Combined detection kit and method

Abstract

A detection kit and a method. The detection kit comprises a first reagent for determining the type of an HLA and a second reagent for examining the integrity of the antigen presentation pathway. Furthermore, the detection kit can also determine whether HLA alleles have lost heterozygosity. The kit and the method for jointly evaluating HLA subtype compatibility and antigen presentation function integrity enable systematic assessment of antigen presentation capacity, selection of biomarkers for predicting immunotherapy sensitivity, identification of patient populations suitable for receiving immunotherapy, and assistance in evaluating treatment response, thereby improving the precision and effectiveness of clinical treatment.

Description

A combined detection kit and method Technical Field

[0001] This application relates to the field of biomedicine, specifically to a detection kit and method for simultaneously detecting HLA typing, loss of heterozygosity, and integrity of antigen presentation function. Background Technology

[0002] T cells are central to the adaptive immune response, and their activation depends on the recognition of antigen-presenting cells (APCs) by antigen peptides presented via the major histocompatibility complex (MHC) molecules. T cells can only recognize antigens bound to MHC molecules in peptide form. Endogenous antigens, after degradation by the proteasome, are transported into the endoplasmic reticulum via TAP, bind to MHC class I molecules, and are presented on the cell surface for CD8 uptake. + Cytotoxic T-cell recognition. Oncolytic viruses (OVs) are a class of viruses that can selectively infect and lyse tumor cells while exhibiting low cytotoxicity to normal cells, making them an important research direction in the field of tumor immunotherapy. These viruses can exist naturally or be modified through genetic engineering to enhance their targeting and immunogenicity. Compared with traditional radiotherapy and chemotherapy, oncolytic virus therapy has many advantages, including high specificity, fewer adverse reactions, and the ability to induce anti-tumor immune responses. Currently, the mechanisms of oncolytic virus therapy for tumors mainly include: firstly, viral replication within tumor cells and directly causing cell lysis; secondly, the release of tumor-associated antigens and immunostimulatory molecules during lysis, activating the body's adaptive immune system, especially inducing specific T-cell responses, thereby achieving sustained immune control of the tumor.

[0003] T cells mediate anti-tumor immune responses by recognizing antigenic peptides presented by the major histocompatibility complex (HLA / MHC) molecules on the surface of tumor cells via their receptors (TCRs). Therefore, the effectiveness of tumor cells in processing and presenting tumor antigens to their cell surface is crucial to the efficacy of the aforementioned immunotherapy strategies. Any abnormality affecting antigen processing or HLA molecule expression may impair T cell recognition, leading to immune escape and reduced treatment response rates.

[0004] HLA class I molecules are a core component of the antigen presentation machinery (APM), widely expressed on the surface of all nucleated cells. Their main function is to present endogenous antigenic peptides (such as viral peptides and tumor neoantigens) to CD8+ cytotoxic T lymphocytes (CTLs), thereby activating a specific immune response. HLA (human leukocyte antigen) class I molecules are central to the presentation of tumor neoantigens and activation of CD8+ T cells by oral veno-viral (OV). Effective antigen presentation is crucial for inducing T cell immune responses, depending on tumor cells expressing specific antigens and possessing matching HLA subtypes. Furthermore, during tumorigenesis, loss of heterozygosity (LOH) is a common immune escape mechanism, leading to reduced expression of HLA class I molecules on the surface of tumor cells, hindering the effective presentation of viral peptides and tumor neoantigens. Simultaneously, although viral peptides are produced in large quantities during OV treatment, due to HLA class I OH, the CTL response is weak, viral replication cannot be amplified by the immune system, resulting in poor treatment efficacy and OV treatment resistance. On the other hand, the process of antigens being processed from tumor cells, loaded onto MHC molecules, and presented to the cell surface involves multiple functional molecules, such as TAP and β2-microglobulin. The integrity of the expression and function of these molecules determines the effectiveness of the antigen presentation pathway. Even if HLA type matches and there is no LOH, if the function of the antigen presentation pathway (APM) genes is impaired, immune recognition will still fail.

[0005] However, due to the prevalent gene mutations and epigenetic abnormalities within tumor cells, the loss or dysfunction of antigen presentation pathway-related molecules often occurs, resulting in the so-called Antigen Presentation Defect (APD). Furthermore, existing technologies, due to their fragmented detection dimensions, insufficient sensitivity, and lack of multi-dimensional integration, cannot meet the needs for accurate prediction in immunotherapy. On the one hand, due to the extreme complexity of the HLA gene system itself, the same gene has many different alleles in different individuals, and many HLA alleles may differ by only one or a few bases. Traditional Sanger sequencing or qPCR only detects HLA type and cannot assess HLA-ALOH and antigen presentation pathway (APM) gene mutations, leading to a high false-positive rate in prediction results. On the other hand, for HLA LOH, existing STR-PCR or microarray technologies rely on pre-defined sites and have low sensitivity. In addition, for APM gene mutation detection, traditional methods require separate detection of multiple genes such as TAP1 / 2, PSMB9, and ERA P1 / 2, and only detect hotspot mutations, making the operation cumbersome and consuming large amounts of samples. Summary of the Invention

[0006] This application provides a method for simultaneous detection of HLA typing, LOH, and APM mutations. By combining a kit and method to assess HLA subtype adaptability and antigen-presenting functional integrity, it systematically detects antigen-presenting ability, screens biomarkers to predict immunotherapy sensitivity, identifies suitable patient populations for immunotherapy, and assists in judging treatment response to improve the accuracy and effectiveness of clinical treatment. This application also provides a specific hybridization capture probe primer combination that can simultaneously detect HLA typing, loss of heterozygosity, and antigen-presenting gene mutations.

[0007] The kit provided in this application has the following advantages: 1) It is universal, capable of detecting all HLA-I genotypes in the IPD-IMGT / HLA database; 2) It has high sensitivity, accurately determining the HLA genotype and subtype of a sample; 3) It has high specificity, capable of detecting loss of heterozygosity; 4) It is systematic, capable of detecting multiple genes at once when detecting antigen presentation pathways, with simple operation, and determining the integrity of antigen presentation pathways by whether antigen presentation genes are mutated; 5) It is accurate, with 100% consistency with the gold standard PCR-SBT (Sanger) method.

[0008] On the one hand, this application provides a test kit comprising a first reagent for detecting HLA typing and a second reagent for detecting the integrity of antigen presentation pathways.

[0009] In some embodiments, the second reagent for detecting the integrity of the antigen presentation pathway includes detecting whether there is a genetic mutation in the antigen presentation gene, which includes antigen processing genes and / or antigen transport genes.

[0010] In some embodiments, the antigen-presenting gene is selected from one or more of the following group: B2M, JAK2, PSMB8, CALR, NFKB1, PSMB9, CANX, NLRC5, STAT1, ERAP1, PDIA3, TAP1, ERAP2, PSMB10, TAP2, IRF1, PSMB5, ​​TAPBP, IRF2, PSMB6, JAK1, and PSMB7.

[0011] In some embodiments, the second reagent for detecting the integrity of the antigen presentation pathway includes probe primers for detecting whether the antigen-presenting gene has mutated.

[0012] In some embodiments, the probe primer sequences for detecting whether the antigen-presenting gene is mutated include the sequences shown in SEQ ID NO:184 to SEQ ID NO:338.

[0013] In some implementations, the HLA typing is derived from the IPD-IMGT / HLA database.

[0014] In some implementations, the HLA typing is HLA-I class molecules.

[0015] In some implementations, the HLA typing includes A01, A02, A03, A11, A23, A24, A25, A26, A29, A30, A31, A32, A33, A34, A36, A43, A66, A68, A69, A74, A80, B07, B08, B13, B14, B15, B18, B27, B35, B37, B38, B39, B40, B4 1. B42, B44, B45, B46, B47, B48, B49, B50, B51, B52, B53, B54, B55, B56, B57, B58, B59, B67, B73, B78, B81, B82, B83, C01, C02, C03, C04, C05, C06, C07, C08, C12, C14, C15, C16, C17 and / or C18.

[0016] In some implementations, the HLA typing is A01, A02, A03, A11, A24, A31, A32, A68, B07, B35, B45, B51, B52 and / or C12 typing.

[0017] In some implementations, the first reagent for detecting HLA typing can also detect whether HLA alleles are lacking heterozygosity.

[0018] In some embodiments, the first reagent for detecting HLA typing includes probe primers for detecting HLA typing.

[0019] In some embodiments, the reagent for detecting whether HLA alleles are heterozygous includes probe primers for detecting whether HLA alleles are heterozygous.

[0020] In some embodiments, the probe primer sequences for detecting whether HLA alleles are heterozygous include the sequences shown in SEQ ID NO:1 to SEQ ID NO:183.

[0021] In some embodiments, the probe primer sequences for detecting HLA typing include the sequences shown in SEQ ID NO:1 to SEQ ID NO:183.

[0022] On the other hand, this application provides a set of nucleic acid libraries, which include nucleic acid molecules for detecting HLA typing and whether HLA alleles are lacking heterozygosity, and / or nucleic acid molecules for detecting the integrity of antigen presentation pathways.

[0023] In some embodiments, the nucleic acid molecule used to detect the integrity of the antigen presentation pathway includes a nucleic acid molecule that detects whether the antigen presentation gene is mutated, the antigen presentation gene including antigen processing genes and / or antigen transport genes.

[0024] In some embodiments, the antigen-presenting gene is selected from one or more of the following group: B2M, JAK2, PSMB8, CALR, NFKB1, PSMB9, CANX, NLRC5, STAT1, ERAP1, PDIA3, TAP1, ERAP2, PSMB10, TAP2, IRF1, PSMB5, ​​TAPBP, IRF2, PSMB6, JAK1, and PSMB7.

[0025] In some embodiments, the nucleic acid molecule used to detect whether the antigen-presenting gene has mutated includes a probe primer for detecting whether the antigen-presenting gene has mutated.

[0026] In some implementations, the nucleic acid molecules used to detect HLA typing are capable of detecting HLA typing from the IPD-IMGT / HLA database.

[0027] In some implementations, the nucleic acid molecule used for HLA typing includes probe primers for HLA typing.

[0028] In some embodiments, the nucleic acid molecule used to detect whether HLA alleles are heterozygous includes probe primers for detecting whether HLA alleles are heterozygous.

[0029] In some implementations, the nucleic acid molecule used for HLA typing includes probe primers for HLA typing.

[0030] In some embodiments, the nucleic acid molecule used to detect whether HLA alleles are heterozygous includes probe primers for detecting whether HLA alleles are heterozygous.

[0031] In some embodiments, the probe primer sequences for detecting whether the antigen-presenting gene is mutated include the sequences shown in SEQ ID NO:184 to SEQ ID NO:338.

