTCACCGTCTCCTCT (SEQ ID NO. 48).

[0156] Light chain FWR1 : AIQMTQSPSSLSASIGDRVTITC (SEQ ID NO. 49).

[0157] Light chain FWR1 coding sequence:

[0158] GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTATTGGAGACAGAGTC ACCATCACTTGT (SEQ ID NO. 50).

[0159] Light chain CDR1 : RASQGLRDELA (SEQ ID NO. 51 ).

[0160] Light chain CDR1 encoded sequence:

[0161] CGGGCGAGTCAGGGCCTTAGGGATGAGTTAGCC (SEQ ID NO. 52).

[0162] Light chain FWR2:WYQQKPGQAPKLLIY (SEQ ID NO.53).

[0163] Light chain FWR2 coding sequence: TGGTATCAGCAGAAACCAGGGCAAGCCCCTAAGCTCCTGATATAT (SEQ ID NO. 54).

[0164] Light chain CDR2: GVSTLQS (SEQ ID NO.55).

[0165] Light chain CDR2 encoding sequence: GGCGTATCCACTTTACAGAGT (SEQ ID NO.56).

[0166] Light chain FWR3:GVPSRFSGSGSGTDFTLTITSLQPEDFATYY (SEQ ID NO.57).

[0167] Light chain FWR3 encoding sequence:

[0168] GGAGTCCCCTCAAGGTTCAGCGGCAGTGGATCTGGCACAGATTTCACTCTCACCATCACCAGCCTGCAGCCTGAAGATTTTGCAACTTACTAT (SEQ ID NO. 58).

[0169] Light chain CDR3: CLQGFNYPKTF (SEQ ID NO.59).

[0170] Light chain CDR3 encoding sequence: TGTCTACAGGGTTTCAATTACCCCAAGACGTTC (SEQ ID NO.60).

[0171] Light chain FWR4:GPGTKVEIR (SEQ ID NO.61).

[0172] Light chain FWR4 coding sequence: GGCCCCGGGACCAAGGTCGAAATCAGG (SEQ ID NO.62).

[0173] Light chain variable region sequence:

[0174] AIQMTQSPSSLSASIGDRVTITCRASQGLRDELAWYQQKPGQAPKLLIYGVSTLQSGVPS RFSGSGSGTDFTLTITSLQPEDFATYYCLQGFNYPKTFGPGTKVEIR (SEQ ID NO. 63).

[0175] Heavy chain CDR1-encoding sequence:

[0176] GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTATTGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCCTTAGGGATGAGTTAGCCTGGTATCAGCAGAAACCAGGGCAAGCCCCTAAGCTCCTGATATATGGCGTATCCACTTTACAGAGTGGAGTCCCCTCAAGGTTCAGCGGCAGTGGATCTGGCACAGATTTCACTCTCACCATCACCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCTACAGGGTTTCAATTACCCCAAGACGTTCGGCCCCGGGACCAAGGTCGAAATCAGG (SEQ ID NO. 64).

[0177] mAb40 antibody:

[0178] Heavy chain FWR1: QLLLQSGGGLVKPGGHMRLSCEGS (SEQ ID NO. 65).

[0179] Heavy chain FWR1-encoding sequence:

[0180] CAACTACTACTACTGCAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGCACATGAGACTCTCCTGTGAAGGCTCT (SEQ ID NO. 66).

[0181] Heavy chain CDR1: GFTFGDY (SEQ ID NO. 67).

[0182] Heavy chain CDR1-encoding sequence: GGATTCACATTTGGTGACTAC (SEQ ID NO. 68).

[0183] Heavy chain FWR2: YMNWVRQAPGKGLEWIASM (SEQ ID NO. 69).

[0184] Heavy chain FWR2 coding sequence:

[0185] TACATGAATTGGGTCCGCCAGGCTCCAGGAAAGGGACTCGAATGGATCGCATCCATG (SEQ ID NO. 70).

[0186] Heavy chain CDR2: SPRETA (SEQ ID NO. 71).

[0187] Heavy chain CDR2 coding sequence: AGTCCTCGGGAAACCGCC (SEQ ID NO. 72).

[0188] Heavy chain FWR3: TYYADVVRGRFIISRDNAQQSTYLQMNFLRGDDSAVYY (SEQ ID NO. 73).

