A kit for in vitro quantitative hepatitis B virus large surface protein assay, as well as a biomarker kit and monoclonal antibody kit for treating liver diseases.

Abstract

This invention discloses a kit for in vitro quantitative detection of hepatitis B virus large surface protein (LHBS). This kit includes multiple monoclonal antibodies, each with its own binding specificity to specific regions of LHBS, thereby improving the sensitivity and dynamic range of LHBS detection in biological samples. Secondly, this invention also provides a set of biological markers targeting specific regions of LHBS, and the monoclonal antibodies can specifically identify this set of biomarkers, enabling non-invasive analysis of HBV infection stages and liver cancer prognosis in biological samples. Furthermore, this invention also provides a set of monoclonal antibodies that, by detecting biomarkers in desired subjects, can predict, diagnose, or treat chronic liver disease.

Description

Technical Field

[0001] This invention relates to a kit and method for detecting hepatitis B virus (HBV) infection stages, particularly to a kit for in vitro quantitative analysis of HBV large surface protein, a set of biomarkers for analyzing HBV infection stages and liver cancer prognosis, and a set of monoclonal antibodies for predicting, diagnosing, or treating chronic liver diseases. Background Technology

[0002] Chronic hepatitis B (CHB) virus infection is a leading cause of hepatocellular carcinoma (HCC) worldwide, and the most significant cause in Asia. Early diagnosis and effective treatment of hepatitis B virus (HBV) are crucial for preventing advanced liver disease. Although viral DNA titer is a sensitive early biomarker for antiviral treatment efficacy, the experimental methods for this detection require DNA extraction and real-time polymerase chain reaction (PCR), which is time-consuming and costly. Therefore, developing new biomarkers to detect early changes in viral replication and predict the effectiveness of antiviral treatment is essential.

[0003] Recently, large HBV surface protein (LHBS) has been identified as a crucial marker of the HBV lifecycle. LHBS comprises the pre-S1, pre-S2, and S domains of the surface protein and is an integral component of the viral particle sheath. Given the high correlation between serum LHBS levels and HBV DNA replication rate, it is reasonable to hypothesize that serum LHBS levels have the potential to serve as a biomarker for viral replication (e.g., HBV DNA). Compared to detecting HBV DNA, detecting serum LHBS is more time- and cost-effective. Therefore, there is an urgent need to develop LHBS-related biomarkers to improve the sensitivity, dynamic range, and analysis of HBV infection stages. Summary of the Invention

[0004] Therefore, one aspect of the present invention is to provide a kit for in vitro quantitative analysis of hepatitis B virus large surface protein (LHBS) in biological samples, which includes a set of monoclonal antibodies that have their own binding specificity to specific regions of LHBS, so as to improve the sensitivity and dynamic range of LHBS detection.

[0005] Secondly, another aspect of the present invention is to provide a set of biomarkers for analyzing the HBV infection stage and liver cancer prognosis in biological samples, wherein the set of biomarkers includes multiple biomarkers, each having a specific region of LHBS.

[0006] Furthermore, another aspect of the present invention is to provide a monoclonal antibody group for predicting, diagnosing, or treating chronic liver disease using biomarkers of subjects in need.

[0007] According to the above-described form of the present invention, a kit for in vitro quantitative determination of hepatitis B virus large surface protein (LHBS) in biological samples is proposed. In one embodiment, this kit may include a blocking solution; a set of monoclonal antibodies for detecting biomarkers in biological samples, wherein the set of monoclonal antibodies includes a first monoclonal antibody immobilized at multiple individual sites on a solid support and immersed in the blocking solution; at least one of a second monoclonal antibody and a third monoclonal antibody, wherein the second monoclonal antibody and the third monoclonal antibody are respectively linked to a label; and a detection reagent capable of reacting with the label to form a detectable product.

[0008] In the above embodiments, the first monoclonal antibody has binding specificity to the first polypeptide listed in sequence identification number (SEQ ID NO): 1 in the biological sample, the second monoclonal antibody has binding specificity to the second polypeptide listed in SEQ ID NO: 2 in the biological sample, and the third monoclonal antibody has binding specificity to the third polypeptide listed in SEQ ID NO: 3 in the biological sample.

[0009] According to another aspect of the present invention, a set of biomarkers for analyzing the HBV infection stage and liver cancer prognosis in biological samples is proposed, which may include a first biomarker having a first polypeptide of SEQ ID NO: 1, a second biomarker having a second polypeptide of SEQ ID NO: 2, and / or a third biomarker having a third polypeptide of SEQ ID NO: 3.

[0010] According to another embodiment of the present invention, a monoclonal antibody suite is provided for predicting, diagnosing, or treating chronic liver disease based on biomarkers of a subject in need. In one embodiment, the aforementioned monoclonal antibody suite may include a first monoclonal antibody or a first antigen-binding fragment thereof, and at least one of a second monoclonal antibody or a second antigen-binding fragment thereof and a third monoclonal antibody or a third monoclonal antibody thereof. The first monoclonal antibody specifically detects a first biomarker having a first polypeptide of SEQ ID NO: 1, and the first monoclonal antibody or its first antigen-binding fragment comprises heavy chain CDR1 to CDR3 sequences composed of SEQ ID NOs: 4 to 6 and light chain CDR1 to CDR3 sequences composed of SEQ ID NOs: 7 to 9. The second monoclonal antibody or its second antigen-binding fragment specifically detects a second biomarker having a second polypeptide of SEQ ID NO: 2, and the second antibody or its second antigen-binding fragment comprises heavy chain CDR1 to CDR3 sequences composed of SEQ ID NOs: 10 to 12 and light chain CDR1 to CDR3 sequences composed of SEQ ID NOs: 13 to 15. The third monoclonal antibody or the third antigen binding fragment can specifically detect the third biomarker having the third polypeptide of SEQ ID NO: 3, and the third monoclonal antibody or the third antigen binding fragment contains the heavy chain CDR1 to CDR3 sequences composed of SEQ ID NOs: 16 to 18 and the light chain CDR1 to CDR3 sequences composed of SEQ ID NOs: 19 to 21.

[0011] In the above embodiments, the aforementioned biomarkers include a first biomarker having a first polypeptide of SEQ ID NO: 1, a second biomarker having a second polypeptide of SEQ ID NO: 2, and a third biomarker having a third polypeptide of SEQ ID NO: 3.

[0012] In the above embodiments, the aforementioned first monoclonal antibody or its first antigen-binding fragment comprises a heavy chain sequence as shown in SEQ ID NO: 22 and a light chain sequence as shown in SEQ ID NO: 23.

[0013] In the above embodiments, the aforementioned second monoclonal antibody or its second antigen-binding fragment comprises a heavy chain sequence as shown in SEQ ID NO: 24 and a light chain sequence as shown in SEQ ID NO: 25.

[0014] In the above embodiments, the aforementioned third monoclonal antibody or its third antigen-binding fragment comprises a heavy chain sequence as shown in SEQ ID NO: 26 and a light chain sequence as shown in SEQ ID NO: 27.

[0015] In the above embodiments, the aforementioned first monoclonal antibody comprises a heavy chain encoded by the sequence listed in SEQ ID NO: 28 and a light chain encoded by the sequence listed in SEQ ID NO: 29.

[0016] In the above embodiments, the aforementioned second monoclonal antibody comprises a heavy chain encoded by the sequence listed in SEQ ID NO: 30 and a light chain encoded by the sequence listed in SEQ ID NO: 31.

[0017] In the above embodiments, the aforementioned third monoclonal antibody comprises a heavy chain encoded by the sequence listed in SEQ ID NO: 32 and a light chain encoded by the sequence listed in SEQ ID NO: 33.

[0018] In the above embodiments, the aforementioned first monoclonal antibody or its first antigen-binding fragment, the second monoclonal antibody or its second antigen-binding fragment, and the third monoclonal antibody or its third antigen-binding fragment are chimeric antigen receptors (CARs) of engineered T cells.

