Modified enhanced immunocyte targeting HPV and pharmaceutical use thereof

Abstract

Disclosed in the present application are a modified enhanced immunocyte targeting HPV and the pharmaceutical use thereof. Provided in the present invention are a T-cell receptor or a fragment thereof targeting the E7 antigen of HPV, and the pharmaceutical use thereof. In addition, further provided are a modified immunocyte simultaneously expressing HPV-targeting TCR and converting a costimulatory molecule, and the pharmaceutical use thereof. In the modified immunocyte of the present application, the intracellular activation signaling domain of a costimulatory molecule is used to replace the intracellular inhibitory signaling domain of an immunosuppressive receptor, so that the inhibitory signal induced by the binding of immunosuppressive factors or ligands is converted into an activation signal, which can effectively block cell depletion induced by immunosuppressive molecules, and thus increase cytokine release, and improve the cell proliferation ability and the tumor cell killing ability. The present invention is expected to overcome a series of problems faced by adoptive T-cell immunotherapy, such as tumor heterogeneity, poor tumor barrier permeability, short duration of action, and local immunosuppressive microenvironment.

Description

Modified HPV-targeted enhanced immune cells and their medical uses Technical Field

[0001] The present application relates to the field of immunotherapy technology, and specifically to a modified immune cell and its medical use. At the same time, the present application also provides a T cell receptor or a fragment thereof targeting the HPV E7 antigen and its related applications. Background Art

[0002] 1.1 Molecular biological characteristics and subtypes of HPV

[0003] HPV is a DNA virus. Its genomic DNA is approximately 8 kb in size and consists of three parts: the early protein-coding region, the late protein-coding region, and the upstream regulatory region. The early coding region, consisting of 4500 base pairs, encodes six early regulatory proteins (E1 / E2 / E4 / E5 / E6 / E7), which are involved in DNA replication, transcription, translation regulation, and cellular transformation. The late coding region, consisting of 2500 base pairs, encodes the major capsid protein L1 and the minor capsid protein L2. The upstream regulatory region, consisting of 1000 base pairs, contains the HPV genome's replication origin and gene expression regulatory elements, regulating viral transcription and replication. Furthermore, the HPV genome does not encode the polymerase or other enzymes necessary for viral replication. Therefore, HPV must rely on host cell replication proteins to mediate viral DNA synthesis. HPV is classified based on the homology of the E6, E7, and L1 gene sequences. Using 90% homology as the standard, over 200 subtypes have been identified. Based on their pathogenicity, they can be divided into high-risk types (hrHPV) and low-risk types. There are approximately 14 high-risk HPV types, including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68. High-risk HPV can cause a variety of cancers, including almost all cervical cancers, most anal cancers, and many oropharyngeal cancers, vaginal cancers, vulvar cancers, and penile cancers. Among them, high-risk HPV16 and HPV18 subtypes have been found to be associated with most HPV-related cancers. 70% of cervical cancers are caused by persistent infection with HPV16 and HPV18, and 84.1% of invasive cervical cancers are also mainly caused by the two.

[0004] Human leukocyte antigens (HLA) are the most polymorphic gene complex in the human genome. Genotyping includes HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1. Studies have found that HLA-A alleles play a more important role in the Han Chinese population than other HLA genes. The most common alleles in China are HLA-A*24:02, HLA-A*11:01, and HLA-A*02:01.

[0005] 1.2 HPV carcinogenic pathway

[0006] Taking cervical cancer as an example, 80% of sexually active women have HPV infection, of which about 90% can rely on their own immune system to naturally resolve within 1-2 years, and a small number of them become persistently infected. Ultimately, about 1% of patients develop cervical cancer. Usually, it takes 5-10 years of high-risk HPV infection to cause abnormal cytological changes and induce cervical cancer. The precursor lesion of cervical cancer / atypical hyperplasia of the cervical epithelium is called cervical intraepithelial neoplasia (CIN), which can be divided into the following three levels according to the severity:

[0007] CIN1: The lesion is limited to the lower 1 / 3 of the epithelium, which is mild cervical atypical hyperplasia;

[0008] CIN2: The lesion is limited to 1 / 2-2 / 3 of the epithelial layer, which is moderate cervical atypical hyperplasia;

[0009] CIN3: The lesion involves almost all epithelial layers, with only 1-2 layers of normal squamous epithelium remaining on the surface, which is severe cervical atypical hyperplasia and cervical carcinoma in situ.

[0010] All levels of precancerous lesions have the potential to develop into invasive cancer, with higher levels increasing the likelihood of progression. Studies have found that HPV-DNA integration is found in 83% of cervical cancers, and HPV genes play an important role in the development and progression of HPV-related tumors.