[0032] In some embodiments, the probe primer sequences for detecting HLA typing include the sequences shown in SEQ ID NO:1 to SEQ ID NO:183.

[0033] In some embodiments, the probe primer sequences for detecting whether HLA alleles are heterozygous include the sequences shown in SEQ ID NO:1 to SEQ ID NO:183.

[0034] In some implementations, the nucleic acid library also includes target DNA.

[0035] In some implementations, the target DNA includes a adapter.

[0036] On the other hand, this application also provides a method for constructing a nucleic acid library, wherein the nucleic acid library includes any of the nucleic acid libraries described above, and the method includes:

[0037] a) Template DNA fragmentation,

[0038] b) The fragmented DNA solution is ligated with the sequencing library adapters.

[0039] c) Amplify and enrich the adapter ligation products of the DNA sequencing library obtained in b) and

[0040] d) Probe and primer liquid phase hybridization capture and amplification to obtain the final library.

[0041] On the other hand, this application provides the use of the kits and / or nucleic acid libraries described in any of the above claims in the manufacture of medicaments for stimulating immune responses in subjects or for tumor immunotherapy in subjects.

[0042] In some implementations, the tumor immunotherapy includes TCR-T cell therapy, oncolytic virus therapy, and / or immune checkpoint inhibitor therapy.

[0043] In some implementations, the immune checkpoint inhibitor therapy includes the use of PD-1 / PD-L1 inhibitors.

[0044] In some implementations, the tumor immunotherapy is oncolytic virus therapy.

[0045] In some implementations, the tumor immunotherapy includes a combination therapy of oncolytic viruses and immune checkpoint inhibitors.

[0046] In some implementations, the tumor immunotherapy is a combination therapy of oncolytic virus and PD-1 / PD-L1 inhibitor.

[0047] In some implementations, the tumor is a solid tumor and / or a hematoma.

[0048] In some implementations, the tumors include soft tissue sarcoma, head and neck tumors, skin cancer, melanoma, liver cancer, stomach cancer, lung cancer, kidney cancer, thyroid cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, gynecological and genital tumors, myeloma, lymphoma, and / or leukemia.

[0049] In some implementations, the tumors include synovial sarcoma, myxosarcoma, liposarcoma, adenoid cystic carcinoma, thymic carcinoma, oropharyngeal carcinoma, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, renal cell carcinoma, metastatic renal cell carcinoma, clear renal cell carcinoma, glioma, recurrent glioma, pancreatic ductal carcinoma, triple-negative breast cancer, bladder cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, prostate cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, gastroesophageal junction cancer, multiple myeloma, Hodgkin's lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia, acute myeloid leukemia, and / or acute lymphoblastic leukemia.

[0050] On the other hand, this application provides a system for predicting the population for tumor immunotherapy, the system comprising the detection kit described in any of the above claims and / or the nucleic acid library described in any of the above claims.

[0051] In some implementations, the tumor immunotherapy includes TCR-T cell therapy, oncolytic virus therapy, and / or immune checkpoint inhibitor therapy.

[0052] In some implementations, the immune checkpoint inhibitor therapy includes the use of PD-1 / PD-L1 inhibitors.

[0053] In some implementations, the tumor immunotherapy is oncolytic virus therapy.

[0054] In some implementations, the tumor immunotherapy includes a combination therapy of oncolytic viruses and immune checkpoint inhibitors.

[0055] In some implementations, the tumor immunotherapy is a combination therapy of oncolytic virus and PD-1 / PD-L1 inhibitor.

[0056] In some implementations, the tumor is a solid tumor and / or a hematoma.

[0057] In some implementations, the tumors include soft tissue sarcoma, head and neck tumors, skin cancer, melanoma, liver cancer, stomach cancer, lung cancer, kidney cancer, thyroid cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, gynecological and genital tumors, myeloma, lymphoma, and / or leukemia.

[0058] In some implementations, the tumors include synovial sarcoma, myxosarcoma, liposarcoma, adenoid cystic carcinoma, thymic carcinoma, oropharyngeal carcinoma, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, renal cell carcinoma, metastatic renal cell carcinoma, clear renal cell carcinoma, glioma, recurrent glioma, pancreatic ductal carcinoma, triple-negative breast cancer, bladder cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, prostate cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, gastroesophageal junction cancer, multiple myeloma, Hodgkin's lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia, acute myeloid leukemia, and / or acute lymphoblastic leukemia.

[0059] In some implementations, the system also carries

[0060] 1) Comparison software;

[0061] 2) Output module for HLA-A, B, and C typing results of the output samples;

[0062] 3) An output module that outputs the heterozygous loss state of HLA genes; and / or

[0063] 4) Output module for outputting antigen-presenting gene variation results.

[0064] In some embodiments, the alignment software includes alignment software for comparing HLA allele sequences in the template with HLA allele sequences in the IPD-IMGT / HLA database.

[0065] On the other hand, this application provides an anti-tumor drug delivery platform, which includes the reagent kit and / or the nucleic acid library described in any of the above claims, and a drug module for tumor immunotherapy.

[0066] In some implementations, the tumor is a solid tumor and / or a hematoma.

[0067] In some implementations, the tumors include soft tissue sarcoma, head and neck tumors, skin cancer, melanoma, liver cancer, stomach cancer, lung cancer, kidney cancer, thyroid cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, gynecological and genital tumors, myeloma, lymphoma, and / or leukemia.

[0068] In some implementations, the tumors include synovial sarcoma, myxosarcoma, liposarcoma, adenoid cystic carcinoma, thymic carcinoma, oropharyngeal carcinoma, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, renal cell carcinoma, metastatic renal cell carcinoma, clear renal cell carcinoma, glioma, recurrent glioma, pancreatic ductal carcinoma, triple-negative breast cancer, bladder cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, prostate cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, gastroesophageal junction cancer, multiple myeloma, Hodgkin's lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia, acute myeloid leukemia, and / or acute lymphoblastic leukemia.

[0069] In some implementations, the tumor immunotherapy is oncolytic virus therapy.

[0070] In some implementations, the tumor immunotherapy includes a combination therapy of oncolytic viruses and immune checkpoint inhibitors.

[0071] In some implementations, the tumor immunotherapy is a combination therapy of oncolytic virus and PD-1 / PD-L1 inhibitor.

[0072] On the other hand, this application provides a method for screening biomarkers, including amplifying the HLA gene and antigen-presenting gene of the sample to be tested using the kit described in any of the above claims, the nucleic acid library described in any of the above claims, or the method for constructing the nucleic acid library described in the above claims, then sequencing the amplicon, comparing the sequencing results with a reference genome to determine the HLA type, whether the HLA allele is lost heterozygosity, and whether the antigen presentation pathway is intact, wherein the HLA type is an HLA-I class molecule, and the detection of the integrity of the antigen presentation pathway is to detect whether the antigen presentation-related gene is mutated.

[0073] On the other hand, this application provides the use of biomarkers in the preparation of tumor immunotherapy prediction systems and / or anti-tumor drug screening platforms. The biomarkers include HLA typing, HLA alleles and / or antigen-presenting genes. The tumor immunotherapy includes oncolytic virus therapy, TCR therapy and / or immunosuppressant therapy. The HLA typing is an HLA-I molecule. The detection of antigen presentation pathway integrity is to detect whether antigen presentation-related genes are mutated.

[0074] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0075] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0076] Figure 1 shows the detection process of the reagent kit described in this application. Detailed Implementation

[0077] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0078] Terminology Definition

[0079] In this application, the terms "presentation" and "submission" are used interchangeably and refer to the binding between an antigenic peptide and HLA. The term "antigenic peptide" refers to a limited number of different peptides produced during the processing of protein antigens in antigen-presenting cells.

[0080] In this application, the terms "T cell response" and "immune response" are used interchangeably and refer to the activation of T cells and / or the induction of one or more effector functions following the binding of a specific HLA-antigen peptide. These effector functions include cytotoxicity, cytokine production, and proliferation.

[0081] In this application, the terms "variation" and "mutation" are used interchangeably to refer to a change in at least one nucleotide or amino acid, such as changing from A to T or from histidine to alanine, resulting in the loss of the protein's original function. For example, a gene / protein that could normally present an antigen may lose its original function after a mutation.

[0082] In this application, the term "antigen-presenting gene" refers to a gene that processes and transports antigens during antigen presentation. It can also refer to an important component gene in MHC I molecules, and more broadly, it can include signaling pathway molecules (signal transduction molecules) that regulate the presentation pathway, including but not limited to transcription factors and pattern recognition receptors. An important component gene of MHC I molecules, such as the β2-microglobulin gene (B2M), encodes a light chain of HLA-I class molecules. Antigen-presenting genes can be antigen-presenting genes extracted from the human reference genome hg19, including but not limited to: B2M, JAK2, PSMB8, CALR, NFKB1, PSMB9, CANX, NLRC5, STAT1, ERAP1, PDIA3, TAP1, ERAP2, PSMB10, TAP2, IRF1, PSMB5, ​​TAPBP, IRF2, PSMB6, JAK1, and PSMB7. The term "antigen processing gene" refers to a proteasome gene that processes antigens into antigenic peptides that facilitate the binding of MHC molecules (also known as HLA molecules), helping MHC I molecules on the cell surface bind to high-affinity peptides. This antigen processing gene may include, but is not limited to, proteasome subunit β family protein genes (PSMB) and TAP-associated proteins (Tapasin, TAPBPL), such as PSMB5, ​​PSMB6, PSMB8, PSMB9, or PSMB10. The term "antigen transport gene" refers to a gene that participates in the modification and presentation of endogenous antigenic peptides. These genes are typically located on the endoplasmic reticulum, enabling them to bind to MHC class I molecules, forming MHC I-antigen peptide complexes, which are then presented to the cell surface for recognition by T cells, generating a specific immune response. This antigen transport gene includes, but is not limited to, molecular chaperone proteins, endoplasmic reticulum aminopeptidase genes (ERAP), and antigen processing-associated transporters (TAPs), such as ERAP1, ERAP2, CANX, PDIA3, CALR, TAPBP, TAP1, and TAP2. Signaling pathway molecules that regulate the presentation pathway include, but are not limited to, JAK kinases (JAK2, JAK2), STAT transcription factors (STAT1), NF-κB signaling pathway molecules (NFKB1), and transcription regulators (NLRC5, IRF1, IRF2).

[0083] In this application, the "antigen presentation pathway (APM)" involves multiple steps, including antigen uptake, processing, peptide synthesis, peptide binding to MHC molecules, and finally display on the cell surface to T cells.