[0189] Heavy chain FWR3 coding sequence:

[0190] ACATACTACGCAGACGTTGTGAGGGGTCGATTCATTATCTCTAGGGACAACGCCCAGCAGTCAACTTATCTGCAAATGAACTTCCTGCGCGGCGACGATTCGGCCGTCTATTAC (SEQ ID NO. 74).

[0191] Heavy chain CDR3: CARGVNSRSSNYVYGFEVW (SEQ ID NO. 75).

[0192] Heavy chain CDR3 coding sequence:

[0193] TGTGCGCGAGGGGTTAACAGCCGCTCAAGTAACTACGTCTACGGTTTTGAAGTCTGG (SEQ ID NO. 76).

[0194] Heavy chain FWR4: GQGTTVTVSS (SEQ ID NO. 77).

[0195] Heavy chain FWR4 coding sequence: GGCCAGGGGACCACGGTCACCGTCTCTTCA (SEQ ID NO. 78).

[0196] Heavy chain variable region sequence:

[0197] QLLLLQSGGGLVKPGGHMRLSCEGSGFTFGDYYMNWVRQAPGKGLEWIASMSPRETATYYADVVRGRFIISRDNAQQSTYLQMNFLRGDDSAVYYCARGVNSRSSNYVYGFEVWGQGTTVTVSS (SEQ ID NO. 79).

[0198] Heavy chain variable region encoding sequence:

[0199] CAACTACTACTACTGCAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGCACATGAGACTCTCCTGTGAAGGCTCTGGATTCACATTTGGTGACTACTACATGAATTGGGTCCGCCAGGCTCCAGGAAAGGGACTCGAATGGATCGCATCCATGAGTCCTCGGGAAACCGCCACATACTACGCAGACGTTGTGAGGGGTCGATTCATTATCTCTAGGGACAACGCCCAGCAGTCAACTTATCTGCAAATGAACTTCCTGCGCGGCGACGATTCGGCCGTCTATTACTGTGCGCGAGGGGTTAACAGCCGCTCAAGTAACTACGTCTACGGTTTTGAAGTCTGGGGCCAGGGGACCACGGTCACCGTCTCTTCA (SEQ ID NO. 80).

[0200] Light chain FWR1 : EVVLTQSPGTLSLFPGDRATLSC (SEQ ID NO. 81 ).

[0201] Light chain FWR1 encoding sequence:

[0202] GAAGTTGTGTTGACGCAGTCTCCGGGCACCCTGTCTTTGTTTCCGGGGGATAGGGCCACCCTCTCCTGC (SEQ ID NO. 82).

[0203] Light chain CDR1 : RASRTIDNTYLA (SEQ ID NO. 83).

[0204] Light chain CDR1 encoding sequence: AGGGCCAGTCGGACGATTGACAACACCTACCTAGCC (SEQ ID NO. 84).

[0205] Heavy chain FWR2: WIRQPPGKAPRLLIY (SEQ ID NO. 82).

[0206] Heavy chain FWR2 encoding sequence: GAGGTGCAGCTGGTGGAGTCTGGGGCTGCCTGGTCAAAGCCTATACTGTATGCAACCCCAGAACCCTAAAGGGCTACAGCCATGGACTACAGCCCTCTGGGTTTCTGGTGAAGCAAGGTGGAAATCAAACGGGTGC (SEQ ID NO. 83).

[0207] Heavy chain CDR2: PATDSDTVNYLN (SEQ ID NO. 84).

[0208] Heavy chain CDR2 encoding sequence: GAGGTGCAGCTGGTGGAGTCTGGGGCTGCCTGGTCAAAGCCTATACTGTATGCAACCCCAGAACCCTAAAGGGCTACAGCCATGGACTACAGCCCTCTGGGTTTCTGGTGAAGCAAGGTGGAAATCAAACGGGTGC (SEQ ID NO. 83).

[0209] Heavy chain FWR3: RYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR (SEQ ID NO. 85).

[0210] Heavy chain FWR3 encoding sequence: GAGGTGCAGCTGGTGGAGTCTGGGGCTGCCTGGTCAAAGCCTATACTGTATGCAACCCCAGAACCCTAAAGGGCTACAGCCATGGACTACAGCCCTCTGGGTTTCTGGTGAAGCAAGGTGGAAATCAAACGGGTGC (SEQ ID NO. 83).