[0019] In the above embodiments, the aforementioned first monoclonal antibody or its first antigen-binding fragment, the second monoclonal antibody or its second antigen-binding fragment, and the third monoclonal antibody or its third antigen-binding fragment are antibody-drug conjugates (ADCs).

[0020] The kits for in vitro quantitative hepatitis B virus large surface protein (LHBS) of the present invention are used to analyze biomarkers for HBV infection stages and liver cancer prognosis, as well as monoclonal antibody kits for predicting, diagnosing, or treating chronic liver diseases. The antibodies have their own binding specificity to specific regions of LHBS, thereby improving the sensitivity and dynamic range of LHBS detection in biological samples, analyzing HBV infection stages and liver cancer prognosis in biological samples, and developing antigen-binding domains and antibody-drug complexes (ADCs) for engineered T cell CARs.

[0021] It is understood that the above overview and the following detailed description are merely illustrative examples, mainly to provide further explanation of the claimed invention. Attached Figure Description

[0022] To make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, a detailed description of the accompanying drawings is provided below:

[0023] Figure 1 A schematic diagram showing the large HBV surface (LHBS), medium HBV surface (MHBS), and HBV surface (HBS) proteins according to an embodiment of the present invention.

[0024] Figures 2A to 2C This describes a pre-S1 monoclonal antibody against different regions of LHBS according to an embodiment of the present invention. Figure 2A ), pre-S2 monoclonal antibody ( Figure 2B ) and HBS monoclonal antibody ( Figure 2C The affinity of combining )

[0025] Figure 2D This diagram illustrates the recognition regions of the pre-S1 monoclonal antibody, pre-S2 monoclonal antibody, and HBS monoclonal antibody against LHBS according to an embodiment of the present invention.

[0026] Figures 3A to 3C This describes a preS1 monoclonal antibody against a specific recombinant protein according to an embodiment of the present invention. Figure 3A ), pre-S2 monoclonal antibody ( Figure 3B ) and HBS monoclonal antibody ( Figure 3C The curves showing the detection sensitivity and dynamic range of the device.

[0027] Figures 4A to 4B This illustrates the use of a pre-S2 monoclonal antibody according to an embodiment of the present invention. Figure 4A ) or HBS monoclonal antibody ( Figure 4B The detection sensitivity and dynamic range of the LHBS chemiluminescent sandwich ELISA system for detecting antibodies in serum.

[0028] Figure 5 This is a bar chart showing the serum LHBS levels of different patient groups at different stages of HBV infection detected by an LHBS chemiluminescence sandwich ELISA system according to an embodiment of the present invention.

[0029] Figures 6A to 6C This invention illustrates the expression levels of major HBS and LHBS in the peritumoral and tumor regions of HCC patients according to an embodiment of the present invention, using IHC staining ( Figure 6A and Figure 6B ) and Western ink dot method ( Figure 6C ) to conduct analysis.

[0030] Figure 7A and Figure 7B This presents the results of a Kaplan-Meier analysis showing the correlation between tissue LHB staining characteristics and disease-free survival (DFS) and overall survival (OS) in a clinical age-matched HCC cohort according to an embodiment of the present invention.

[0031] Figure 8A and Figure 8BThis presents the Kaplan-Meier analysis results of serum LHBS performance detected by ELISA in an age-matched HCC clinical group according to another embodiment of the present invention, which correlated with disease-free survival (DFS) and overall survival (OS).

[0032] Figure 9 This shows the analysis results of LHBS in HBV(+) HepAD38 live cells using flow cytometry according to an embodiment of the present invention.

[0033] In the attached figures, the following labels are used:

[0034] 901, 903, 905, 907: Curves Detailed Implementation

[0035] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The same reference numerals used in the drawings and description refer to the same or similar parts wherever possible.

[0036] As mentioned above, the present invention provides a kit for in vitro quantitative detection of hepatitis B virus large surface protein (LHBS) and an immunoassay method, wherein the kit and immunoassay method include a variety of monoclonal antibodies, each having its own binding specificity (also known as binding specificity) for specific regions of LHBS, thereby improving the sensitivity and dynamic range of LHBS detection in biological samples.

[0037] It has been found that even when these patients continue to receive antiviral treatment, LHBS in their serum remains positive, which can be used to predict HBV DNA conversion. Real-time PCR detection of HBV DNA is both time-consuming and costly; therefore, quantitative detection of LHBS has the potential to replace quantitative detection of HBV DNA.

[0038] In general, the term "monoclonal antibody" as used herein refers to a monoclonal antibody that has specific binding specificity to a specific region of LHBS, including but not limited to a first monoclonal antibody that has specific binding specificity to the first polypeptide of SEQ ID NO: 1, a second monoclonal antibody that has specific binding specificity to the second polypeptide of SEQ ID NO: 2, and a third monoclonal antibody that has specific binding specificity to the third polypeptide of SEQ ID NO: 3.

[0039] Generally, the first monoclonal antibody or its antigen-binding fragment or capturing antibody has binding specificity to the first polypeptide of SEQ ID NO: 1, which corresponds to the pre-S1 region of LHBS. In one embodiment, the first monoclonal antibody may be immobilized on a surface for specifically capturing the pre-S1 region of LHBS protein in a biological sample. In some examples, the biological sample may include, but is not limited to, tissue and / or liquid samples. In some examples, tissue may include solid tissue and / or soft tissue. In some specific examples, liquid samples may include serum, blood, urine, semen, cerebrospinal fluid (CSF), and saliva.

[0040] In some embodiments, the first monoclonal antibody or its antigen-binding fragment may comprise a heavy chain CDR sequence and a light chain CDR sequence, wherein the heavy chain CDR sequence is selected from the sequences listed in SEQ ID NOs: 4 to 6, and the light chain CDR sequence is selected from the sequences listed in SEQ ID NOs: 7 to 9. In other embodiments, the first monoclonal antibody or its antigen-binding fragment may comprise a heavy chain sequence as listed in SEQ ID NO: 22 and a light chain sequence as listed in SEQ ID NO: 23. In some specific embodiments, the first monoclonal antibody or its antigen-binding fragment may comprise a heavy chain sequence encoded by the sequence listed in SEQ ID NO: 28 and a light chain sequence encoded by the sequence listed in SEQ ID NO: 29.

[0041] Generally, the second monoclonal antibody or its antigen-binding fragment or detection antibody has binding specificity to the second polypeptide of SEQ ID NO: 2, which corresponds to the pre-S2 region of LHBS. The second monoclonal antibody or detection antibody can be designed in the reaction mixture to capture the pre-S2 region of LHBS protein or its immune complex, thereby significantly expanding the dynamic range of LHBS detection.

[0042] In some embodiments, the second monoclonal antibody or its antigen-binding fragment may include a heavy chain CDR sequence and a light chain CDR sequence, wherein the heavy chain CDR sequence is selected from the sequences listed in SEQ ID NOs: 10 to 12, and the light chain CDR sequence is selected from the sequences listed in SEQ ID NOs: 13 to 15. In other embodiments, the second monoclonal antibody or its antigen-binding fragment may include a heavy chain sequence as shown in SEQ ID NO: 24 and a light chain sequence as shown in SEQ ID NO: 25. In a particular embodiment, the second monoclonal antibody or its antigen-binding fragment may include a heavy chain sequence encoded by the sequence listed in SEQ ID NO: 30 and a light chain sequence encoded by the sequence listed in SEQ ID NO: 31.

[0043] Generally, a third monoclonal antibody or its antigen-binding fragment, or another detection antibody, has binding specificity to the third polypeptide of SEQ ID NO: 3, which corresponds to the S region of LHBS. The third monoclonal antibody or other detection antibody can be designed in the reaction mixture to capture the S region of the LHBS protein or its immune complex, thereby significantly expanding the dynamic range of LHBS detection.