[0084] In this application, the term "HLA" refers to a group of genes located on human chromosome 6 that encode proteins that can bind to antigenic peptides to form HLA-antigen peptide complexes, presenting tumor antigens to T cells. HLA is divided into HLA-I class molecules and HLA-II class molecules. Based on allele differences, HLA-I class molecules can be further divided into the HLA-A gene family, HLA-B gene family, and HLA-C gene family, while HLA-II class molecules can be divided into the HLA-DR gene family, HLA-DQ gene family, and HLA-DP gene family. In this application, the HLA-I class molecules are HLA-I class gene molecules from the IPD-IMGT / HLA Database.

[0085] In this application, the term "loss of heterozygosity (LOH)" or "loss of HLA allele heterozygosity" refers to the loss of an allele at an HLA locus in tumor cells, resulting in homozygosity at that locus. This is one of the common immune escape mechanisms in tumorigenesis, leading to reduced expression of HLA class molecules on the surface of tumor cells, thereby reducing the probability of tumor cells being recognized by the immune system. In addition, loss of HLA heterozygosity may also affect the presentation of tumor antigens, making it difficult for the immune system to recognize and attack tumor cells.

[0086] In this application, the terms "tumor immunotherapy," "immunotherapy," or "tumor immunotherapy" refer to the treatment of a disease or condition by inducing or enhancing an immune response in an individual. In some embodiments, immunotherapy refers to a therapy that includes administering one or more drugs to an individual to elicit a T-cell response. In this application, immunotherapy includes, but is not limited to: TCR-T cell therapy, oncolytic virus therapy, immune checkpoint inhibitor therapy (e.g., PD-1 / PD-L1 therapy), or any combination of the above.

[0087] In this application, the term "TCR-T cell therapy (T Cell Receptor-Engineered T Cell Therapy, abbreviated as TCR-T)" refers to a cellular immunotherapy method. This therapy involves introducing the T cell receptor (TCR) gene, which specifically recognizes tumor antigens, into the patient's own T cells, enabling them to target and kill tumors, thereby achieving immune clearance of the tumor. The term "immune checkpoint" refers to a series of molecules expressed on immune cells that regulate the degree of immune activation. They play a major role in preventing autoimmune reactions (abnormal immune function leading to attacks on normal cells). Tumor cells express substances that activate immune checkpoints. Once activated, these checkpoints act like a "brake," preventing antigens from being presented to T cells and blocking the antigen presentation process in the tumor immune chain. This suppresses the immune function of T cells, allowing the tumor cells to escape surveillance and survive. The term "immune checkpoint inhibitor" refers to the phenomenon where immune cells produce small protein molecules that inhibit themselves. Tumor cells utilize this mechanism to suppress immune cells, escaping from the body's immune system and surviving. Immune checkpoint inhibitor drugs can relieve this inhibition, allowing immune cells to reactivate and eliminate cancer cells. In this application, immune checkpoint inhibitors include, but are not limited to, CTLA-4 inhibitors and PD-1 inhibitors (PD-1 / PD-L1 inhibitors), wherein PD-1 inhibitors (PD-1 / PD-L1 inhibitors) include PD-1 antibodies (PD-1 inhibitors) and PD-L1 antibodies (PD-L1 inhibitors).

[0088] In this application, the terms "CTLA-4," "CTLA4," "CD152," "differentiation cluster 152," or "cytotoxic T-lymphocyte-associated protein 4" refer to an immunosuppressive receptor that primarily inhibits CD28-mediated co-stimulatory signals by competitively binding to B7 molecules (CD80 / CD86) on the surface of antigen-presenting cells during the early stages of T cell activation, thereby reducing T cell activation and proliferation. CTLA-4 antibodies relieve T cell suppression, enhance naive T cell activation, and promote anti-tumor immune responses by blocking the interaction between CTLA-4 and B7. This mechanism primarily acts on the immune initiation phase in lymph nodes. The terms "PD-1," "CD729," "differentiation cluster 729," or "programmed death receptor 1" refer to an inhibitory receptor expressed on the surface of activated T cells, whose ligand PD-L1 is highly expressed in various tumor cells. PD-1 binding to PD-L1 inhibits T cell activation, causing T cell dysfunction and allowing tumors to evade immune surveillance. PD-1 monoclonal antibodies enhance the body's anti-tumor immune response by blocking the interaction between PD-1 and PD-L1, thereby relieving T cell suppression and restoring their proliferation and cytotoxic activity. This therapy has been widely used in the treatment of various solid tumors and hematological malignancies.

[0089] In this application, the term "oncolytic virus" refers to a natural or recombinant virus capable of selectively infecting and killing tumor cells without harming normal cells. The oncolytic viruses include, but are not limited to, adenovirus (AdV), herpes simplex virus (HSV), Newcastle disease virus (NDV), measles virus (MV), vesicular stomatitis virus (VSV), poliovirus, Coxsackie virus, reovirus, and vaccinia virus (VV). The oncolytic viruses of this application can also be used in combination with immune checkpoint inhibitors, such as PD-L1 / PD-L2 inhibitors. In one embodiment, the oncolytic viruses of this application can be used in combination with a PD-1 antibody, wherein the PD-1 antibody can be any known PD-1 antibody. This combination can be administered simultaneously with an immune checkpoint inhibitor or separately from the immune checkpoint inhibitor.

[0090] In this application, "treatment population" refers to a patient population that has been screened to a specific HLA subtype.

[0091] In this application, the term "biomarker" refers to any information (or any parameter) about a biomolecule (e.g., a gene or protein), cancer (e.g., tumor type), or subject (e.g., the subject's age) that can be used to predict whether a treatment will be effective or ineffective in the subject. Therefore, as used herein, "biomarker information" or "biomarker value" refers to any information associated with a biomarker. As a non-limiting example, a biomarker may be an HLA typing, HLA allele, HLA allele loss of heterozygosity, or antigen-presenting gene; for example, for a cancer patient with HLA-A*02:25, the biomarker may be HLA-A*02 or HLA-A*02:25; for example, a biomarker may be B2M.

[0092] In this application, the term "adapter" refers to a short nucleotide fragment that serves as a bridge connecting the DNA fragment to be tested to the sequencing chip. Adaptors include, but are not limited to, UDI adapters and UMI adapters.

[0093] In this application, the term "heterozygote" refers to two distinct alleles on homologous chromosomes. "Homozygote" refers to two identical alleles on homologous chromosomes. In this application, HLA allele homozygote means, for example: if allele 1 of tissue sample A is HLA-A*01:01 and allele 2 is HLA-A*01:01, then A is an HLA allele homozygote; if allele 1 of tissue sample B is HLA-A*01:01 and allele 2 is HLA-A*02:01, then A is an HLA allele heterozygote.

[0094] In this application, the term "about" means ±50% of the corresponding value.

[0095] Invention Details

[0096] On one hand, this application provides a kit comprising a first reagent for detecting HLA genotyping and a second reagent for detecting the integrity of antigen presentation pathways. In some embodiments, the first reagent for detecting HLA genotyping can also detect whether HLA alleles are lacking heterozygosity.

[0097] HLA

[0098] In antigen-presenting cells (APCs), protein antigens are processed into peptides. These peptides bind to HLA and are presented as peptide-HLA complexes to the cell surface of T cells. Different individuals express different HLA molecules, and different HLA molecules present different peptides. Therefore, according to the prior art, if a peptide or fragment of a larger polypeptide is presented by an HLA molecule expressed by a subject, it is identified as immunogenic to that specific human subject. In other words, the prior art describes immunogenic peptides as HLA-restricted epitopes. However, HLA-restricted epitopes induce T cell responses only in a subset of individuals expressing HLA molecules. Despite HLA allele matching, a peptide that activates a T cell response in one individual may be inactive in others. Therefore, it is unknown how an individual's HLA molecules present antigen-derived epitopes that positively activate a T cell response.

[0099] HLA is encoded by most polymorphic genes in the human genome. Each person possesses three HLA class I molecules (HLA-A*, HLA-B*, HLA-C*) and four HLA class II molecules (HLA-DP*, HLA-DQ*, HLA-DRB1*, HLA-DRB3* / 4* / 5*), which present different epitopes from the same protein antigen. The function of HLA molecules is to regulate T cell responses. However, how human HLA regulates T cell activation remains unknown.

[0100] The nomenclature used to represent the amino acid sequence of an HLA molecule is as follows: Gene Name * Allele: Protein Number, for example, it could look like: HLA-A*02:25. In this example, "02" refers to the allele. In this application, it can also be written as HLA-A02 or HLA-A2. In most cases, the allele is defined by serotype C, meaning that proteins of a given allele will not react with each other in serological assays. Protein numbers are assigned sequentially upon protein discovery. Any protein with a different amino acid sequence is assigned a new protein number (e.g., even a change in one amino acid in the sequence is considered a different protein number). Further information about the nucleic acid sequence of a given locus can be appended to the HLA nomenclature, but such information is not required for the methods described herein.

[0101] The mouse major histocompatibility complex (MHC) and the human MHC (HLA) share a high degree of structural and functional similarity. Immunologically, both activate T cells by recognizing and presenting antigen fragments. Structurally, MHC molecules consist of heavy and light chains forming antigen-binding grooves for loading and presenting antigen fragments to T cells. Regarding antigen presentation mechanisms, MHC molecules participate in antigen presentation in both humans and mice. Class I molecules are responsible for presenting intracellular pathogen antigen fragments, while class II molecules primarily present extracellular antigens; this mechanism is conserved across species. The properties of MHC genes are also highly similar. The MHC gene complex consists of multiple closely adjacent gene loci whose encoded products have the same or similar functions. This polymorphism and polygenicity are present in both the mouse MHC (H-2 complex) and the human HLA system.

[0102] In this application, HLA typing is derived from HLA-I typing (HLA-A, HLA-B, HLA-C) in the IPD-IMGT / HLA Database. In some embodiments, the first reagent for detecting HLA typing may include detecting HLA typing from the IPD-IMGT / HLA Database. In some embodiments, the first reagent for detecting HLA typing may include detecting HLA-I typing from the IPD-IMGT / HLA Database. In some implementations, the first reagent for detecting HLA typing may include the reagent for detecting HLA typing of A01, A02, A03, A11, A23, A24, A25, A26, A29, A30, A31, A32, A33, A34, A36, A43, A66, A68, A69, A74, A80, B07, B08, B13, B14, B15, B18, B27, B35, B37, B38, B39, B4 The first reagent for detecting HLA typing includes HLA types A01, B41, B42, B44, B45, B46, B47, B48, B49, B50, B51, B52, B53, B54, B55, B56, B57, B58, B59, B67, B73, B78, B81, B82, B83, CO1, CO2, CO3, CO4, CO5, CO6, CO7, CO8, C12, C14, C15, C16, C17, and / or C18. In some embodiments, the first reagent for detecting HLA typing may include a first reagent for detecting HLA types A01, A02, A03, A11, A24, A31, A32, A68, B07, B35, B45, B51, B52, and / or C12.