[0211] GGCGTCCCAGATAGGTTCAGTGGCGGTGGGTCTGGGACCGACTTCACTCTCACCATCCGCAGACTGGAGCCTGAAGATTATGCAGTGTATTAC (SEQ ID NO. 90).

[0212] Heavy chain CDR3: WGQGTLVTVSS (SEQ ID NO. 86).

[0213] Heavy chain CDR3 encoding sequence: GAGGTGCAGCTGGTGGAGTCTGGGGCTGCCTGGTCAAAGCCTATACTGTATGCAACCCCAGAACCCTAAAGGGCTACAGCCATGGACTACAGCCCTCTGGGTTTCTGGTGAAGCAAGGTGGAAATCAAACGGGTGC (SEQ ID NO. 83).

[0214] Heavy chain FWR4: WGQGTLVTVSS (SEQ ID NO. 87).

[0215] Heavy chain FWR4 encoding sequence: GAGGTGCAGCTGGTGGAGTCTGGGGCTGCCTGGTCAAAGCCTATACTGTATGCAACCCCAGAACCCTAAAGGGCTACAGCCATGGACTACAGCCCTCTGGGTTTCTGGTGAAGCAAGGTGGAAATCAAACGGGTGC (SEQ ID NO. 83).

[0216] Heavy chain variable region sequence:

[0217] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMDWVRQAPGKGLEWVSAISSGSDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAWGQGTLVTVSS (SEQ ID NO. 88).

[0218] Light chain variable region encoding sequence:

[0219] GAAGTTGTGTTGACGCAGTCTCCGGGCACCCTGTCTTTGTTTCCGGGGGATAGGGCCACCCTCTCCTGCAGGGCCAGTCGGACGATTGACAACACCTACCTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGGAGCATCCAGCAGGGCCACCGGCGTCCCAGATAGGTTCAGTGGCGGTGGGTCTGGGACCGACTTCACTCTCACCATCCGCAGACTGGAGCCTGAAGATTATGCAGTGTATTACTGTCAACAGTACGCCTCACCTCCGTACACTTTCGGCCGGGGGACCCAGTTGGAAATCAAA (SEQ ID NO. 96).

[0220] wherein the mouse IgG2a backbone sequence in the above antibody is as follows:

[0221] GCCAAGACAACAGCTCCTTCCGTGTACCCTCTGGCCCCCGTGTGCGGCGACACCACCGGCTCTTCTGTGACCCTGGGCTGCCTGGTGAAGGGCTACTTTCCTGAGCCCGTCACCCTGACCTGGAACTCCGGATCTCTGTCCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCGATCTGTACACCCTGTCCTCCAGCGTGACCGTGACCTCTTCTACATGGCCTTCCCAGTCTATCACCTGCAACGTGGCTCATCCTGCCAGCTCTACCAAAGTGGACAAGAAGATCGAGCCTAGAGGCCCTACAATCAAGCCCTGTCCTCCTTGCAAGTGCCCTGCTCCTAACCTGCTGGGAGGCCCCTCTGTGTTCATCTTCCCTCCTAAGATCAAGGACGTGCTGATGATCTCCCTGTCTCCTATCGTGACCTGTGTGGTGGTCGATGTGTCTGAGGACGACCCTGACGTGCAGATCTCTTGGTTCGTGAACAATGTGGAAGTGCACACCGCCCAGACCCAGACCCACAGAGAGGACTACAACAGCACACTGAGAGTGGTGTCCGCCCTGCCTATCCAGCACCAGGACTGGATGTCCGGCAAAGAGTTCAAGTGCAAGGTGAACAACAAGGACCTGCCCGCCCCCATCGAGCGGACCATCTCCAAGCCTAAAGGCTCCGTGCGGGCCCCTCAAGTGTACGTGCTGCCTCCACCAGAGGAAGAGATGACCAAGAAGCAGGTGACACTGACCTGCATGGTGACCGACTTCATGCCTGAGGATATCTACGTGGAATGGACCAACAACGGCAAGACCGAGCTGAACTACAAGAACACCGAGCCCGTTCTGGACTCCGACGGCTCTTACTTCATGTACTCCAAGCTGAGAGTGGAGAAGAAAAACTGGGTGGAACGGAACTCCTACTCTTGCTCCGTCGTGCACGAGGGCCTGCACAACCACCACACCACCAAGTCCTTCTCCAGAACCCCTGGCAAG (SEQ ID NO.97)..