[0044] Both the second and third monoclonal antibodies can be designed to detect the pre-S2 and S regions of the LHBS protein, thereby increasing the dynamic breadth of LHBS detection. In one embodiment, the second and third monoclonal antibodies can be respectively ligated to a label for subsequent reaction with a reporting substrate.

[0045] It is important to note that monoclonal antibodies can be chimerized or humanized for different applications. Furthermore, if the primary monoclonal antibody is not responsible for capturing the pre-S1 region of the LHBS protein in a biological sample, it is difficult to achieve the default sensitivity and dynamic range for LHBS detection.

[0046] The aforementioned monoclonal antibodies can be applied to kits and immunoassays for in vitro quantitative LHBS. In some embodiments, the kit may include a blocking solution. A first monoclonal antibody may be immobilized at multiple individual sites on a solid support and partially immersed in the blocking solution. A second monoclonal antibody is conjugated to a label, a third monoclonal antibody is conjugated to the label, and a detection reagent is included, which reacts with the label to form a detectable product.

[0047] In some embodiments, the in vitro quantitative immunoassay for LHBS can be performed as follows: First, a first monoclonal antibody is immobilized on a surface and immersed in a blocking solution. Next, a biological sample solution reacts with the first monoclonal antibody to form a first immune complex. Then, a second and a third monoclonal antibody react with the first immune complex to form a second immune complex. Subsequently, a detection reagent reacts with a label to form a detectable product. This detectable product is then quantified, wherein the dynamic range of LHBS is between 1.7 ng / mL and 108.5 ng / mL.

[0048] In some embodiments, the analytical sensitivity of LHBS by the above immunoassay is no greater than 0.1 ng / mL.

[0049] In some embodiments, the aforementioned surfaces, blocking solutions, detection reagents, markers, and detectable products are not particularly limited.

[0050] In some examples, the aforementioned surface may be a solid surface suitable for linking monoclonal antibodies. Specific examples of the aforementioned solid surface may include, but are not limited to, particles (including but not limited to agarose or latex beads or granules or magnetic particles), beads, nanoparticles, polymers, substrates, glass slides, coverslips, plates, dishes, pores, membranes, and / or grafting. Solid surfaces may include many different materials, including but not limited to polymers, plastics, resins, polysaccharides, silicon or silica-based materials, carbon, metals, inorganic glasses, and membranes.

[0051] In some embodiments, the blocking solution and detection reagent described above may be commonly used buffer solutions or commercially available buffer solutions, and therefore will not be described in detail.

[0052] The term "label" as used herein refers to the detection (e.g., measurement and / or determination) of the presence or absence of a molecule or functional group using spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical methods, such as fluorescence, chemifluorescence, or chemiluminescence, or any other suitable method. Such labels may include, for example, light-absorbing dyes, fluorescent dyes, or radioactive labels. The aforementioned labels, methods for detecting labels, and methods for adding labels to reagents (e.g., antibodies and nucleic acid probes) are well known in the art.

[0053] The label used in the methods described in this invention can be a primary label (where the label contains or generates a directly detectable functional group) or a secondary label (where the detectable label binds to another functional group to generate a detectable signal; for example, immunolabeling reactions using secondary and tertiary antibodies are common). The label can be covalently or non-covalently linked to the reagent. Alternatively, the label can, for example, utilize the direct labeling of a molecule that binds to the reagent via ligand receptor binding pairs or other such specific recognizing molecules. Labels can include, but are not limited to, radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

[0054] In some embodiments, the aforementioned markers may be enzymes, including but not limited to horseradish peroxidase (HRP) and alkaline phosphatase. Enzyme-type markers can produce detectable products, such as chemiluminescent, color, or fluorescent signals. Enzymes considered as markers may include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, δ-V-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose hexaphosphate dehydrogenase, glucose amylase, and acetylcholinesterase.

[0055] In some embodiments, commercially available detection systems, such as the biotin-streptavidin system, can also be used. In this system, the antibody that can react with the target biomarker (i.e., is specific) is biotinylated. The quantity of biotinylated antibody bound to the biomarker can be determined using a streptavidin-peroxidase conjugate and a chromogenic substrate.

[0056] Generally, the term "biomarker" as used herein may include, but is not limited to, a single biomarker or a group of biomarkers comprising multiple biomarkers. In one example, a biomarker may include, but is not limited to, a first biomarker having a first polypeptide of SEQ ID NO: 1, a second biomarker having a second polypeptide of SEQ ID NO: 2, and / or a third biomarker having a third polypeptide. In other examples, a group of biomarkers may include, but is not limited to, a first biomarker, and at least one of a second and a third biomarker.

[0057] In application, the aforementioned biomarkers or biomarker groups can be used to manage liver diseases, such as non-invasively analyzing the stage of HBV infection and the prognosis of liver cancer in biological samples, or to predict or diagnose the severity of chronic liver disease in biological samples.

[0058] In other applications, the aforementioned monoclonal antibody set can also be used to treat liver diseases, such as monoclonal antibodies for detecting the aforementioned biomarkers or monoclonal antibody sets for detecting the aforementioned biomarker sets. These biomarkers in subjects in need can be used to predict, diagnose, or treat chronic liver disease. In these examples, the monoclonal antibody or monoclonal antibody set may include a first, second, and / or third monoclonal antibody or its antigen-binding fragment.

[0059] Adoptive chimeric antigen receptor-T cell (CAR-T cell) immunotherapy is associated with improved tumor targeting efficacy. Modulated T cells express the constructs of genetically engineered CARs, which contain single-chain variable (ScFv) fragments of immunoglobulins that recognize tumor antigens, enabling them to attack target cells without priming via antigen presentation. T cells can be engineered using vectors expressing ScFvs that target HCC-associated antigens (e.g., HBV LHBS protein). In some embodiments, the first monoclonal antibody or its first antigen-binding fragment, the second monoclonal antibody or its second antigen-binding fragment, and the third monoclonal antibody or its third antigen-binding fragment may be antigen-binding domains of chimeric antigen receptors (CARs) on engineered T cells. In other examples, the first monoclonal antibody or its first antigen-binding fragment, the second monoclonal antibody or its second antigen-binding fragment, and the third monoclonal antibody or its third antigen-binding fragment may be the antigen-binding domain of an antibody-drug conjugate (ADC).

[0060] It is understood that the specific configurations, viewpoints, examples, terms and embodiments described below are intended to illustrate the application of the present invention, but are not intended to limit the present invention. Those who have common knowledge in the technical field of the present invention may make various modifications and refinements to suit different uses and conditions without departing from the spirit and scope of the present invention.

[0061] Example 1: Characteristics of monoclonal antibodies

[0062] 1.1 Patient group

[0063] This study recruited 49 HBV carriers from National Cheng Kung University Hospital in Taiwan between 2010 and 2017. After obtaining informed consent, these individuals received drug treatment and were followed up. Serum samples from these patients were tested for various HBV biomarkers, including HBsAg, DNA titer, and LHBS. The HBV infection stage of the patients was determined according to the clinical guidelines published by the European Association for the Study of the Liver (EASL) in 2017 (J. Hepatol. 67(2):370-398, 2017 Aug.).

[0064] 1.2 Preparation of antigens for mouse immunization

[0065] In this embodiment, to generate mouse antibodies specifically recognizing the pre-S region of HBV LHBS, the pre-S region gene was selected and inserted into the pET21b plasmid vector, and the expression of *E. coli* strain BL21 was induced using the inducing chemical isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.2 mM). The purified recombinant pre-S region protein was injected into BALB / c mice to generate pre-S region antibodies. To generate antibodies recognizing HBS, recombinant HBS protein purchased from Leadgene, Inc. was injected into mice to generate HBS-specific antibodies. Figure 1 Mice with the highest serum affinity for HBS were selected for further studies.