[0103] In some embodiments, the first reagent for detecting HLA genotyping includes probe primers for detecting HLA genotyping; in some embodiments, the probe primers for detecting HLA genotyping may include those for detecting A01, A02, A03, A11, A23, A24, A25, A26, A29, A30, A31, A32, A33, A34, A36, A43, A66, A68, A69, A74, A80, B07, B08, B13, B14, B15, B18, B27, B35, B37, B38, B39, B 40, B41, B42, B44, B45, B46, B47, B48, B49, B50, B51, B52, B53, B54, B55, B56, B57, B58, B59, B67, B73, B78, B81, B82, B83, C01, C02, C03, C04, C05, C06, C07, C08, C12, C14, C15, C16, C17 and / or C18 genotyping probe primers; in some embodiments, the probe primer sequences for detecting HLA genotyping exemplaryly include the sequences shown in SEQ ID NO:1 to SEQ ID NO:183.

[0104] In some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting HLA-A; in some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting A01 genotyping; specifically, the probe primer sequences exemplarily include the sequences shown in SEQ ID NO:1 to SEQ ID NO:14. In some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting A02 genotyping; specifically, the probe primer sequences exemplarily include the sequences shown in SEQ ID NO:15 to SEQ ID NO:41. In some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting A03 genotyping; specifically, the probe primer sequences exemplarily include the sequences shown in SEQ ID NO:42 to SEQ ID NO:65. In some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting the A11 genotype; specifically, the probe primer sequences typically include the sequences shown in SEQ ID NO:66 to SEQ ID NO:75. In some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting the A24 genotype; specifically, the probe primer sequences typically include the sequences shown in SEQ ID NO:76 to SEQ ID NO:90. In some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting the A31 genotype; specifically, the probe primer sequences typically include the sequences shown in SEQ ID NO:91 to SEQ ID NO:94. In some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting the A32 genotype; specifically, the probe primer sequences typically include the sequences shown in SEQ ID NO:95 to SEQ ID NO:107. In some embodiments, the probe primers for detecting HLA typing may include probe primers for detecting A68 typing; specifically, the probe primer sequences exemplary include the sequences shown in SEQ ID NO:108 to SEQ ID NO:122.

[0105] In some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting HLA-B; in some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting B51; specifically, the probe primer sequences exemplarily include the sequences shown in SEQ ID NO:123 to SEQ ID NO:144. In some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting B52; specifically, the probe primer sequences exemplarily include the sequences shown in SEQ ID NO:145 to SEQ ID NO:149. In some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting B35; specifically, the probe primer sequences exemplarily include the sequences shown in SEQ ID NO:150 to SEQ ID NO:162. In some embodiments, the probe primers for detecting HLA genotyping may include probe primers for detecting B07; specifically, the probe primer sequences exemplarily include the sequences shown in SEQ ID NO:163 to SEQ ID NO:177.

[0106] In some embodiments, the probe primers for detecting HLA typing may include probe primers for detecting HLA-C typing; in some embodiments, the probe primers for detecting HLA typing may include probe primers for detecting C12 typing; specifically, the probe primer sequences exemplary include the sequences shown in SEQ ID NO:178 to SEQ ID NO:183.

[0107] It should be understood that the sequences listed in this application are merely exemplary sequences of the present invention. In the kits of this application, all HLA genotyping-related probe and primer sequences described herein can be used; therefore, this application is not limited to the exact sequences shown.

[0108] Antigen presentation pathway integrity

[0109] Antigen presentation is a crucial process by which the immune system recognizes and responds to endogenous and exogenous antigens (such as viral antigens or tumor antigens). This process involves multiple steps, including antigen uptake, processing, peptide synthesis, peptide binding to MHC molecules, and finally, presentation to T cells on the cell surface. Therefore, the integrity of the antigen presentation pathway affects HLA-presented antigens. This application demonstrates pathway integrity by detecting the expression of key genes in the antigen presentation pathway.

[0110] During antigen presentation, genes / proteins such as B2M (β2-Microglobulin), JAK1 (Janus kinase 1), JAK2 (Janus kinase2), CALR (calreticulin), NFKB1 (nuclear factor kappa B subunit 1), CANX (Calnexin), NLRC5 (NLR family, CARD domain containing 5), STAT1 (Signal Transducer and Activator of Transcription 1), PDIA3 (Protein disulfide isomerase family Amember 3), IRF1 (Interferon regulatory factor 1), IRF2 (Interferon regulatory factor 2), PSMB10 (proteasome(prosome,macropain)subunit,beta type,10), PSMB5 (proteasome(prosome,macropain)subunit,beta type,5), PSMB6 (proteasome(prosome,macropain)subunit,beta type,6), PSMB7 (proteasome(prosome,macropain)subunit,beta type,7), PSMB8 (proteasome(prosome,macropain)subunit,beta type,8), PSMB9 (proteasome(prosome,macropain)subunit,beta type,9), TAPBP (TAP binding protein, Tapasin), ERAP1 (Endoplasmic Reticulum Aminopeptidase 1), ERAP2 (Endoplasmic Reticulum Aminopeptidase 2), TAP1 (Transporter associated with antigen processing 1) and TAP2 (Transporter associated with antigen processing 2) all play crucial roles.They play an indispensable role in ensuring that antigens can be effectively processed by cells and presented to T cells.

[0111] TAP1 and TAP2 are key transport proteins in antigen processing and presentation. They form heterodimers responsible for transporting cytoplasmic peptides to the endoplasmic reticulum (ER) so that these peptides can bind to MHC class I molecules. TAPs are essential for maintaining the stability of MHC class I molecules and presenting antigen peptides to CD8+ T cells. TAPBPL, CALR, PDIA3, and CANX are molecular chaperones in the ER that facilitate the binding of MHC class I molecules to antigen peptides and are crucial for the proper folding and stable expression of MHC class I molecules on the cell surface. PSMB5, ​​PSMB6, PSMB7, PSMB8, PSMB9, and PSMB10 are catalytic subunits of the immunoproteasome. Under the induction of cytokines such as interferon-γ (IFN-γ), they replace the corresponding subunits in the standard proteasome to form the immunoproteasome. The immunoproteasome is more efficient in processing antigen peptides, generating peptides more suitable for binding to MHC class I molecules, thereby enhancing antigen presentation. ERAP1 and ERAP2 are endoplasmic reticulum aminopeptidases that prune the N-terminal amino acids of antigenic peptides to ensure that the peptide length and sequence are suitable for the binding groove of MHC class I molecules. B2M encodes the light chain of HLA-I molecules and is crucial for the stable structure and cell surface expression of the entire molecule. IRF1 and IRF2 act as tumor suppressor factors because they can inhibit tumor cell growth and activate anti-tumor immune responses, and can act as commanders to initiate the MHC class I antigen presentation pathway. NLRC5 regulates at the gene transcription level, determining how many MHC class I molecules a cell can produce, and is responsible for the transcriptional activation of MHC class I genes and related antigen processing genes. NFKB1 can regulate the expression of MHC molecules and is the hub connecting innate and adaptive immunity. JAK1, JAK2, and STAT1 can upregulate the expression of MHC class I molecules, B2M, TAP, and proteasome subunits.

[0112] Dysfunction or loss of these genes / proteins may lead to reduced antigen presentation efficiency, affecting T cell recognition of antigens and thus weakening the body's immune response. For example, loss of function of TAP1 and TAP2 can lead to reduced expression of MHC class I molecules, interfering with the formation of cytotoxic T lymphocytes and affecting immune responses to certain pathogens (Ian Mantel et al., Nature reviews. Clinical oncology, 2022 Jan 11.).

[0113] However, detecting only one or a few antigen-presenting-related genes cannot fully demonstrate the integrity of the antigen presentation pathway. For example, B2M is crucial for the correct assembly and stable expression of MHC-I molecules. If B2M is mutated, MHC-I molecules cannot be assembled, antigen recognition and presentation cannot occur, and the immune response cannot be activated. Conversely, if B2M is functional, but other key steps and molecules in the antigen presentation pathway (such as the antigen processing gene TAP1) are mutated, the immune response will also fail to activate. For another example, it is generally believed in the art that the absence of B2M in cells leads to impaired MHC I pathway function because the MHC I heavy chain itself lacks the correct conformation required to display peptides. TAP-deficient cells are considered to have severe MHC I antigen presentation defects because they cannot deliver cytoplasmic peptides to MHC I molecules in the endoplasmic reticulum (ER). However, studies have shown that highly active memory CD8+ T cells can still recognize cells completely lacking B2M or TAP (Cancer Immunol Res. 2025 Jan 9; 13(1):98-108. doi:10.1158 / 2326-6066.CIR-24-0320.). Therefore, variations in one or several antigen-presenting genes do not necessarily indicate impaired antigen presentation function. Similarly, normal function of one or several antigen-presenting genes does not necessarily indicate the integrity of the antigen presentation pathway. Therefore, comprehensive detection of antigen-presenting-related genes is indispensable in determining the integrity of the antigen presentation pathway.

[0114] In this application, the second reagent for detecting the integrity of the antigen presentation pathway includes probe primers for detecting the integrity of the antigen presentation pathway; in some embodiments, the second reagent includes probe primers for detecting whether the antigen-presenting gene extracted from the human genome hg19 has been mutated, wherein the antigen-presenting gene may be an antigen processing gene, an antigen transport gene, and / or a signaling pathway molecule, and the antigen-presenting gene includes, but is not limited to, B2M, JAK2, PSMB8, CALR, NFKB1, PSMB9, CANX, NLRC5, STAT1, ERAP1, PDIA3, TAP1, ERAP2, PSMB10, TAP2, IRF1, PSMB5, ​​TAPBP, IRF2, PSMB6, JAK1, and PSMB7; the probe primer sequence for detecting whether the antigen-presenting gene has been mutated may include the sequences shown in SEQ ID NO:184 to SEQ ID NO:338. In some embodiments, the probe primers for detecting whether the antigen-presenting gene is mutated may include probe primers for detecting whether the TAP2 gene is mutated; specifically, the probe primer sequences exemplarily include the sequences shown in SEQ ID NO:184 to SEQ ID NO:208. In some embodiments, the probe primers for detecting whether the antigen-presenting gene is mutated may include probe primers for detecting whether the TAP1 gene is mutated; specifically, the probe primer sequences exemplarily include the sequences shown in SEQ ID NO:209 to SEQ ID NO:234. In some embodiments, the probe primers for detecting whether the antigen-presenting gene is mutated may include probe primers for detecting whether the ERAP1 gene is mutated; specifically, the probe primer sequences exemplarily include the sequences shown in SEQ ID NO:235 to SEQ ID NO:270. In some embodiments, the probe primers for detecting whether the antigen-presenting gene is mutated may include probe primers for detecting whether the ERAP2 gene is mutated; specifically, the probe primer sequences exemplarily include the sequences shown in SEQ ID NO:271 to SEQ ID NO:306. In some embodiments, the probe primers for detecting whether the antigen-presenting gene has mutated may include probe primers for detecting whether the PSMB10 gene has mutated; specifically, the probe primer sequences exemplary include the sequences shown in SEQ ID NO:307 to SEQ ID NO:316. In some embodiments, the probe primers for detecting whether the antigen-presenting gene has mutated may include probe primers for detecting whether the PSMB8 gene has mutated; specifically, the probe primer sequences exemplary include the sequences shown in SEQ ID NO:317 to SEQ ID NO:328.In some embodiments, the probe primers for detecting whether the antigen-presenting gene is mutated may include probe primers for detecting whether the PSMB9 gene is mutated; in some embodiments, the probe primer sequences exemplary include the sequences shown in SEQ ID NO:329 to SEQ ID NO:338.