[0222] Killing effect of HBcAg antibody on hepatocytes

[0223] First, the binding ability of the antibody to hepatitis B-related hepatocellular carcinoma tumor cells was detected, and the tumor paraffin-embedded tissues of hepatitis B-related hepatocellular carcinoma patients were sectioned and stained using multiplex immunofluorescence staining technology. The specific steps are as follows: the paraffin tissue section is placed in a constant temperature oven at 65°C for 2h, and then dewaxed with xylene and gradient alcohol hydration; add Tris-EDTA antigen repair solution in the section box, open the water in the pressure cooker and boil, then put the paraffin section box into the pressure cooker, close the lid, and start timing when the pressure cooker is heated to the exhaust valve rotates for 2.5min, turn off the power and cool naturally at room temperature. Wash with PBS buffer for 3 times, 1 min each time, put the whole glass slide into 0.3% Triton X-100 solution for 20 min, wash with PBS buffer for 3 times, 1 min each time, and then wash with PBST solution for 1 time, 3 min each time. Draw the tissue area with an immunohistochemical pen, add 5% BSA to block at room temperature for 30 min. Dilute the primary antibody mixed solution with 1% BSA, i.e. GPC3 (Cat#AF2119-SP, R&D, 1:40), HBcAg (Cat#ab115992, abcam, 1:200), and APR-mAbs (25μg / ml), drop on the tissue section, and incubate the section in a wet box at 4°C overnight. The next day, place the wet box at room temperature for 1h, wash with PBST solution for 3 times, 5 min each time. Use 1% BSA to configure the secondary antibody solution (Cat#A21436, Cat#ab6718 and Cat#A32787, Thermo Fisher Scientific), and stain at room temperature for 1 hour, then stain with DAPI solution (Cat#0012100500, Panovue) for 15 minutes. Use a laser confocal scanning microscope to scan the glass slide at 40x magnification and analyze. The results show that the antibody provided by the application, such as mAb40, can bind to tumor cells of HBV-positive hepatocellular carcinoma patients (Fig. 4B), indicating that the antibody can bind to tumor cells of hepatitis B-related hepatocellular carcinoma patients. Figure 3 ), indicating that the antibody can bind to tumor cells of hepatitis B-related hepatocellular carcinoma patients.

[0224] Further, human hepatoma cell line HepG2.2.15 stably transfected with full-length genome of HBV and human hepatoma cell line Huh7 stably transfected with HBcAg antigen genome were selected and co-cultured with the antibody provided by the application in vitro, and after 24 hours, the cells in the supernatant of the culture medium were collected, and the adherent cells were trypsinized, centrifuged, resuspended with binding buffer to obtain a cell suspension, then the cell suspension was incubated with PI and Annexin-V antibody in the apoptosis detection kit at room temperature for 15 minutes, and after adding an appropriate amount of binding buffer, Attune Flow Cytometers (ThermoFisher) were used to obtain fluorescence measurement results, and FlowJo software (Tree Star) was used to analyze the data. The results suggest that the HBcAg specific antibody provided by the application can promote the apoptosis of HBV+ hepatoma cells to a higher level than the antibody that cannot bind HBcAg. Figure 4

[0225] To further characterize the anti-HBcAg antibody provided by the application has the function of killing tumors, and has the potential to enhance the application of anti-PD-1 monoclonal antibody drugs, the tumor size change curve of HBcAg+ hepatoma mouse subcutaneous tumor during treatment and the weight of the tumor after treatment were determined to evaluate the therapeutic effect of the antibody.