[0066] 1.3 Screening and purification of monoclonal antibodies

[0067] In this specific example, the mice injected with the recombinant protein described in the previous section were sacrificed. Spleen cells were harvested to prepare fusion tumors. The antibody titer of the fusion tumor cultures containing antibodies against the pre-S region and HBS protein was detected using ELISA. Fusion tumor cells exhibiting antibodies recognizing the target protein could be further subjected to serial limiting dilution procedures to generate monoclonal antibody fusion tumor cell lines. The monoclonal antibodies were tested for sensitivity / specificity in recognizing LHBS, HBS, and the pre-S1 peptide spanning amino acids 21 to 47, a region previously reported to be highly antigenic. After purifying fusion tumor cell lines exhibiting satisfactory sensitivity and specificity to the target protein, they were intraperitoneally injected into mice to induce ascites. The antibody-rich ascites was then collected, and monoclonal antibodies were purified using IgG beads. A fraction of these antibody samples was directly bound to biotin and used as detection antibodies in a sandwich ELISA system.

[0068] 1.4 Antigen Recognition Region of Monoclonal Antibodies

[0069] This specific example analyzes the antigen recognition region of the generated monoclonal antibody. Please refer to [link / reference]. Figures 2A to 2C This describes a pre-S1 monoclonal antibody according to an embodiment of the present invention ( Figure 2A ), pre-S2 monoclonal antibody ( Figure 2B ) and HBS monoclonal antibody ( Figure 2C The binding affinity of LHBS in different regions of the embodiment.

[0070] like Figure 2AAs shown, for the pre-S1 antibody, different oligopeptides spanning the N-terminal amino acid region 21 to 47 (corresponding to the 1st to 7th amino acid residues of the pre-S1 peptide) were synthesized and detected. This region exhibits high antigenicity, and the relative affinity for this peptide was determined using the pre-S1 antibody. The results showed that, compared to adjacent protein regions, the pre-S1 antibody could specifically recognize peptides containing amino acids 25-38 (sequence FPDHQLDPAFGANS; SEQ ID NO: 1).

[0071] For the pre-S2 antibody, 293T cells were transfected with the LHBS gene, in which the LHBS gene had partial deletions of different pre-S regions, and the cell lysates were identified by the pre-S2 antibody using ELISA. Figure 2B The results showed that the pre-S2 antibody could recognize full-length and various truncated LHBS constructs, except for peptides with missing amino acid residues at 152-163 and 164-174 in the pre-S2 region, indicating that the epitope of the pre-S2 monoclonal antibody is likely located near amino acid residues 152-174 in the pre-S2 region (SEQ ID NO: 2).

[0072] like Figure 2C In the example of the HBS antibody shown, this antibody can recognize all LHBS truncated proteins except for the deletion of amino acid residues 251 to 288 (SEQ ID NO: 3), so this region is considered to be the recognition region of the HBS antibody.

[0073] Please see Figure 2D This is a schematic diagram illustrating the recognition regions of the pre-S1 monoclonal antibody, pre-S2 monoclonal antibody, and HBS monoclonal antibody against LHBS according to an embodiment of the present invention.

[0074] 1.5 DNA Sequencing of Antibody Variable Genes

[0075] DNA sequencing of the antibody variable (V) gene in pre-S1, pre-S2, and HBS antibody-generating fusion tumor cell lines was performed by Leadgene, Inc. Monoclonal antibody was confirmed for each cell line, and the DNA sequences of the framework region (FR) and complementary determining region (CDR) of the antibody V gene were identified. The corresponding amino acid residues in these regions were determined based on the DNA sequences.

[0076] The amino acid sequences of the heavy and light chains of the pre-S1 antibody are listed in SEQ ID NO: 22 (heavy chain) and SEQ ID NO: 23 (κ light chain), respectively. Their respective CDR 1 to CDR 3 are listed in SEQ ID NO: 4 to 6 (heavy chain) and SEQ ID NO: 7 to 9 (κ light chain), respectively, and their respective DNA sequences are listed in SEQ ID NO: 28 (heavy chain) and SEQ ID NO: 29 (κ light chain).

[0077] The amino acid sequences of the heavy and light chains of the pre-S2 antibody are listed in SEQ ID NO: 24 (heavy chain) and SEQ ID NO: 25 (κ light chain), respectively. Their respective CDR 1 to CDR 3 are listed in SEQ ID NO: 10 to 12 (heavy chain) and SEQ ID NO: 13 to 15 (κ light chain), respectively, and their respective DNA sequences are listed in SEQ ID NO: 30 (heavy chain) and SEQ ID NO: 31 (κ light chain).

[0078] The amino acid sequences of the HBS antibody heavy and light chains are shown in SEQ ID NO: 26 (heavy chain) and SEQ ID NO: 27 (κ light chain), respectively. Their respective CDR 1 to CDR 3 are shown in SEQ ID NO: 16 to 18 (heavy chain) and SEQ ID NO: 19 to 21 (κ light chain), respectively. Their respective DNA sequences are shown in SEQ ID NO: 32 (heavy chain) and SEQ ID NO: 33 (κ light chain).

[0079] Example 2: Characteristics of monoclonal antibodies

[0080] 2.1 Determination of antibody sensitivity

[0081] To determine antibody titers, 0.1 ng / μL of each antigen protein or peptide was plated onto each well of a 96-well ELISA tray and reacted overnight at 4°C. The following day, the purified monoclonal antibody was sequence-diluted 10-fold and added to the pre-coated antigen-lined ELISA wells, followed by reaction with... A standard ELISA wash was performed using 20 μL phosphate-buffered saline (PBST, pH 7.4). The signal of the antigen-antibody complex was detected using a horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody, followed by chemiluminescence HRP acceptor tetramethylbenzidine (TMB) and signal measurement using an ELISA chemiluminescence reader.

[0082] Furthermore, to determine the antigen detection limits of the antibodies, various antigen proteins were serially diluted 10-fold and then plated onto the wells of an ELISA culture plate. Antibodies (diluted 1:2000 in PBST) were then added, followed by standard chemiluminescence detection. Calibration curves were plotted to determine the limits of detection (LOD), limits of quantification (LOQ), linear range, and limits of linearity (LOL) for each antibody against its specific antigen proteins (Armbruster & Pry, Clin. Biochem. Rev. Vol. 29, Suppl(i)., 2008).

[0083] In this embodiment, the sensitivity of mouse monoclonal antibodies against the recombinant target protein was tested for the pre-S1, pre-S2, and HBS antibodies.

[0084] Please see Figures 3A to 3C This describes the pre-S1 monoclonal antibody according to several embodiments of the present invention ( Figure 3A ), pre-S2 monoclonal antibody ( Figure 3B ) and HBS monoclonal antibody ( Figure 3C The graphs show the detection sensitivity and dynamic range of their respective recombinant proteins.

[0085] Figure 3A and Figure 3B The results showed that the preS1 antibody and the preS2 antibody identified the preS recombinant protein in E. coli with good performance.

[0086] like Figure 3A As shown, for the pre-S1 antibody, the limit of quantitation (LOQ) and limit of linearity (LOL) values ​​are 0.09 ng / mL and 12.5 ng / mL, respectively, representing a detection dynamic range of 0.09 ng / mL to 12.5 ng / mL.

[0087] like Figure 3B As shown, the preS2 antibody also exhibited high sensitivity to the preS region protein, with LOQ and LOL values ​​of 0.04 ng / mL and 1.56 ng / mL, respectively.

[0088] As for Figure 3C The HBS antibody shown has a LOQ and LOL of 0.06 ng / mL and 50 ng / mL for the recombinant HBS protein, respectively. Because the above-mentioned monoclonal antibody exhibits satisfactory sensitivity and specificity to its target protein, it can be used for large-scale multiplication using the produced ascites fluid, followed by purification using an IgG column and conjugation with biotin for use in a sandwich assay.