[0011] HPV infects the body through epithelial tissue damage, and its life cycle is closely related to the differentiation process of keratinocytes. During the process of keratinocytes developing into the spinous layer, viral DNA replication and gene expression occur. The virus is internalized into the cell through endocytosis and penetrates into the cell nucleus to initiate the replication process. Specifically, the E1 and E2 proteins play a key role in the initial replication and transcription of the HPV virus, and the E4 and E5 proteins regulate the functions of the late stage of the viral life cycle. They will cause cell cycle regulation disorders by downregulating the P21 gene, causing HPV-infected cells to become malignant cells. In addition, HPV-DNA integration occurs in the E2 gene coding region, and the inactivation of this gene expression causes upregulation of E6 and E7 gene transcription. E6 and E7 are the only two viral genes consistently found in cervical cancer tissues, suggesting that they are directly related to the occurrence of HPV-related tumors. Studies have found that upon binding to the cellular ubiquitin ligase E6AP, the E6 protein undergoes a conformational change, specifically binding to the tumor suppressor protein p53, forming the E6 / E6AP / p53 complex. This leads to the ubiquitination and degradation of p53, thereby inhibiting apoptosis. Similarly, the E7 protein can bind to and inactivate pRb, promoting transcriptional activation of genes by the transcription factor family E2F, leading to cell cycle abnormalities. Inactivation of the tumor suppressor genes p53 and pRb leads to cell cycle deregulation, inhibiting apoptosis, and causing excessive cell proliferation and cancer. Furthermore, E7 can bind to the DREAM domain of RB-like, E2F4, and MuyB proteins, leading to their degradation via the proteasome, activating downstream p53 signaling pathways, and causing cell cycle abnormalities. In other words, the E6 and E7 proteins are key factors in the oncogenic transformation and malignant lesions of HPV-infected cells.

[0012] In summary, HPV infection triggers changes in host cell gene expression, involving the activation of numerous signaling pathways regulating apoptosis and the cell cycle, thereby conferring tumorigenic characteristics. Persistent HPV infection promotes chronic inflammation, leading to the production and release of reactive oxygen species (ROS), which in turn damage DNA and cause malignant transformation of infected cells. E5, E6, and E7 proteins promote cyclooxygenase 2 expression, triggering the release of large amounts of prostaglandins, which participate in cell proliferation, angiogenesis, and apoptosis inhibition, adversely affecting cervical tissue. Furthermore, E6 and E7 proteins inhibit the NF-κB signaling pathway, helping HPV evade immune surveillance. E7 protein also inhibits the expression of apoptosis genes Bcl-xL, Fas, and Bad, promoting the malignant proliferation of infected cells, supporting HPV replication, and thus promoting viral persistence. This chronic, persistent immune response manifests pathologically as cervical hyperplasia and ultimately leads to cervical cancer.

[0013] Because the initial development and subsequent progression of cervical cancer are completely dependent on the constitutive expression of two major HPV oncogenes, E6 and E7, the oncoproteins E6 and E7 serve as biomarkers for cervical cancer cells. If the expression and function of E6 and / or E7 are inhibited, tumor cell proliferation arrests and apoptosis occurs. When target antigens such as E6 and E7 are delivered to the body in various forms, MHC class I and class II molecules present intracellular and extracellular antigens, respectively, activating CD8+ T cells and CD4+ T cells, inducing specific cellular immune responses against HPV. Following intracellular antigen presentation, under the synergistic action of cytokines, effector CD8+ T cells kill HPV-infected cells or tumor cells expressing the target antigen genes. Therefore, the E6 and E7 proteins are ideal targets for the treatment of HPV-related inflammation and tumors, and have broad application development prospects.

[0014] 1.3 Global prevalence of HPV

[0015] HPV, short for human papillomavirus (HPV), is a non-enveloped, double-stranded circular DNA virus with high host specificity and affinity. It is primarily transmitted sexually, from mother to child, or through skin-to-skin contact. Eighty percent of sexually active women are infected with HPV. A molecular epidemiological survey found that HPV infection rates are highest in Africa and South America, reaching 22.9% and 18.6%, respectively. Globally, the most common subtype is high-risk HPV16, followed by HPV18, HPV58, HPV52, HPV31, HPV33, and HPV45. High-risk HPV DNA is found in nine malignancies, including oropharyngeal, cervical, vulvar, vaginal, penile, and anal cancers. According to Global Cancer Statistics 2020, global annual new cases of HPV-related cancers reached 852,620, accounting for 4.40% of all cancers, and annual deaths reached 447,900, accounting for 4.50% of all cancers. In 2022, an estimated 127,804 new cases of HPV-related cancers and 68,370 deaths were reported in China by gender. This has become a serious public health issue threatening women's health.

[0016] 1.4 Current status of treatment for HPV-related cancers

[0017] HPV-related oropharyngeal, cervical, vulvar, vaginal, penile, and anal cancers, once in their advanced stages, are incurable and difficult to remit, with no effective treatments currently available. While chemotherapy can provide some relief for these tumors, the remissions are typically short-lived.

[0018] Cervical cancer accounts for the highest proportion of HPV-related cancers, accounting for approximately 70%. Thanks to widespread cervical cancer screening and the promotion of HPV vaccines, cervical cancer has become largely preventable. However, the current five-year survival rate for cervical cancer patients remains only around 60%. For early-stage, non-metastatic cervical cancer, surgery and chemotherapy are the mainstays of treatment. For patients with FIGO stage IB-IIA disease and no lymph node metastasis, the five-year survival rate can reach 88%-95%. However, for metastatic or recurrent cervical cancer, traditional treatments have not been effective. While the use of targeted therapies and immunotherapies, such as anti-angiogenic drugs and immune checkpoint inhibitors, has significantly improved survival for these patients, a definitive cure remains elusive. Further molecular research is urgently needed to uncover new therapeutic targets and treatments to address these unmet clinical needs.