[0226] For this assay, first establish HBcAg+ hepatoma cell line (HBcAg+ Hepa1-6), and inject HBcAg+ Hepa1-6 (5x106 cells / each) subcutaneously on the back of 6-8 week old male C57BL / 6 mice. Then the mice were randomly divided into 5 groups, 6 in each group, and received tumor periphery drug injection every 2 days, a total of 4 times, including PBS group, isotype control antibody (#B115101, abinvivo) group, anti-PD-1 monoclonal antibody group (100 μg / each), HBcAg specific antibody group (200 ug / each) and HBcAg-mAb and PD-1 monomer combination group. Measure the length and width of the tumor with a vernier caliper every 2-3 days, calculate the tumor volume according to the formula V=(length x width)2 / 2, and draw the tumor growth curve. By comparing the tumor growth of the 4 groups of mice, it was found that the HBcAg specific antibody mAb40, mAb32 and mAb41 provided by the application can kill HBcAg+ hepatoma, and the therapeutic effect is better than that of simple HBcAg antibody treatment and simple PD-1 monoclonal antibody treatment when combined with PD-1 monoclonal antibody.

[0227] Example 5 Analysis and identification of the target of the HBcAg antibody on liver cells

[0228] ​In terms of the specificity of the target point where the antibody is used and the safety of subsequent use, the applicant has made explorations. On the one hand, the applicant carries out immunofluorescence staining on fresh frozen sections of tumor tissues and paracancerous tissues of patients with hepatitis B related liver cancer. The specific operation steps are as follows: after the frozen section is thawed and restored to room temperature, PBS is added for rehydration, and 1% BSA is added for blocking for 1 hour. After the blocking solution is absorbed, the primary antibody buffer solution (1% BSA) is added for overnight incubation. After washing 5 times with PBS, the secondary antibody buffer solution (0.25% BSA) is added for 2 hours of incubation. Finally, after being fixed with 4% paraformaldehyde, nuclear staining is carried out with DAPI, and the section is photographed. The results suggest that, compared with paracancerous liver cells, tumor cells of hepatitis B related liver cancer significantly overexpress HBcAg, and there is more HBcAg protein binding / expression on the cell membrane Figure 6 a-b). On the other hand, fresh specimens of tumor tissues and paracancerous tissues of patients with hepatitis B related liver cancer are dissociated into single cell suspension and subjected to flow cytometry detection. The specific operation is as follows: after the cell suspension is washed with PBS+1% FBS solution, it is configured to a concentration of 3*10^6 cells / ml, 100ul of cell suspension is added to 50ug / ml of synthetic antibody, and incubated at room temperature for 30 minutes. After the incubation of the cells with the primary antibody (Cat# ab8637), the supernatant is removed after washing twice with PBS+1% FBS solution, and fluorescent secondary antibody (donkey anti-mouse AF647, Cat# A32787, 1:500, 200ul) is added, and incubated at room temperature for 30 minutes. After incubation, wash once and fix with 1% paraformaldehyde. After fixing, the flow cytometry analyzer is used to detect the binding of tumor cells and liver cell membranes to the antibody Figure 6 c).

[0229] Further, the inventors performed immunoelectron microscopy staining on tissue sections of hepatitis B-related liver cancer to observe the specific location of HBcAg protein expression. The steps are as follows: fresh tumor tissue is cut into small tissue blocks of 1 mm3, and placed in an immunoelectron microscopy fixing solution. After being rinsed three times with 0.1M phosphate buffer PB (pH 7.4) pre-cooled at 4°C, the sample is dehydrated with pre-cooled gradient alcohol. Then, the sample is permeated with different gradient alcohols and resin solutions. After permeation with pure resin, the sample is dropped into an embedding capsule, and then placed in pure resin and capped with a capsule cap for embedding. After polymerization at -20°C for 48 hours using a low-temperature ultraviolet polymerization instrument, the sample is restored to room temperature, and the resin block is removed for use. The sample is cut into 70-80 nm ultrathin sections using an ultramicrotome, and the sections are collected on a coated nickel mesh, and stored at 4°C for immunolabeling. After rehydration, TBS room temperature washing, and 1% BSA / TBS blocking, the antibody and blocking solution are added and incubated overnight at 4°C. After rehydration, the sample is washed three times with TBS, and then the secondary antibody and secondary antibody diluent are added and incubated at room temperature for 20 minutes, and then dried. After multiple washes, the sample is stained with uranium, and then dried. The sample is observed under a transmission electron microscope and images are collected. The black 10 nm gold particles are positive expression. The results show that in the cancer-adjacent liver cells, the HBcAg protein is mainly expressed on the endoplasmic reticulum, while in the tumor cells, the HBcAg protein is scattered in the cytoplasm, and the expression amount is significantly higher than that in the cancer-adjacent liver cells. At the same time, in the tumor tissue, the distribution of HBcAg protein can be observed in the intercellular space and on the surface of the cell membrane. Figure 7 ) of the HBcAg protein on the cell membrane can be directly recognized and combined by the antibody, and induce the subsequent antibody killing function.