[0089] 2.2 Detection of LHBS in serum using ELISA

[0090] The monoclonal antibody generated in this embodiment can be used to establish a sandwich ELISA system for detecting LHBS in serum. The LHBS-specific antibody targeting the pre-S1 region is plated onto ELISA wells and incubated overnight at 4°C. The next day, standard normal serum or HBsAg(+) serum (5 μL serum + 98 μL 2% bovine serum albumin / well) is added to the wells. After a series of washing steps, detection antibodies recognizing the pre-S2 or HBS region are added. Finally, chemiluminescence detection is used to visualize the LHBS signal. Using different concentrations of the pre-S region recombinant protein, the resulting signal values ​​are plotted as a standard curve to quantify the LHBS content in serum.

[0091] Please see Figures 4A to 4B This describes the use of pre-S2 monoclonal antibodies according to several embodiments of the present invention. Figure 4A ) or HBS monoclonal antibody ( Figure 4B Line graph showing the detection sensitivity and dynamic range of the LHBS chemical cold light sandwich ELISA system in serum as the detection antibody.

[0092] In the LHBS ELISA system, the pre-S1 antibody, which specifically identifies LHBS, is used as the coating antibody, while the pre-S2 or HBS antibody is used as the detection antibody. In the ELISA analysis, pooled sera from multiple HBsAg(+) cases with known LHBS concentrations are tested. The results show that the LHBS sandwich ELISA assay using the pre-S2 detection antibody presents a LOQ value of 13.5 ng / mL, with a linear range of 13.5 ng / mL to 108.5 ng / mL. Figure 4A Platform 1). The LOQ value of the HBS antibody-detecting ELISA was 1.7 ng / mL, with a linear range of 1.7 to 108.5 ng / mL. Figure 4B Platform 2). The above data confirms that the LHBS sandwich ELISA system using pre-S2 or HBS detection antibodies can sensitively detect LHBS in serum.

[0093] Example 3: Detection of LHBS at different stages of HBV infection using sandwich ELISA

[0094] To determine the correlation between LHBS levels and HBV infection stages, this example collected serum samples from 49 individuals at different HBV infection stages.

[0095] Please see Figure 5 The diagram illustrates a histogram showing serum LHBS levels in different patient groups at different stages of HBV infection detected by an LHBS chemiluminescent sandwich ELISA system according to an embodiment of the present invention.

[0096] Figure 5 The results of Kruskal-Wallis test analysis indicated that the group in the immune tolerance stage was characterized by normal ALT but high viral titer, exhibiting the highest LHBS content. HBeAg(+) chronic HBV (CHB) carriers showed a lower viral titer, exhibiting moderate viral titer. The HBeAg(-) CHB, cirrhosis, and HCC groups typically showed very low viral titers, and their LHBS content was significantly lower than the previous two groups (p < 0.002). Figure 5 ).

[0097] Furthermore, Pearson correlation analysis showed that LHBS levels in these patients were correlated with viral DNA valence (r = 0.36). These results indicate that serum LHBS levels are an indicator of the stage of viral infection and can serve as an effective biomarker.

[0098] Please see Figures 6A to 6C This describes the expression levels of major HBS and LHBS in the peritumoral and tumor regions of HCC patients according to an embodiment of the present invention, using IHC staining ( Figure 6A and Figure 6B ) and Western ink dot method ( Figure 6C ) to conduct analysis. Figure 6A The images show IHC images from two representative cases, among which... Figure 6A Subfigures a, b, e, f, i, and j show the IHC results of Case 1, while subfigures c, d, g, h, k, and l show the IHC results of Case 2. Subfigures a through d show the scan views. Subfigures a and c show that major hepatocytes (HBS) are present in non-tumor areas but not in tumors, with non-tumor areas showing clusters of ground-glass hepatocytes (GGH). Subfigures b and d show that large hepatocytes (LHBS) are present in both non-tumor and tumor areas. Subfigures e through h show magnified views of GGH in non-tumor liver (corresponding to the areas indicated by the red arrows in the scan views of subfigures a through d). Subfigures i through 1 show magnified views of the tumor (corresponding to the areas indicated by the black arrows in subfigures a through d). Figure 6B This table summarizes the IHC results for HCC cases (n=12), showing the percentage (median 6SEM) of positively stained cells for type I GGH (scattered, Is), clustered, Ic, and type II GGH (II), as well as the tumor region (T). For major HBS and LHBS, the IHC results for antibodies identifying the major S region and pre-S1 region are shown. The horizontal line represents the median of each data set. Figure 6C Western ink dot spectrophotometry was used to visualize LHBS in the peritumoral and tumor regions of HCC cases (n=22). Wild-type (n=10) and pre-S2 mutant (n=12) cases were analyzed and pre-identified using pre-S1 gene chip analysis. Human 293T cells were transfected with wild-type, pre-S1, and pre-S2 mutant HBS genes to detect the specificity of monoclonal antibodies for HBS. Figure 6C The arrow points to the LHBS of p39 / gp42 kDa. β-actin served as an internal control group. Figures 6A to 6C The abbreviations are as follows: C, control group; Ic, type I GGH clusters; II, type II GGH; Is, type I sporadic GGH; N, non-neoplastic; T, neoplastic; WT, wild type.

[0099] Please see Figure 7A and Figure 7B The results show the Kaplan-Meier analysis of the correlation between tissue LHB staining patterns and disease-free survival (DFS) and overall survival (OS) in an age-matched HCC clinical group according to an embodiment of the present invention. *P<0.05. High LHB staining patterns in liver tissue were significantly correlated with worsening DFS (P=0.011) and OS (P=0.004).

[0100] Please see Figure 8A and Figure 8B The results show the Kaplan-Meier analysis of serum LHBS levels detected by ELISA in an age-matched HCC clinical group according to another embodiment of the present invention, and its correlation with disease-free survival (DFS) and overall survival (OS). *P<0.05. High serum LHBS levels were also significantly correlated with worsening DFS (P=0.042) and OS (P=0.017).

[0101] The prognostic importance of clinicopathological parameters, HBsAg manifestations, and HBV serological profiles for disease-free survival in hepatocellular carcinoma patients is listed in Table 1. Univariate analysis showed that viral load (P = 0.043), tumor size (P = 0.001), vascular invasion (P = 0.044), AJCC stage (P = 0.024), serum LHBS (P = 0.048), and tissue LHBS staining (P = 0.014) were significant predictors of disease-free survival (DFS) progression (Table 1). Multivariate analysis showed that tissue LHBS staining (P = 0.022, HR = 2.650, CI = 1.154–6.087) and tumor size (P = 0.001, HR = 3.979, CI = 1.750–9.048) were independently associated with DFS (Table 1).

[0102] Table 1. Prognostic significance of clinicopathological indicators, HBsAg manifestations and HBV serum profiles for disease-free survival in hepatocellular carcinoma patients.

[0103]

[0104] Table 2. Clinical and pathological indicators, HBsAg manifestations, and HBV serum profiles (excluding LHBS staining) of hepatocellular carcinoma patients are of prognostic importance for disease-free survival.

[0105]

[0106] As shown in Table 2, since serum was obtained using a non-invasive method, the effectiveness of serum LHBS detection, rather than tissue LHBS staining, could be evaluated in multivariate analysis. Multivariate analysis showed that serum LHBS (P = 0.049, HR = 2.267, CI = 1.001–5.137), tumor size (P = 0.001, HR = 4.443, CI = 1.907–10.353), and vascular invasion (P = 0.021, HR = 2.590, CI = 1.157–5.797) were independently associated with disease-free survival (DFS) (Table 2).

[0107] Example 4: LHBS has the potential for targeted therapy via CAR-T cells or T-cell conjugating antibodies.