[0115] It should be understood that the sequences listed in this application are merely exemplary sequences of the present invention. In the kits of this application, probe and primer sequences associated with all antigen-presenting genes described herein can be used; therefore, this application is not limited to the exact sequences shown.

[0116] HLA allele heterozygosity loss

[0117] HLA loss of heterozygosity (LOH, HLA-LOH) refers to the loss of an allele at an HLA locus in tumor cells, resulting in homozygosity at that locus. It is one of the common immune escape mechanisms in tumorigenesis, leading to reduced expression of HLA class molecules on the surface of tumor cells, thereby reducing the probability of tumor cells being recognized by the immune system. In addition, HLA loss of heterozygosity may also affect the presentation of tumor antigens, making it difficult for the immune system to recognize and attack tumor cells.

[0118] Currently, detecting HLA loss of heterozygosity is quite difficult because identifying it requires counting the copy number of each allele. This necessitates that the probe has sufficient coverage (e.g., ≥95%) for each exon of each HLA gene to avoid alleles being unsequential due to probe deletion. Conventional low-resolution HLA typing methods struggle to distinguish between true homozygosity (where an individual genuinely inherits two identical alleles) and pseudohomozygosity caused by loss of heterozygosity.

[0119] In this application, tools such as LOHHLA and HLA-Loss can be used to calculate the copy number of HLA regions to analyze whether HLA has a heterozygous loss.

[0120] In this application, the high-resolution kit used can detect whether HLA alleles have lost heterozygosity; in some embodiments, the first reagent for detecting HLA typing can also detect whether HLA alleles have lost heterozygosity, and the reagent includes probe primers for detecting whether HLA alleles have lost heterozygosity; in some embodiments, the probe primer sequence includes the sequences shown in SEQ ID NO:1 to SEQ ID NO:183.

[0121] Tumor immunotherapy

[0122] In this application, tumor immunotherapy may be oncolytic virus therapy, immune checkpoint inhibitor therapy, and / or TCR-T cell therapy. In some embodiments, the tumor immunotherapy may be an oncolytic virus. Regardless of the type of oncolytic virus used or whether it is genetically modified, it will promote HLA presentation. The oncolytic virus may be any known oncolytic virus, including but not limited to adenovirus (AdV), herpes simplex virus (HSV), Newcastle disease virus (NDV), measles virus (MV), vesicular stomatitis virus (VSV), poliovirus, Coxsackie virus, reovirus, and vaccinia virus (VV). In some embodiments, the tumor immunotherapy may be an immune checkpoint inhibitor. The immune checkpoint inhibitor may be any known immune checkpoint inhibitor, including but not limited to PD-1 inhibitors, PD-L1 inhibitors, PD-1 / PD-L1 inhibitors, and CTLA-4 inhibitors. The PD-1 inhibitor includes PD-1 antibodies (PD-1 inhibitors) and PD-L1 antibodies (PD-L1 inhibitors). In some embodiments, the tumor immunotherapy may be TCR-T cell therapy. The TCR-T cell therapy may be any known TCR-T cell therapy, including but not limited to Tebentafusp and Afami-cel. In some embodiments, tumor immunotherapy may be a combination of oncolytic virus and immune checkpoint inhibitor therapy; in specific embodiments, tumor immunotherapy may be a combination of oncolytic virus and PD-1 / PD-L1 inhibitor therapy; in specific embodiments, the oncolytic virus of this application may be used in combination with a PD-1 antibody, wherein the PD-1 antibody may be any known PD-1 antibody. The combination may be administered simultaneously with an immune checkpoint inhibitor or separately from an immune checkpoint inhibitor.

[0123] In this application, the tumor may include, but is not limited to, any tumor related to HLA typing and / or tumors that can be treated with the aforementioned tumor immunotherapy methods. In some embodiments, the tumor may be a solid tumor or a hematologic malignancy; in some embodiments, the tumor may be soft tissue sarcoma, head and neck tumor, skin cancer, melanoma, liver cancer, gastric cancer, lung cancer, kidney cancer, thyroid cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, gynecological or genital tumors, myeloma, lymphoma, and / or leukemia; more specifically, the tumor may be synovial sarcoma, myxosarcoma, liposarcoma, adenoid cystic carcinoma, thymic carcinoma, oropharyngeal carcinoma, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, non-small cell lung cancer, etc. Cellular lung cancer, small cell lung cancer, squamous cell lung cancer, renal cell carcinoma, metastatic renal cell carcinoma, clear renal cell carcinoma, glioma, recurrent glioma, pancreatic ductal carcinoma, triple-negative breast cancer, bladder cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, prostate cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, gastroesophageal junction cancer, multiple myeloma, Hodgkin's lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia, acute myeloid leukemia and / or acute lymphoblastic leukemia.

[0124] In some implementations, tumor immunotherapy may be oncolytic virus therapy, and the tumor may be a solid tumor; further, the tumor may be soft tissue sarcoma, head and neck tumor, skin cancer, melanoma, liver cancer, gastric cancer, gynecological and / or genital tumors; even further, the tumor may be soft tissue sarcoma, head and neck tumor, skin cancer, melanoma, liver cancer, gastric cancer, lung cancer, kidney cancer, thyroid cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, lymphoma, leukemia, gynecological and / or genital tumors; more specifically, the tumor may be synovial sarcoma, myxoid sarcoma, liposarcoma, adenoid cystic carcinoma, thymic carcinoma, oropharyngeal carcinoma, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, bladder cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, ovarian cancer, hepatocellular carcinoma and / or gastric cancer.

[0125] In some embodiments, tumor immunotherapy may be immune checkpoint inhibitor therapy, such as PD-1 / PD-L1 inhibitors or CTLA-4 inhibitors, wherein the tumor may be a solid tumor or a hematologic malignancy; in some embodiments, the tumor may be soft tissue sarcoma, head and neck tumors, skin cancer, melanoma, liver cancer, gastric cancer, lung cancer, kidney cancer, thyroid cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, lymphoma, leukemia, gynecological and / or genital tumors; more specifically, the tumor may be synovial sarcoma, myxosarcoma, liposarcoma, adenoid cystic carcinoma, thymic carcinoma, etc. Oropharyngeal cancer, squamous cell carcinoma of the head and neck, skin cancer, malignant melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, renal cell carcinoma, metastatic renal cell carcinoma, clear renal cell carcinoma, glioma, recurrent glioma, pancreatic ductal carcinoma, triple-negative breast cancer, bladder cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, prostate cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, gastroesophageal junction cancer, multiple myeloma, Hodgkin's lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia, acute myeloid leukemia and / or acute lymphoblastic leukemia.

[0126] In some embodiments, tumor immunotherapy may be TCR-T cell therapy, and the tumor may be a solid tumor or a hematologic malignancy; in some embodiments, the tumor may be soft tissue sarcoma, head and neck tumor, skin cancer, melanoma, liver cancer, gastric cancer, lung cancer, kidney cancer, thyroid cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, lymphoma, leukemia, gynecological and / or genital tumors; more specifically, the tumor may be synovial sarcoma, myxoid sarcoma, liposarcoma, adenoid cystic carcinoma, thymic carcinoma, oropharyngeal carcinoma, head and neck squamous cell carcinoma, skin cancer, Malignant melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, renal cell carcinoma, metastatic renal cell carcinoma, clear renal cell carcinoma, glioma, recurrent glioma, pancreatic ductal carcinoma, triple-negative breast cancer, bladder cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, prostate cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, gastroesophageal junction cancer, multiple myeloma, Hodgkin's lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia, acute myeloid leukemia and / or acute lymphoblastic leukemia.

[0127] According to the embodiments provided in this application, the kit of this application can simultaneously and accurately detect HLA typing-LOH-APM mutations. Therefore, when a specific HLA typing-LOH-APM mutation is detected in a patient, immunotherapy can be selected based on the detection results to promote its antigen presentation function and thereby enhance the immune response.

[0128] Nucleic acid library

[0129] On the other hand, this application provides a set of nucleic acid libraries, the libraries including the aforementioned nucleic acid molecules for detecting HLA typing and nucleic acid molecules for detecting the integrity of antigen presentation pathways. The nucleic acid molecules for detecting the integrity of antigen presentation pathways include probe primers for detecting whether antigen-presenting genes extracted from the human genome hg19 are mutated. The antigen-presenting genes can be antigen processing genes, antigen transport genes, and / or signaling pathway molecules. Antigen-presenting genes include, but are not limited to, B2M, JAK2, PSMB8, CALR, NFKB1, PSMB9, CANX, NLRC5, STAT1, ERAP1, PDIA3, TAP1, ERAP2, PSMB10, TAP2, IRF1, PSMB5, ​​TAPBP, IRF2, PSMB6, JAK1, and PSMB7. In some embodiments, the library further includes nucleic acid molecules capable of detecting whether HLA alleles are lacking heterozygosity. In some embodiments, the nucleic acid molecules for detecting HLA typing can detect HLA typing from the IPD-IMGT / HLA database. HLA typing can be A01, A02, A03, A11, A23, A24, A25, A26, A29, A30, A31, A32, A33, A34, A36, A43, A66, A68, A69, A74, A80, B07, B08, B13, B14, B15, B18, B27, B35, B37, B38, B39, B40, B41, B42, B44, B45, B46, B47, B48, B49, B50, B51, B52, B53 The HLA typing can be A01, A02, A03, A11, A24, A31, A32, A68, B07, B35, B45, B51, B52, and / or C12. In some embodiments, the HLA typing can be A01, A02, A03, A11, A24, A31, A32, A68, B07, B35, B45, B51, B52, and / or C12.