[0230] Example 6: Identification of the HBcAg antibody binding epitope

[0231] Based on the above in vivo and in vitro results, it can be seen that different antibody sequences have different killing and binding abilities on hepatitis B liver cancer cells, which shows that the binding epitope of the antibody on the HBcAg protein is very important, and a specific binding site may be required to ensure high affinity binding and activate the downstream anti-tumor function. In order to further clarify the best HBcAg antibody binding epitope, we used cryo-EM to identify the binding epitope of the mAb40 antibody Fab with the HBcAg antigen dimer, which had the best tumor killing effect in the in vivo experiment. HBcAg is a dimer, and the sequences of its A and B chains are the same, and the two chains are exactly the same, and the sequences are as follows:

[0232] MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKFRQLLWFHVSCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO: 98).

[0233] By local classification and refinement, the resulting Horizontal density map Figure 8 a-c) Suggestion: Fab binds to the top of the dimer, covering 78-80 residues, which are defined as human epitope 4 (he4). Three complementarity determining regions (CDRs) on the heavy chain and one CDR on the light chain are involved in the interaction with the HBcAg dimer. The buried surface area of the epitope on the heavy and light chains is and Six potential hydrogen bonds are formed between mAb40 Fab (Y33, T56, Y59, Y107, Y109) and HBcAg dimer (E77, D78, P79). In addition, positively charged residues R54 on the heavy chain and R27 on the light chain form salt bridges with negatively charged residues E77 on both chains of the HBcAg antigen dimer Figure 8 d).

[0234] The present application is illustrated by the above examples, but the present application is not limited to the above process steps, that is, it does not mean that the present application must rely on the above process steps to be implemented. It should be clear to those skilled in the art that any improvement on the present application, equivalent replacement of the raw materials selected by the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the scope of protection and disclosure of the present application.

Claims

1. An isolated monoclonal antibody, characterized in that, The monoclonal antibody binds to an HBcAg antigen, the monoclonal antibody binds to at least one amino acid residue selected from the group consisting of E77 and D78 and P79 of the HBcAg dimer A / B chain.

2. The monoclonal antibody of claim 1, wherein, the sequence of the A chain and the sequence of the B chain are as set forth in SEQ ID NO. 98, respectively.

3. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is a humanized antibody.

4. The monoclonal antibody of claim 1, wherein, The monoclonal antibody binds to the K D value is less than or equal to 1.593E-9 M.

5. The monoclonal antibody of claim 1, wherein, The monoclonal antibody binds to the K D value is less than or equal to 2.780E-10 M.

6. The monoclonal antibody of claim 1, wherein, The monoclonal antibody binds to at least the residue E77 of the A chain.

7. The monoclonal antibody of claim 1, wherein, The monoclonal antibody binds to at least the residue D78 of the A chain.

8. The monoclonal antibody of claim 1, wherein, The monoclonal antibody binds to at least the residue D78 of the B chain.

9. The monoclonal antibody of claim 1, wherein, The monoclonal antibody binds to at least the residue E77 of the B chain.

10. The monoclonal antibody of claim 1, wherein, The monoclonal antibody binds to at least the residue P79 of the B chain.

11. The monoclonal antibody of claim 1, wherein, The monoclonal antibody binds to at least one amino acid residue selected from the group consisting of E77 and D78 and P79 of the HBcAg dimer A / B chain.

12. The monoclonal antibody of claim 1, wherein, The monoclonal antibody is mAb40, the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of the mAb40 comprise the sequences as set forth in SEQ ID NO. 67, SEQ ID NO. 71 and SEQ ID NO. 76, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of the mAb40 comprise the sequences as set forth in SEQ ID NO. 83, SEQ ID NO. 87 and SEQ ID NO. 91, respectively.