[0108] The single-stranded variable region fragment (ScFv) gene of the monoclonal antibody generated in this embodiment was selected and colonized into a CAR-T vector to construct CAR-T plasmids expressing the pre-S1 ScFv. The pre-S1 ScFv CAR-T constructs were transfected into T cells. After mixing LHBS(+) hepatocytes with pre-S1 ScFv(+) T cells, flow cytometry analysis showed that the T cells could recognize LHBS(+) hepatocytes, as shown in the results. Figure 9 As shown.

[0109] Please see Figure 9 This illustrates the analytical results of LHBS analysis of HBV(+) HepAD38 live cells using flow cytometry with an LHBS monoclonal antibody, according to an embodiment of the present invention. Figure 9In the diagram, curve 901 represents the results detected using autologous antibodies, curve 903 represents the results detected using secondary antibody 488 (as a negative control), curve 905 represents the results detected using LHBS monoclonal antibody clone "7-21-5", and curve 907 represents the results detected using LHBS monoclonal antibody clone "8-17-1". The latter two target peptides spanning amino acids 21 to 47 of the LHBS pre-S1 region. FL1-H refers to fluorescence intensity. Figure 9 As shown, LHBS antibodies can bind to LHBS, indicating that LHBS protrudes from the outer surface of HBV(+) viable hepatocytes, and T cells can recognize LHBS(+) hepatocytes. Therefore, LHBS has the potential to be a target molecule for CAR-T cell therapy.

[0110] Hepatocellular carcinoma (HCC) is one of the deadliest cancers in the world, and timely antiviral treatment is the most important method for preventing HBV-related HCC. To date, the most common biomarker for the efficacy of antiviral treatment is viral DNA titer, but its detection requires a cumbersome experimental process, including DNA extraction and real-time PCR. In the specific examples mentioned above, it was found that viral LHBS is highly correlated with viral replication activity in CHB carriers, with the highest levels during the immune tolerance phase and the lowest levels in cirrhosis and HCC. Viral LHBS also showed a high correlation with DNA titer. A sensitive and simple ELISA method was developed to detect LHBS as an indicator of viral titer in serum. This method, using a chemiluminescent immunoassay in conjunction with an ELISA system, can measure LHBS with high sensitivity and quantification. The dynamic range of this method reaches approximately 100-fold, providing reliable measurements for samples with a wide concentration range.

[0111] By directly detecting proteins in serum, the aforementioned in vitro quantitative LHBS kit and immunoassay method is more time- and cost-effective than viral DNA titering. Therefore, serum LHBS can serve as a valuable early biomarker for the efficacy of antiviral treatment. For example, a set of biomarkers for the non-invasive analysis of HBV infection stages in biological samples can be provided, comprising a first biomarker having a first polypeptide of SEQ ID NO: 1, a second biomarker having a second polypeptide of SEQ ID NO: 2, and a third biomarker having a third polypeptide of SEQ ID NO: 3.

[0112] In summary, the above examples only illustrate the kitting and immunoassay methods for in vitro quantitative LHBS and biomarkers using specific sequences of nucleic acids and amino acids, specific antigens, specific patient populations, specific analytical models, or specific evaluation methods, for non-invasive analysis of HBV infection stages in biological samples. However, those skilled in the art will understand that other nucleic acid and amino acid sequences, other antigens, other patient populations, other analytical models, or other evaluation methods can also be used in the kitting and immunoassay methods for in vitro quantitative LHBS to non-invasively analyze biomarkers of HBV infection stages in biological samples, without departing from the spirit and scope of the invention, nor being limited by the above. For example, monoclonal antibodies can be chimeric or humanized for different applications, thereby beneficially improving the sensitivity and dynamic range of LHBS detection. In other examples, the aforementioned monoclonal antibody kits can also predict, diagnose, or treat chronic liver disease by detecting the aforementioned biomarkers in subjects in need.

[0113] According to the above embodiments of the present invention, the in vitro quantitative LHBS kit, immunoassay method, and biomarker set of the present invention include monoclonal antibodies with binding specificity to specific regions, thereby non-invasively analyzing the HBV infection stage in biological samples, thus improving the sensitivity and dynamic range of LHBS detection in biological samples. The present invention also provides a set of biomarkers corresponding to specific regions of LHBS, which can be specifically identified by monoclonal antibodies to analyze the HBV infection stage and liver cancer prognosis in biological samples.