[0130] In some embodiments, the nucleic acid library comprises nucleic acid molecules represented by sequences SEQ ID NO:1 to SEQ ID NO:338.

[0131] It should be understood that the sequences listed in this application are merely exemplary sequences of the present invention. In the nucleic acid library of this application, all HLA genotyping-related probe and primer sequences described herein can be used; therefore, this application is not limited to the exact sequences shown.

[0132] In some embodiments, the nucleic acid library further includes a target DNA nucleic acid molecule; specifically, the target DNA nucleic acid also includes an adapter.

[0133] On the other hand, this application provides a method for constructing the above-mentioned nucleic acid library, the method comprising: 1) fragmenting template DNA; 2) ligating the fragmented DNA solution with a sequencing library adapter; 3) amplifying and enriching the DNA sequencing library adapter ligation product obtained in 2); and / or 4) probe-primer hybridization capture.

[0134] In some embodiments, the template DNA fragmentation method includes any method known in the art, including but not limited to sonication, atomization, restriction endonuclease, transposase, DNase I, exonuclease / endonuclease hybrid method, and metal ion catalytic hydrolysis; specifically, DNA fragmentation is achieved through FEA Enzyme and DNA Damage Repair Enzyme.

[0135] In some embodiments, the adapter includes any adapter known in the art, including but not limited to single-end / double-end adapters, UMI adapters, UDI adapters, transposase adapters, and complete / incomplete adapters; specifically, the adapter is a UDI adapter.

[0136] In some embodiments, the method further includes a purification method, which can be any purification means known in the art, including but not limited to: organic solvent extraction, column purification and magnetic bead purification; specifically, the purification method is magnetic bead purification.

[0137] In some embodiments, the probe-primer hybridization capture is performed via liquid-phase hybridization capture using probe primers and hybridization capture reagents.

[0138] biomarkers

[0139] This application is capable of detecting biomarkers, including patient HLA typing, HLA alleles and / or antigen presentation pathway genes.

[0140] HLA testing can be any known testing method in the field, including but not limited to methods based on liquid-phase DNA probe hybridization capture technology and next-generation sequencing.

[0141] In some implementations, this application detects HLA using next-generation sequencing.

[0142] The detection method for antigen-presenting genes can be any known detection method in the field, including but not limited to liquid-phase DNA probe hybridization capture technology and gene sequencing detection.

[0143] In some embodiments, this application can detect antigen presentation pathway genes by gene sequencing.

[0144] system

[0145] This application provides a system comprising a first module for detecting a patient's HLA typing and detecting whether HLA alleles exhibit loss of heterozygosity, and / or a second module for detecting the integrity of an antigen presentation pathway. The first module can detect patient HLA using any known method, including but not limited to liquid-phase DNA probe hybridization capture technology and next-generation sequencing (NGS)-based methods. The second module can detect the integrity of an antigen presentation pathway using any known method, including but not limited to liquid-phase DNA probe hybridization capture technology and next-generation sequencing (NGS)-based methods. In some embodiments, the system includes the aforementioned kit and / or the aforementioned nucleic acid library.

[0146] In the system described in this application, the first module and the second module can be run in any order. For example, the first module can be run first, followed by the second module.

[0147] The system described in this application may further include a storage module configured to store data on the class I and / or class II HLA genotypes of each subject in a model population including patients, and the amino acid sequences of one or more test peptides; wherein the aforementioned model population represents the target human population for testing.

[0148] The system described in this application may also be equipped with alignment software, which can compare HLA data of patient samples with HLA sequences in the IPD-IMGT / HLA database, wherein the patients include the aforementioned tumor patients. In some embodiments, the alignment software may include any alignment software known in the art, including but not limited to BWA (Burrows-Wheeler Alignment), Bowtie2 (Multiple Sequence Comparison by Log-Expectation), Razers3, Muscle, MAFFT, and MUMmer; specifically, the alignment software may be Razers3 and BWA.

[0149] The system described in this application may further include a computational module configured to identify and / or quantify amino acid sequences of multiple class I HLA molecules in one or more test peptides capable of binding to each subject in the aforementioned model population. In some embodiments, the computational module may further include extraction software; specifically, the extraction software may extract the sequencing sequences of aligned HLA alleles from HLA allele alignment results. In some embodiments, the computational module may include any known extraction software. For example, the extraction software may be samtools. In some embodiments, the computational module may also be configured to simultaneously select two, three, or four alleles for each HLA class I gene locus (A, B, C); specifically, the computational module may include OptiType; in some embodiments, the computational module may also perform imbalance detection operations on HLA alleles and / or copy number detection operations on HLA alleles.

[0150] The system described in this application may further include a data analysis and interpretation module. This data analysis and interpretation module can submit HLA alleles and antigen-presenting genes in batches, and it may include any known analysis software for processing, such as TypeLoader2.

[0151] The system described in this application may also include a function prediction and coding region annotation module, which can submit antigen-presenting genes in batches and may include any known function prediction software for processing, such as SIFT and PolyPhen-2.

[0152] The system described in this application may also include a report generation module, wherein the report may include a detailed description of HLA alleles, matching suggestions, etc.

[0153] The system described in this application may also include an output module configured to display the values ​​of any output predictions or treatment options or recommendations described herein, or any pharmacodynamic biomarkers described herein. For example, it may output HLA typing, HLA alleles, whether HLA alleles are lost heterozygous, and / or whether antigen-presenting genes are mutated.

[0154] platform

[0155] On the other hand, this application also provides an anti-tumor drug delivery platform, which includes a drug module for tumor immunotherapy, a screening module for HLA typing in a patient population, and / or a screening module for the integrity of antigen presentation pathways. The drug delivery platform provided by this application can also screen for whether HLA alleles lack heterozygosity.

[0156] In some embodiments, the drug module of the tumor immunotherapy includes TCR-T cell therapy, oncolytic virus therapy, and / or immune checkpoint inhibitor therapy. In some embodiments, the drug module of the tumor immunotherapy may be oncolytic virus therapy, wherein the oncolytic virus can be any known oncolytic virus, such as, but not limited to, adenovirus (AdV), herpes simplex virus (HSV), Newcastle disease virus (NDV), measles virus (MV), vesicular stomatitis virus (VSV), poliovirus, Coxsackie virus, reovirus, and vaccinia virus (VV). In some embodiments, the drug module of the tumor immunotherapy may be an immune checkpoint inhibitor, such as a PD-1 / PD-L1 inhibitor or a CTLA-4 inhibitor, wherein the PD-1 / PD-L1 inhibitor can be any known inhibitor, including but not limited to any known PD-1 antibody and PD-L1 antibody. In some embodiments, the drug module of the tumor immunotherapy may be oncolytic virus combined with immune checkpoint inhibitor therapy, specifically, oncolytic virus combined with PD-1 / PD-L1 inhibitor therapy, and more specifically, oncolytic virus combined with PD-1 antibody therapy.

[0157] Without being limited by any theory, the embodiments described below are merely for illustrating the various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0158] Example

[0159] Example 1: Reagent Kit Screening Strategy

[0160] Example 1.1 HLA typing and HLA allele heterozygosity detection method

[0161] HLA detection can be performed using any known method in the field, including but not limited to methods based on liquid-phase DNA probe hybridization capture technology and next-generation sequencing. The specific design is shown in Figure 1, and includes the following steps:

[0162] a. Download the coding sequence (CDS) sequences of all HLA-I type genes (HLA-A, HLA-B, HLA-C) from the IPD-IMGT / HLA database;

[0163] b. The CDS sequences of the HLA-A, HLA-B, and HLA-C genes were deduplicated based on sequence similarity ≥95%, preserving HLA polymorphic sequence information as much as possible; and

[0164] c. For the deduplication HLA polymorphic sequences in b above, design and synthesize hybridization capture probes of 120 nt in length to obtain probe primers for HLA typing and HLA allele heterozygosity detection.

[0165] Example 1.2 Method for detecting the integrity of antigen presentation pathway

[0166] The detection method for antigen presentation pathway genes can be any known detection method in the art, including but not limited to liquid-phase DNA probe hybridization capture technology and gene sequencing. Specifically, this includes the design of hybridization capture probe primers for detecting antigen presentation pathway-related gene variants: CDS sequences of 22 antigen presentation pathway-related genes (B2M, JAK2, PSMB8, CALR, NFKB1, PSMB9, CANX, NLRC5, STAT1, ERAP1, PDIA3, TAP1, ERAP2, PSMB10, TAP2, IRF1, PSMB5, ​​TAPBP, IRF2, PSMB6, JAK1, and PSMB7) were extracted from the human reference genome hg19, and 120nt hybridization capture probes were designed and synthesized to obtain probe primers for detecting antigen presentation gene variants.

[0167] Example 1.3 Nucleic acid extraction and library construction from tumor tissue and control samples

[0168] Nucleic acid extraction and library construction from tumor tissue and control samples can be performed using any known detection method in the art. This application provides an exemplary method for nucleic acid extraction and library construction from tumor tissue and control samples, as shown in Figure 1. The specific steps include:

[0169] a. DNA was extracted from tumor tissue and normal control samples using DNA extraction reagents (magnetic bead pre-separation, Kangwei Century);

[0170] b. Use DNA library construction reagent (ND627-C10, Novizan) to construct libraries from the extracted tumor tissue DNA and normal control sample DNA, respectively. Detailed reagents and operating procedures are as follows:

[0171] I. Nucleic acid fragmentation: Prepare the DNA fragmentation reaction mixture according to Table 1, and set the PCR program according to Table 2 to complete the DNA fragmentation;

[0172] Table 1 DNA Fragment Reaction Mixture

[0173] Table 2. Sequence of DNA fragmentation reactions

[0174] II. DNA sequencing library adapter ligation: Prepare the adapter ligation reaction mixture according to Table 3, set the PCR program according to Table 4, and finally purify the DNA sequencing library using purification magnetic beads to complete the DNA sequencing library adapter ligation.

[0175] Table 3 DNA sequencing library adapter ligation reaction mixture

[0176] Table 4 DNA fragmentation reaction procedure

[0177] III. DNA Sequencing Library: Prepare the library enrichment reaction mixture according to Table 5, and set up the PCR run according to Table 6.

[0178] The procedure concludes with purification using magnetic beads to complete the enrichment of the DNA sequencing library.