13. The monoclonal antibody of claim 1, wherein, The monoclonal antibody is mAb40, the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of the mAb40 comprise the sequences as set forth in SEQ ID NO. 67, SEQ ID NO. 71 and SEQ ID NO. 76, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of the mAb40 comprise the sequences as set forth in SEQ ID NO. 83, SEQ ID NO. 87 and SEQ ID NO. 91, respectively.

14. The monoclonal antibody according to claim 13, characterized in that, The monoclonal antibody is mAb40, the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of the mAb40 comprise the sequences as set forth in SEQ ID NO. 67, SEQ ID NO. 71 and SEQ ID NO. 76, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of the mAb40 comprise the sequences as set forth in SEQ ID NO. 83, SEQ ID NO. 87 and SEQ ID NO. 91, respectively.

15. The monoclonal antibody of claim 1, wherein, The monoclonal antibody is mAb32, the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of the mAb32 comprise the sequences as set forth in SEQ ID NO. 3, SEQ ID NO. 7 and SEQ ID NO. 11, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of the mAb32 comprise the sequences as set forth in SEQ ID NO. 19, SEQ ID NO. 23 and SEQ ID NO. 27, respectively.

16. The monoclonal antibody of claim 15, wherein, The monoclonal antibody is mAb32, the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of the mAb32 comprise the sequences as set forth in SEQ ID NO. 3, SEQ ID NO. 7 and SEQ ID NO. 11, respectively, and the amino acid sequences of the light chain CDR1, CDR2 and CDR3 of the mAb32 comprise the sequences as set forth in SEQ ID NO. 19, SEQ ID NO. 23 and SEQ ID NO. 27, respectively.

17. The monoclonal antibody of claim 1, wherein, The monoclonal antibody is mAb41, the amino acid coding sequences of the heavy chain CDR1, CDR2 and CDR3 of the mAb41 comprise the sequences as shown in SEQ ID NO. 35, SEQ ID NO. 39 and SEQ ID NO. 43 respectively, and the amino acid coding sequences of the light chain CDR1, CDR2 and CDR3 of the mAb41 comprise the sequences as shown in SEQ ID NO. 51, SEQ ID NO. 55 and SEQ ID NO. 59 respectively.

18. The monoclonal antibody of claim 17, wherein, The heavy chain variable region sequence of the monoclonal mAb41 comprises the sequence as shown in SEQ ID NO. 47, and the light chain variable region sequence of mAb41 comprises the sequence as shown in SEQ ID NO.

63.

19. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody of any one of claims 1-18.

20. A vector or expression cassette, characterized in that, The vector or expression cassette comprises the nucleic acid molecule of claim 19.

21. A cell, characterized in that, The cell comprises the nucleic acid molecule of claim 19 and / or the vector or expression cassette of claim 20.

22. A monoclonal antibody characterized in that, The monoclonal antibody is expressed by the vector or expression cassette of claim 20.

23. A monoclonal antibody characterized in that, The monoclonal antibody is expressed by the cell of claim 21.

24. The monoclonal antibody of any one of claims 1-18 and 22-23, wherein, The monoclonal antibody is used for treating liver cancer.

25. The monoclonal antibody of claim 24, wherein the liver cancer is hepatocellular carcinoma.

26. The monoclonal antibody of claim 25, wherein the hepatocellular carcinoma is caused by hepatitis B virus.

27. A composition characterized in that, The composition comprises the monoclonal antibody of any one of claims 1-18 and 22-23 and / or the nucleic acid of claim 19 and / or the vector or expression cassette of claim 22 and / or the cell of claim 21.

28. The composition of claim 27, wherein, The composition further comprises other anti-tumor components.

29. The composition of claim 27, wherein, The anti-tumor component is PD-1.

30. The composition of claim 28, wherein, The composition comprises the monoclonal antibody mAb40 and PD-1.

31. The composition of claim 28, wherein, The composition comprises the monoclonal antibody mAb32 and PD-1.

32. The composition of claim 28, wherein, The composition comprises the monoclonal antibody mAb41 and PD-1.

33. Use of the monoclonal antibody of any one of claims 1-18 and 22-23 and / or the nucleic acid of claim 25 and / or the vector or expression cassette of claim 20 and / or the cell of claim 21 and / or the composition of any one of claims 27-32 in the preparation of an anti-tumor drug.

34. Use according to claim 33, characterized in that, The tumor is liver cancer.

Images