[0114] While the present invention has been disclosed above with reference to several specific embodiments, various modifications, alterations, and substitutions can be made to the foregoing disclosure. It should be understood that, without departing from the spirit and scope of the invention, certain features of the embodiments of the invention may be used in some cases, but other features may not be used accordingly. Therefore, the spirit and scope of the invention should not be limited to the embodiments described above. sequence list <110> Huang Wenya <120> A kit for in vitro quantitative hepatitis B virus large surface protein assay, as well as a biomarker kit and monoclonal antibody kit for treating liver diseases. <130> none <150> US 63 / 217,300 <151> 2021-07-01 <160> 33 <210> 1 <211> 14 <212> PRT <213> Artificial sequence <220> <223> amino acid sequence of LHBS from amino acid 25 to 38 <400> 1 Phe Pro Asp His Gln Leu Asp Pro Ala Phe Gly Ala Asn Ser 1 5 10 <210> 2 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> amino acid sequence of LHBS from amino acid 152 to 174 <400> 2 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 1 5 10 15 Thr Gly Asp Pro Val Thr Asn 20 <210> 3 <211> 38 <212> PRT <213> Artificial sequence <220> <223> amino acid sequence from LHBS 251 to 288 <400> 3 Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile 1 5 10 15 Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro 20 25 30 Leu Ile Pro Gly Ser Thr 35 <210> 4 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR1 amino acid sequence of the heavy chain of the pre-S1 antibody <400> 4 Gly Tyr Ser Ile Thr Ser Asp Tyr Ala 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> The CDR2 amino acid sequence of the heavy chain of the pre-S1 antibody <400> 5 Ile Arg Tyr Ser Gly Thr Thr 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> The CDR3 amino acid sequence of the heavy chain of the pre-S1 antibody <400> 6 Ala Arg Gly Gly Thr Gly Leu Thr Tyr 1 5 <210> 7 <211> 6 <212> PRT <213> Artificial sequence <220> <223> CDR1 amino acid sequence of the kappa light chain of the pre-S1 antibody <400> 7 Glu Asn Val Gly Thr Tyr 1 5 <210> 8 <211> 3 <212> PRT <213> Artificial sequence <220> <223> The CDR2 amino acid sequence of the kappa light chain of the pre-S1 antibody <400> 8 Gly Ala Ser 1 <210> 9 <211> 9 <212> PRT <213> Artificial sequence <220> <223> The CDR3 amino acid sequence of the kappa light chain of the pre-S1 antibody <400> 9 Gly Gln Thr Tyr Asn Tyr Pro Phe Thr 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR1 amino acid sequence of the heavy chain of the pre-S2 antibody <400> 10 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <220> <223> The CDR2 amino acid sequence of the heavy chain of the pre-S2 antibody <400> 11 Ile Asn Pro Ser Asn Gly Ile Thr 1 5 <210> 12 <211> 15 <212> PRT <213> Artificial sequence <220> <223> The CDR3 amino acid sequence of the heavy chain of the pre-S2 antibody <400> 12 Thr Ile Gly Tyr Asp Tyr Gly Ser Asn Tyr Glu Ala Met Asp Phe 1 5 10 15 <210> 13 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CDR1 amino acid sequence of the kappa light chain of the pre-S2 antibody <400> 13 Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr 1 5 10 <210> 14 <211> 3 <212> PRT <213> Artificial sequence <220> <223> The CDR2 amino acid sequence of the kappa light chain of the pre-S2 antibody <400> 14 Gln Met Ser 1 <210> 15 <211> 9 <212> PRT <213> Artificial sequence <220> <223> The CDR3 amino acid sequence of the kappa light chain of the pre-S2 antibody <400> 15 Ala Gln Asn Leu Glu Leu Pro Trp Thr 1 5 <210> 16 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1 amino acid sequence of the heavy chain of HBS antibody <400> 16 Gly Tyr Thr Leu Thr Asp Tyr Val Ile Gly 1 5 10 <210> 17 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR2 amino acid sequence of the heavy chain of HBS antibody <400> 17 Glu Val Tyr Pro Gly Ser Val Tyr Thr Ser Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 18 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR3 amino acid sequence of the heavy chain of HBS antibody <400> 18 Ala Tyr Asp Gly Tyr Ser Pro Phe Asp Tyr 1 5 10 <210> 19 <211> 11 <212> PRT <213> Artificial sequence <220> <223> The CDR1 amino acid sequence of the kappa light chain of the HBS antibody. <400> 19 Lys Ala Ser Glu Asn Val Gly Thr Tyr Val Ser 1 5 10 <210> 20 <211> 7 <212> PRT <213> Artificial sequence <220> <223> The CDR2 amino acid sequence of the kappa light chain of the HBS antibody. <400> 20 Gly Ala Ser Asn Arg Tyr Ile 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial sequence <220> <223> The CDR3 amino acid sequence of the kappa light chain of the HBS antibody. <400> twenty one Gly Gln Ser Tyr Asn Tyr Pro His Thr 1 5 <210> twenty two <211> 117 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of the heavy chain of the pre-S1 antibody <400> twenty two Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15 Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser 20 25 30 Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu 35 40 45 Trp Met Gly Tyr Ile Arg Tyr Ser Gly Thr Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Thr Gly Leu Thr Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> twenty three <211> 107 <212> PRT <213> Artificial sequence <220> <223> The amino acid sequence of the kappa light chain of the pre-S1 antibody <400> twenty three Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Asn Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Pro Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr Leu Cys Gly Gln Thr Tyr Asn Tyr Pro Phe 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> twenty four <211> 122 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of the heavy chain of the pre-S2 antibody <400> twenty four Gln Val Gln Leu Gln Gln Pro Gly Thr Glu Leu Val Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Leu Arg Pro Gly Gln Gly Phe Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Asn Gly Ile Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Tyr Asp Tyr Gly Ser Asn Tyr Glu Ala Met Asp Phe Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 25 <211> 112 <212> PRT <213> Artificial sequence <220> <223> The amino acid sequence of the kappa light chain of the pre-S2 antibody <400> 25 Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Met Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Ser Gly Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 26 <211> 116 <212> PRT <213> Artificial sequence <220> <223> The amino acid sequence of the heavy chain of HBS antibody <400> 26 Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Asp Tyr Val 20 25 30 Ile Gly Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile Gly 35 40 45 Glu Val Tyr Pro Gly Ser Val Tyr Thr Ser Tyr Asn Glu Lys Phe Lys 50 55 60 Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr Met 65 70 75 80 Gln Leu Ser Gly Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys Ala 85 90 95 Tyr Asp Gly Tyr Ser Pro Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 27 <211> 108 <212> PRT <213> Artificial sequence <220> <223> The amino acid sequence of the kappa light chain of HBS antibody <400> 27 Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr 20 25 30 Val Ser Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Glu Tyr His Cys Gly Gln Ser Tyr Asn Tyr Pro His 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Val Ile Lys Arg 100 105 <210> 28 <211> 351 <212> DNA <213> Artificial sequence <220> <221> CDS <223> Nucleic acid sequence of the heavy chain of the pre-S2 antibody <400> 28 tctgatgtgc agcttcagga gtcgggacct ggcctggtga aaccttctca gtctctgtcc 60 ctcacctgca ctgtcactgg ctactcaatc accagtgatt atgcctggaa ctggatccgg 120 cagtttccag gaaacaaact ggagtggatg ggctacatta ggtacagtgg taccactaac 180 tacaacccat ctctcaaaag tcgaatctct atcactcgag acacatccaa gaaccaattc 240 ttcctgcaat tgaattctgt gactactgag gacacagcca catattactg tgcaagaggg 300 gggacggggc ttacttactg gggccaaggg actctggtca ctgtctctgc a 351 <210> 29 <211> 321 <212> DNA <213> Artificial sequence <220> <221> CDS <223> Nucleic acid sequence of the kappa light chain of the pre-S1 antibody <400> 29 aacattgtta tgacccaatc tcccaaatcc atgtccatgt cactaggaga gagggtcacc 60 ttgacctgca aggccagtga gaatgtgggt acttatgtat cctggtatca acagaaacca 120 gaacagtctc ctaaactcct gatatacggg gcatccaacc ggaacactgg ggtccccgat 180 cgcttcacag gcagtggacc tgcaacagat ttcactctga ccatcagcag tgtgcaggct 240 gaagaccttg cagattatct ctgtggacag acttacaatt atccgttcac gttcggtgct 300 gggaccaagc tggagctgaa a 321 <210> 30 <211> 366 <212> DNA <213> Artificial sequence <220> <221> CDS <223> Nucleic acid sequence of the heavy chain of the pre-S2 antibody <400> 30 caggtccaac tccagcagcc tgggactgaa ctggttaagc ctgggacttc agtgaagttg 60 tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagctgagg 120 tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagctgagg 120 cctggacaag gctttgagtg gattggggag attaatccta gcaatggtat tactaactac 180 cctggacaag gctttgagtg gattggggag attaatccta gcaatggtat tactaactac 180 aatgagaagt tcaagagaaa ggccacactg actgtagaca aatcctccac cacagcctac 240 aatgagaagt tcaagagaaa ggccacactg actgtagaca aatcctccac cacagcctac 240 atgcaactca gcagcctgac atttgaggac tctgcggtct attactgtac aataggctat 300 atgcaactca gcagcctgac atttgaggac tctgcggtct attactgtac aataggctat 300 gactacggta gtaactacga ggctatggac ttctggggtc aaggaacctc agtcaccgtc 360 gactacggta gtaactacga ggctatggac ttctggggtc aaggaacctc agtcaccgtc 360 tcctca 366 tcctca 366 <210> 31<210> 31 <211> 336<211> 336 <212> DNA<212> DNA <213> 人工序列<213> Artificial sequence <220><220> <221> CDS <221> CDS <223> 前S2抗体的kappa轻链的核酸序列 <223> Nucleic acid sequence of kappa light chain of pre-S2 antibody <400> 31 <400> 31 gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac atcagcttcc 60 gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac atcagcttcc 60 atgtcctgca ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg 120 atgtcctgca ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg 120 tatcttcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc 180 tatcttcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc 180 tcaggagtcc cagacaggtt cagtagcggt gggtcaggaa ctgatttcac actgagaatc 240 tcaggagtcc cagacaggtt cagtagcggt gggtcaggaa ctgatttcac actgagaatc 240 agcagagtgg aggctgagga tgtgggtgtt tattactgtg ctcaaaattt agaacttccg 300 tggacgttcg gtggaggcac caagctggaa atcaaa 336 <210> 32 <211> 348 <212> DNA <213> Artificial sequence <220> <221> CDS <223> The nucleic acid sequence of the heavy chain of HBS antibody <400> 32 gttcaactgc agcagtcagg acctgagctg gtgaagcctg gggcttcagt gaagatgtcc 60 tgcaaggctt ctggataacac actcactgac tatgttatag gatgggtgaa gcagagaact 120 ggacagggcc ttgagtggat tggagaggtt tatcctggaa gtgtttatac ttcctacaat 180 gagaagttca agggcaaggc cacactgact gcggacaaat cctccaacac agcctacatg 240 cagctcagcg gcctgacatc tgacgattct gcggtctatt tctgtgcata tgatggttac 300 tccccctttg actactgggg ccaaggcacc actctcacag tctcctca 348 <210> 33 <211> 324 <212> DNA <213> Artificial sequence <220> <221> CDS <223> The nucleic acid sequence of the kappa light chain of the HBS antibody <400> 33 aacattgtaa tgacccaatc tcccaaatcc atgtccatgt cagtaggaga gcgggtcacc 60 ttgacctgca aggccagtga gaatgtgggt acttatgtat cctggtttca acagaaacca 120 gggcagtctc ctaaactgct gatatacggg gcatccaacc ggtatattgg ggtccccgat 180 cgcttcacag gcagtggatc tgcaacagat ttcactctga ccatcagcag tgtgcaggct 240 gaagaccttg cagaatatca ctgtggacag agttacaact atcctcacac gttcggaggg 300 gggaccaagc tggtaataaa acgg 324