[0179] Table 5 DNA sequencing library enrichment reaction mixture

[0180] Table 6 Library enrichment reaction procedure

[0181] Example 1.4 Probe-primer hybridization capture

[0182] The hybridization capture reagent (xGen Hybridization and Wash Kit, IDT) and the hybridization capture probe primer combinations designed and synthesized in 1.1 and 1.2 above were used to perform liquid-phase hybridization capture on the sequencing libraries of tumor tissue DNA and normal control sample DNA obtained in 1.3 above. The detailed reagents and operation steps are as follows:

[0183] a. DNA sequencing library probe hybridization: 500 ng of each DNA sequencing library was added, along with 5 μL of Human Cot DNA and 2 μL of Bloking Oligos for vacuum concentration and drying. The dried library powder was resuspended in Hybridization Master Mix (including 8.5 μL Hybridization Buffer and 2.7 μL Hybridization Buffer Enhancer), and then 4 μL of the hybridization capture probe primer combination designed and synthesized in 1.1 and 1.2 above was added. After thorough mixing, the hybridization reaction was started. Hybridization reaction conditions: 95℃, 30 s; 65℃, 16 h.

[0184] b. Magnetic bead capture: After hybridization, use Capture beads, wash twice at 65°C and three times at room temperature, and resuspend the magnetic beads in 20 μL of nuclease-free water;

[0185] c. Final library PCR and product purification: Add 25 μL HiFi HotStart ReadyMix and 5 μL P5 / P7 primer (5 μM) to the captured library for PCR amplification. The PCR amplification reaction program is shown in Table 7.

[0186] Table 7 Final Library Enrichment Reaction Procedure

[0187] Example 1.5 Bioinformatics Analysis

[0188] The final library obtained in step 1.4 was sequenced using DNBSEQ-T7 according to the official sequencing procedure, and then subjected to bioinformatics analysis.

[0189] a. HLA typing analysis: I. The sequencing data of the tumor tissue and normal control samples obtained above were compared with the HLA allele sequences in the IPD-IMGT / HLA database using Razer 3 software; II. The sequencing sequences of the aligned HLA alleles were extracted from the HLA allele alignment results in the database using Samtools software; III. Using OptiType software and its Integer Linear Programming (ILP) algorithm, considering all the aligned HLA allele sequencing sequences, two alleles were selected for each HLA-I class gene locus (A, B, C), and the HLA-A, B, and C typing results of the tumor tissue samples and normal control samples were finally output.

[0190] b. HLA-LOH Analysis: I. Based on the HLA typing results of tumor tissue samples and normal control samples in a above, HLA allele imbalance detection is performed to obtain the HLA allele imbalance detection results; II. Based on the comparison results of HLA typing of tumor tissue samples and normal control samples in a above, HLA allele copy number detection is performed to obtain the copy number of HLA-specific alleles; III. Based on the allele imbalance in I above and the copy number results in II above, the heterozygous loss status of HLA genes is determined. If HLA allele imbalance occurs and the copy number has not increased, it is determined to be HLA-LOH.

[0191] c. Antigen-presenting gene mutation detection: Sequencing data of tumor tissue and normal control samples obtained above were compared with the human reference genome using bwa-mem and then Vardict was used to detect somatic mutations in antigen-presenting genes. Simultaneously, protein coding region annotation of somatic mutations and SIFT / PolyPhen-2 functional prediction were performed to obtain the antigen-presenting gene mutation results.

[0192] Example 2: Reagent kit for HLA typing of tumor tissue samples

[0193] This embodiment used 15 tumor tissue samples and normal control leukocyte samples to perform HLA typing using the method of the kit of this invention, and compared it with the gold standard first-generation PCR-SBT (Sanger) method to verify the accuracy of the results of the kit's detection method. The detection results of the kit and the comparison method are shown in Table 8 below. In the HLA type genotyping of 15 clinical samples, the detection results of the kit of this invention and the gold standard PCR-SBT (Sanger) method achieved 100% consistency, and could be accurate to a group of alleles expressing the same protein (i.e., containing at least two numerical regions, such as C*04:01). This indicates that the HLA genotyping detection results of the method of this invention have high accuracy and reliability, and can provide detection kits and methods for screening biomarkers of immunotherapy sensitivity. It can be used for HLA typing detection and patient screening of patients suitable for immunotherapy.

[0194] Table 8. Test results of 15 clinical samples using reagent kits and comparative methods.

[0195] Example 3: Screening Strategy Based on Clinical Oncology Patient Validation Kit

[0196] This embodiment provides an exemplary test of the potential for precision tumor immunotherapy in two types of cancer patients using the kit described in this application. On one hand, T cells cannot directly recognize intracellular proteins; they must be captured and presented to the cell surface via HLA molecules to activate the immune system. On the other hand, in immune checkpoint inhibitor therapy, TCR-T therapy, and / or oncolytic virus therapy, HLA is an important biomarker that determines which tumor antigens can be recognized by the immune system, thereby achieving the effect of killing tumors. Besides exemplary bladder cancer and cervical cancer, solid tumors such as lung cancer and liver cancer that can be killed through tumor immunotherapy can also be detected using the kit described in this application. This allows for simultaneous three-dimensional detection of "HLA typing - loss of heterozygosity - antigen-presenting gene variation," thereby obtaining comprehensive results of HLA typing, loss of heterozygosity, and antigen-presenting gene variation in different cancer patients, increasing the possibility of achieving precision tumor immunotherapy. Nine clinical oncology patients underwent HLA typing, loss of heterozygosity, and antigen-presenting gene mutation testing using the kit of this invention on tumor tissue and normal control samples. Immunotherapy was subsequently administered, and follow-up was conducted to confirm the efficacy of the immunotherapy. This kit was used to evaluate the biomarker screening for immunotherapy sensitivity and to assist in assessing the patient's immunotherapy response. Detailed clinical patient information and immunotherapy follow-up results are shown in Table 9 below.

[0197] Table 9. Clinical oncology patient information and immunotherapy follow-up results

[0198] CR: Complete remission, PR: Partial remission, SD: Stable disease; Oncolytic virus immunotherapy: KM1. KM1 is a recombinant vaccinia virus developed by Shenzhen Huayao Kangming Biopharmaceutical Co., Ltd., which uses Listeria vaccinia virus strain as a vector for genomic modification, deleting the TK, L025, and A46R genes and inserting the 4-1BBL and IL-21 genes. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number V201938; deposit date July 4, 2019; strain name: genetically modified Listeria genotype vaccinia virus KM1; depositor: Shenzhen Huayao Kangming Biopharmaceutical Co., Ltd. (applicant).

[0199] The above-mentioned results of the "HLA typing-loss of heterozygosity-antigen presentation gene variation" detection using the kit of the present invention show that...

[0200] 1) In bladder cancer patients, S01002 and S01004 were homozygous for HLA-A*02:06 and HLA-A*33:03, respectively, without loss of heterozygosity of HLA alleles. The event-free survival of these patients after receiving oncolytic virus immunotherapy has reached 9 months and 6 months, respectively. In contrast, bladder cancer patient S01001 was homozygous for HLA-A*02:07 and HLA-A*01:01, also without loss of heterozygosity of HLA alleles. The event-free survival of this patient after receiving oncolytic virus immunotherapy has also reached 9 months.

[0201] No inactivation variants were found in the antigen-presenting genes of the above three patients (i.e., the antigen presentation pathway was intact). The results indicate that this kit can successfully detect HLA typing, loss of heterozygosity of HLA alleles, and integrity of antigen presentation pathway in bladder cancer patients, realizing the successful application of a three-dimensional detection method that simultaneously performs "HLA typing-loss of heterozygosity-antigen presentation gene variants".

[0202] 2) Among cervical cancer patients, the original HLA-A alleles of cervical cancer patient SII1001 were HLA-A*02:07 and HLA-A*11:01, respectively. The tumor cells of this patient simultaneously underwent HLA-LOH loss of the HLA-A*11:01 allele to form HLA-A*02:07 homozygote. After receiving combination therapy with oncolytic virus and PD1, the tumor achieved complete remission and the event-free survival reached 8.9 months. Another cervical cancer patient SB1003, whose HLA-A type was homozygous HLA-A*11:01, also achieved partial remission of tumor after receiving combination therapy with oncolytic virus and PD1, and the event-free survival also reached 5.13 months.

[0203] No inactivation variants were found in the antigen-presenting genes of the two patients mentioned above (i.e., the antigen presentation pathway was intact). The results indicate that this kit can successfully detect HLA typing, loss of heterozygosity of HLA alleles, and integrity of antigen presentation pathways in cervical cancer patients. It achieves the successful application of a three-dimensional detection method that simultaneously performs "HLA typing-loss of heterozygosity-antigen-presenting gene variants". In the two patients in this example, HLA-A*02:07 and HLA-A*11:01 typing may be important biomarkers for patients receiving immunotherapy and affecting their drug sensitivity. Furthermore, when HLA-LOH or homozygosity of these two typings occurs, the immunotherapy effect is better.

[0204] The kit described in this application can be used for screening biomarkers of immunotherapy sensitivity, and can also be used to screen patient populations suitable for receiving immunotherapy, and to help predict the effectiveness of immunotherapy response.

Claims

The test kit contains a first reagent for detecting HLA typing and a second reagent for detecting the integrity of the antigen presentation pathway. According to the detection kit of claim 1, the second reagent for detecting the integrity of the antigen presentation pathway includes detecting whether the antigen presentation gene has a genetic mutation, wherein the antigen presentation gene includes an antigen processing gene and / or an antigen transport gene. According to any one of the detection kits in claims 1-2, the antigen-presenting gene is selected from one or more of the following group: B2M, JAK2, PSMB8, CALR, NFKB1, PSMB9, CANX, NLRC5, STAT1, ERAP1, PDIA3, TAP1, ERAP2, PSMB10, TAP2, IRF1, PSMB5, ​​TAPBP, IRF2, PSMB6, JAK1, and PSMB7. The detection kit according to any one of claims 1-3, wherein the second reagent for detecting the integrity of the antigen presentation pathway comprises a probe primer for detecting whether the antigen presentation gene has mutated. The detection kit according to any one of claims 1-4, wherein the probe primer sequence for detecting whether the antigen-presenting gene is mutated includes the sequences shown in SEQ ID NO:184 to SEQ ID NO:

338. The detection kit according to any one of claims 1-5, wherein the HLA typing is derived from the IPD-IMGT / HLA database. The detection kit according to any one of claims 1-6, wherein the HLA typing is HLA-I class molecule. The detection kit according to any one of claims 1-7, wherein the HLA typing includes A01, A02, A03, A11, A23, A24, A25, A26, A29, A30, A31, A32, A33, A34, A36, A43, A66, A68, A69, A74, A80, B07, B08, B13, B14, B15, B18, B27, B35, B37, B38, and B39. B40, B41, B42, B44, B45, B46, B47, B48, B49, B50, B51, B52, B53, B54, B55, B56, B57, B58, B59, B67, B73, B78, B81, B82, B83, C01, C02, C03, C04, C05, C06, C07, C08, C12, C14, C15, C16, C17 and / or C18. According to any one of the detection kits in claims 1-8, the HLA typing is A01, A02, A03, A11, A24, A31, A32, A68, B07, B35, B45, B51, B52 and / or C12 typing. The detection kit according to any one of claims 1-9, wherein the first reagent for detecting HLA typing can also detect whether HLA alleles are lost heterozygous. The detection kit according to any one of claims 1-10, wherein the first reagent for detecting HLA typing comprises probe primers for detecting HLA typing. The detection kit according to any one of claims 10-11, wherein the first reagent for detecting HLA typing further includes probe primers for detecting whether HLA alleles are heterozygous. The detection kit according to any one of claims 10-12, wherein the probe primer sequence for detecting whether HLA alleles are heterozygous includes the sequences shown in SEQ ID NO:1 to SEQ ID NO:

183. The detection kit according to any one of claims 11-13, wherein the probe primer sequence for detecting HLA typing comprises the sequences shown in SEQ ID NO:1 to SEQ ID NO:

183. A set of nucleic acid libraries, the nucleic acid libraries including nucleic acid molecules for detecting HLA typing and whether HLA alleles are lacking heterozygosity, and / or nucleic acid molecules for detecting the integrity of antigen presentation pathways. According to the nucleic acid library of claim 15, the nucleic acid molecule used to detect the integrity of the antigen presentation pathway includes a nucleic acid molecule for detecting whether the antigen presentation gene is mutated, wherein the antigen presentation gene includes an antigen processing gene and / or an antigen transport gene. According to any one of claims 15-16, the nucleic acid library, wherein the antigen-presenting gene is selected from one or more of the following group: B2M, JAK2, PSMB8, CALR, NFKB1, PSMB9, CANX, NLRC5, STAT1, ERAP1, PDIA3, TAP1, ERAP2, PSMB10, TAP2, IRF1, PSMB5, ​​TAPBP, IRF2, PSMB6, JAK1, and PSMB7. The nucleic acid library according to any one of claims 15-17, wherein the nucleic acid molecule for detecting whether the antigen-presenting gene has mutated includes a probe primer for detecting whether the antigen-presenting gene has mutated. The nucleic acid library according to any one of claims 15-18, wherein the nucleic acid molecule for detecting HLA typing is capable of detecting HLA typing from the IPD-IMGT / HLA database. The nucleic acid library according to any one of claims 15-19, wherein the nucleic acid molecule for detecting HLA typing comprises probe primers for detecting HLA typing. The nucleic acid library according to any one of claims 15-20, wherein the nucleic acid molecule for detecting whether HLA alleles are heterozygous includes probe primers for detecting whether HLA alleles are heterozygous. The nucleic acid library according to any one of claims 15-21, wherein the nucleic acid molecule for detecting HLA typing comprises probe primers for detecting HLA typing. The nucleic acid library according to any one of claims 15-22, wherein the nucleic acid molecule for detecting whether HLA alleles are heterozygous includes probe primers for detecting whether HLA alleles are heterozygous. The nucleic acid library according to any one of claims 18-23, wherein the probe primer sequence for detecting whether the antigen-presenting gene is mutated includes the sequences shown in SEQ ID NO:184 to SEQ ID NO:

338. The nucleic acid library according to any one of claims 22-24, wherein the probe primer sequence for detecting HLA typing comprises the sequences shown in SEQ ID NO:1 to SEQ ID NO:

183. The nucleic acid library according to any one of claims 23-25, wherein the probe primer sequence for detecting whether HLA alleles are heterozygous includes the sequences shown in SEQ ID NO:1 to SEQ ID NO:

183. The nucleic acid library according to any one of claims 15-26 further includes target DNA. The nucleic acid library according to claim 27, wherein the target DNA further includes an adapter. A method for constructing a nucleic acid library, the nucleic acid library comprising any one of claims 15-28, the method comprising: a) Template DNA fragmentation, b) Ligate the fragmented DNA solution to the sequencing library adapters. c) Amplify and enrich the DNA sequencing library adapter ligation products obtained in b). and d) Probe and primer liquid phase hybridization capture and amplification to obtain the final library. Use of the kit according to any one of claims 1-14 and / or the nucleic acid library according to any one of claims 15-28 in the manufacture of a medicament for stimulating an immune response in a subject or for tumor immunotherapy in a subject. According to the use described in claim 30, the tumor immunotherapy includes TCR-T cell therapy, oncolytic virus therapy, and / or immune checkpoint inhibitor therapy. The use according to any one of claims 30-31, wherein the immune checkpoint inhibitor therapy comprises the use of a PD-1 / PD-L1 inhibitor. The tumor immunotherapy is an oncolytic virus therapy according to any one of claims 30-32. The use according to any one of claims 30-33, wherein the tumor immunotherapy comprises a combination therapy of oncolytic viruses and immune checkpoint inhibitors. The use according to any one of claims 30-34, wherein the tumor immunotherapy is a combination therapy of oncolytic virus and PD-1 / PD-L1 inhibitor. The tumor is a solid tumor and / or a hematologic tumor, according to any one of claims 30-35. According to any one of claims 30-36, the tumor includes soft tissue sarcoma, head and neck tumors, skin cancer, melanoma, liver cancer, gastric cancer, lung cancer, kidney cancer, thyroid cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, gynecological and genital tumors, myeloma, lymphoma and / or leukemia. According to any one of claims 30-37, the tumor includes synovial sarcoma, myxosarcoma, liposarcoma, adenoid cystic carcinoma, thymic carcinoma, oropharyngeal carcinoma, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, renal cell carcinoma, metastatic renal cell carcinoma, clear renal cell carcinoma, glioma, recurrent glioma, pancreatic ductal carcinoma, triple-negative breast cancer, bladder cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, prostate cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, gastroesophageal junction cancer, multiple myeloma, Hodgkin's lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia, acute myeloid leukemia, and / or acute lymphoblastic leukemia. A system for predicting populations for tumor immunotherapy, the system comprising a detection kit according to any one of claims 1-14 and / or a nucleic acid library according to any one of claims 15-28. According to the system of claim 39, the tumor immunotherapy includes TCR-T cell therapy, oncolytic virus therapy, and / or immune checkpoint inhibitor therapy. The system according to any one of claims 39-40, wherein the immune checkpoint inhibitor therapy comprises using PD- 1 / PD-L1 inhibitor. The system according to any one of claims 39-41, wherein the tumor immunotherapy is an oncolytic virus therapy. The system according to any one of claims 39-42, wherein the tumor immunotherapy comprises a combination therapy of oncolytic viruses and immune checkpoint inhibitors. The system according to any one of claims 39-43, wherein the tumor immunotherapy is a combination therapy of oncolytic virus and PD-1 / PD-L1 inhibitor. The system according to any one of claims 39-44, wherein the tumor is a solid tumor and / or a hematoma. According to any one of claims 39-45, the tumor includes soft tissue sarcoma, head and neck tumors, skin cancer, melanoma, liver cancer, stomach cancer, lung cancer, kidney cancer, thyroid cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, gynecological and genital tumors, myeloma, lymphoma and / or leukemia. According to any one of claims 39-46, the tumor includes synovial sarcoma, myxosarcoma, liposarcoma, adenoid cystic carcinoma, thymic carcinoma, oropharyngeal carcinoma, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, renal cell carcinoma, metastatic renal cell carcinoma, clear renal cell carcinoma, glioma, recurrent glioma, pancreatic ductal carcinoma, triple-negative breast cancer, bladder cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, prostate cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, gastroesophageal junction cancer, multiple myeloma, Hodgkin's lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia, acute myeloid leukemia, and / or acute lymphoblastic leukemia. The system according to claims 39-47, the system further includes 1) Comparison software; 2) Output module for HLA-A, B, and C typing results of the output samples; 3) Output module for outputting the heterozygous loss state of HLA genes; and / or 4) Output module for outputting antigen-presenting gene variation results. An antitumor drug delivery platform comprising the kits described in claims 1-14 and / or the nucleic acid libraries described in any one of claims 15-28, and a drug module for tumor immunotherapy. According to the platform of claim 49, the tumor is a solid tumor and / or a hematologic tumor. According to any one of claims 49-50, the tumor includes soft tissue sarcoma, head and neck tumors, skin cancer, melanoma, liver cancer, gastric cancer, lung cancer, kidney cancer, thyroid cancer, breast cancer, nasopharyngeal carcinoma, colorectal cancer, pancreatic cancer, gynecological and genital tumors, myeloma, lymphoma and / or leukemia. According to any one of claims 49-51, the tumor includes synovial sarcoma, myxosarcoma, liposarcoma, adenoid cystic carcinoma, thymic carcinoma, oropharyngeal carcinoma, head and neck squamous cell carcinoma, skin cancer, malignant melanoma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, renal cell carcinoma, metastatic renal cell carcinoma, clear renal cell carcinoma, glioma, recurrent glioma, pancreatic ductal carcinoma, triple-negative breast cancer, bladder cancer, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, prostate cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, gastric cancer, gastroesophageal junction cancer, multiple myeloma, Hodgkin's lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia, acute myeloid leukemia, and / or acute lymphoblastic leukemia. The platform according to any one of claims 49-52, wherein the tumor immunotherapy is an oncolytic virus therapy. The platform according to any one of claims 49-53, wherein the tumor immunotherapy comprises a combination therapy of oncolytic viruses and immune checkpoint inhibitors. The platform according to any one of claims 49-54, wherein the tumor immunotherapy is a combination therapy of oncolytic virus and PD-1 / PD-L1 inhibitor. A method for screening biomarkers includes amplifying the HLA gene and antigen-presenting gene of a sample to be tested using a kit as described in any one of claims 1-14, a nucleic acid library as described in any one of claims 15-28, or a method for constructing a nucleic acid library as described in claim 29; then sequencing the amplicon; comparing the sequencing results with a reference genome to determine the HLA genotype, whether the HLA allele is lacking heterozygosity, and whether the antigen-presenting pathway is intact; wherein the HLA genotype is an HLA-I class molecule; and the detection of the integrity of the antigen-presenting pathway involves detecting whether the antigen-presenting related gene is mutated. The use of biomarkers in the preparation of tumor immunotherapy prediction systems and / or anti-tumor drug screening platforms, wherein the biomarkers include HLA typing, HLA alleles and / or antigen-presenting genes, the tumor immunotherapy includes oncolytic virus therapy, TCR therapy and / or immunosuppressant therapy, the HLA typing is HLA-I molecules, and the detection of antigen presentation pathway integrity is to detect whether antigen presentation-related genes are mutated.

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