Claims

1. A kit for in vitro quantitative determination of hepatitis B virus large surface protein (LHBS) in biological samples, characterized in that, Include: One blocking solution; A monoclonal antibody group is used to detect a biomarker in the biological sample, wherein the monoclonal antibody group includes: A first monoclonal antibody is immobilized at multiple discrete sites on a solid support and immersed in the blocking solution, wherein the first monoclonal antibody has binding specificity to a first polypeptide in the biological sample as listed in sequence identification number SEQ ID NO: 1, and the first monoclonal antibody comprises the heavy chain CDR1 sequence of SEQ ID NO: 4, the heavy chain CDR2 sequence of SEQ ID NO: 5, the heavy chain CDR3 sequence of SEQ ID NO: 6, the light chain CDR1 sequence of SEQ ID NO: 7, the light chain CDR2 sequence of SEQ ID NO: 8, and the light chain CDR3 sequence of SEQ ID NO: 9; and At least one of a second monoclonal antibody and a third monoclonal antibody, wherein the second monoclonal antibody and the third monoclonal antibody are each linked to a label, the second monoclonal antibody having binding specificity to a second polypeptide as listed in SEQ ID NO: 2 in the biological sample, the second monoclonal antibody comprising a heavy chain CDR1 sequence composed of SEQ ID NO: 10, a heavy chain CDR2 sequence composed of SEQ ID NO: 11, a heavy chain CDR3 sequence composed of SEQ ID NO: 12, a light chain CDR1 sequence composed of SEQ ID NO: 13, a light chain CDR2 sequence composed of SEQ ID NO: 14, and a light chain CDR3 sequence composed of SEQ ID NO: 15, the third monoclonal antibody having binding specificity to a third polypeptide as listed in SEQ ID NO: 3 in the biological sample, the third monoclonal antibody comprising a heavy chain CDR1 sequence composed of SEQ ID NO: 16, a heavy chain CDR2 sequence composed of SEQ ID NO: 17, a heavy chain CDR3 sequence composed of SEQ ID NO: 18, and a light chain CDR3 sequence composed of SEQ ID NO:

15. The light chain CDR1 sequence composed of SEQ ID NO: 19, the light chain CDR2 sequence composed of SEQ ID NO: 20, and the light chain CDR3 sequence composed of SEQ ID NO: 21; and A detection reagent, wherein the detection reagent can react with the marker to form a detectable product.

2. A monoclonal antibody group of hepatitis B virus large surface protein LHBS in vitro quantitative biological samples, used to predict, diagnose, or treat chronic liver disease based on biomarkers of subjects in need, characterized in that, Include: A first monoclonal antibody or its first antigen-binding fragment for the specific detection of a first polypeptide, wherein the first monoclonal antibody or its first antigen-binding fragment comprises the heavy chain CDR1 sequence of SEQ ID NO: 4, the heavy chain CDR2 sequence of SEQ ID NO: 5, the heavy chain CDR3 sequence of SEQ ID NO: 6, the light chain CDR1 sequence of SEQ ID NO: 7, the light chain CDR2 sequence of SEQ ID NO: 8, and the light chain CDR3 sequence of SEQ ID NO: 9; and At least one of a second monoclonal antibody or a second antigen-binding fragment thereof and a third monoclonal antibody or a third antigen-binding fragment thereof, wherein the second monoclonal antibody or the second antigen-binding fragment thereof is used for specific detection of a second polypeptide, and the third monoclonal antibody or the third antigen-binding fragment thereof is used for specific detection of a third polypeptide. The second monoclonal antibody or its second antigen-binding fragment comprises the heavy chain CDR1 sequence of SEQ ID NO: 10, the heavy chain CDR2 sequence of SEQ ID NO: 11, the heavy chain CDR3 sequence of SEQ ID NO: 12, the light chain CDR1 sequence of SEQ ID NO: 13, the light chain CDR2 sequence of SEQ ID NO: 14, and the light chain CDR3 sequence of SEQ ID NO: 15; and The third monoclonal antibody or its third antigen-binding fragment comprises a heavy chain CDR1 sequence consisting of SEQ ID NO: 16, a heavy chain CDR2 sequence consisting of SEQ ID NO: 17, a heavy chain CDR3 sequence consisting of SEQ ID NO: 18, a light chain CDR1 sequence consisting of SEQ ID NO: 19, a light chain CDR2 sequence consisting of SEQ ID NO: 20, and a light chain CDR3 sequence consisting of SEQ ID NO:

21.

3. The monoclonal antibody group as described in claim 2, characterized in that, The chronic liver disease is selected from a group consisting of latent HBV infection, cirrhosis, and hepatocellular carcinoma.

4. The monoclonal antibody group as described in claim 2, characterized in that, The biomarker includes a first biomarker of the first polypeptide having SEQ ID NO: 1, a second biomarker of the second polypeptide having SEQ ID NO: 2, and a third biomarker of the third polypeptide having SEQ ID NO:

3.

5. The monoclonal antibody group as described in claim 2, characterized in that, The first monoclonal antibody or its first antigen-binding fragment contains a heavy chain sequence as shown in SEQ ID NO: 22 and a light chain sequence as shown in SEQ ID NO:

23.

6. The monoclonal antibody group as described in claim 2, characterized in that, The second monoclonal antibody or its second antigen-binding fragment contains a heavy chain sequence as shown in SEQ ID NO: 24 and a light chain sequence as shown in SEQ ID NO:

25.

7. The monoclonal antibody group as described in claim 2, characterized in that, The third monoclonal antibody or its third antigen-binding fragment contains a heavy chain sequence as shown in SEQ ID NO: 26 and a light chain sequence as shown in SEQ ID NO:

27.

8. The monoclonal antibody group as described in claim 5, characterized in that, The first monoclonal antibody comprises a heavy chain encoded by the sequence shown in SEQ ID NO: 28 and a light chain encoded by the sequence shown in SEQ ID NO:

29.

9. The monoclonal antibody group as described in claim 6, characterized in that, The second monoclonal antibody comprises a heavy chain encoded by the sequence shown in SEQ ID NO: 30 and a light chain encoded by the sequence shown in SEQ ID NO:

31.

10. The monoclonal antibody group as described in claim 7, characterized in that, The third monoclonal antibody comprises a heavy chain encoded by the sequence shown in SEQ ID NO: 32 and a light chain encoded by the sequence shown in SEQ ID NO:

33.

11. The monoclonal antibody group as described in claim 2, characterized in that, The first monoclonal antibody or its first antigen-binding fragment, the second monoclonal antibody or its second antigen-binding fragment, and the third monoclonal antibody or its third antigen-binding fragment are antigen-binding domains of a chimeric antigen receptor (CAR) of an engineered T cell.

12. The monoclonal antibody group as described in claim 2, characterized in that, The first monoclonal antibody or its first antigen-binding fragment, the second monoclonal antibody or its second antigen-binding fragment, and the third monoclonal antibody or its third antigen-binding fragment are antigen-binding domains of the HBS-antibody-drug complex ADC.

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