[0019] In the early stages of cervical cancer treatment, radical surgery is the mainstay, while in the late stages, radiotherapy is the mainstay. With the emergence of new chemotherapy drugs and chemotherapy approaches, concurrent radiotherapy and chemotherapy have become the standard treatment for mid- to late-stage cervical cancer, but local recurrence remains high. Approximately 29% to 38% of cervical cancer patients relapse after treatment, making recurrent cervical cancer a focus of clinical attention. Chemotherapy options for recurrent cervical cancer are very limited, and most cases of recurrent cervical cancer are incurable. According to the Gynecologic Oncology Group (GOG), the best treatment for recurrent or metastatic cervical cancer is a combination of cisplatin, paclitaxel, and bevacizumab, with an overall response rate (RR) of 48% and a median survival of 17 months. However, due to the poor prognosis of this group of patients, there is no consensus on the benefits of second-line chemotherapy for recurrent cervical cancer.

[0020] Immunotherapy is a new approach to treating cancer that relies on the immune system's ability to directly recognize and kill tumor cells. In recent years, several immunotherapy strategies for cervical cancer have achieved significant breakthroughs.

[0021] therapeutic vaccines

[0022] Currently approved prophylactic HPV vaccines are highly effective in preventing HPV infection but appear ineffective in clearing established HPV infection. Research is ongoing on novel therapeutic vaccines for cervical dysplasia and cervical cancer. Preclinical trials using either Pseudomonas exotoxin-HPV16 E70KDEL3 fusion protein or TVGV-1 in combination with two different adjuvants have shown preliminary efficacy in mouse models, associated with prolonged survival and proliferation of HPV16 E7-specific CD8+ T cells. Furthermore, preliminary results have been reported for the therapeutic vaccine pNGVL4a-CRT / E7 in patients with CIN 2 / 3 secondary to HPV16 infection. This vaccine consists of a DNA plasmid (pNGVL4a-A) encoding calreticulin associated with a detoxified form of the HPV16 E7 antigen. This vaccine is designed to increase cytotoxic T cells with the goal of clearing established HPV16 infection. In this first-in-human clinical trial, 27 women received the vaccine, and 30% experienced resolution to CIN 1 or lower, with an increase in cytotoxic T cells compared to the control group.

[0023] Immune checkpoint therapy

[0024] Tumor expression of PD-L1 allows the tumor to evade cellular destruction by CD8+ cytotoxic T cells. Multiple studies have shown that PD-L1 expression is associated with poor prognosis, independent of other influencing factors including stage, tumor size, depth of invasion, lymphatic space invasion, or lymph node metastasis.

[0025] Additionally, checkpoint inhibitors such as CTLA-4, LAG-3, VISTA, and TIGIT are being tested as single agents or in combination with other checkpoint inhibitors for the potential treatment of cervical cancer.

[0026] T cell adoptive therapy

[0027] Adoptive T-Cell Therapy (ACT) involves modifying autologous T cells in vitro, culturing and proliferating them, and specifically recognizing tumor cells. It then massively expands the cells with anti-tumor reactivity and infuses them back into cancer patients, potentially mediating the lasting and complete regression of certain advanced malignant tumors.

[0028] E7 is the main oncogenic protein of HPV. Its overexpression in host cells is a key factor in the occurrence, development and metastasis of cervical cancer. At the same time, as an exogenous viral antigen, it is not expressed in normal tissues. Therefore, TCR-T targeting HPV E7 is an ideal target for the treatment of HPV-related advanced malignancies and has broad application and development prospects.

[0029] Because solid tumors have a wide range of active immune evasion strategies, repeated antigen stimulation and a suppressive tumor microenvironment contribute to T cell exhaustion and compromise the effectiveness of immunotherapy. HPV-targeted anti-exhaustion TCR-T cells are a newly developed, independently developed, anti-exhaustion TCR-T therapy. They transduce a TCR targeting HPV E7 and an anti-exhaustion fusion receptor into T cells, killing tumor cells and simultaneously clearing viral infection by targeting virus-specific antigen expression and DNA integration. The anti-exhaustion fusion receptor acts as a component that counteracts the tumor microenvironment and alleviates immunosuppression, enabling TCR-T cells to continuously exert their tumor-killing and proliferation capabilities, thereby inducing a sustained anti-tumor immune response. This therapy is suitable for a variety of malignancies caused by human papillomavirus (HPV), including cervical cancer, oropharyngeal cancer, head and neck cancer, vaginal cancer, vulvar cancer, and penile cancer.

[0030] LN-145 is an ongoing clinical trial (NCT03108495), which is a product extracted and expanded from TILs. These TILs are then fused after myeloablative chemotherapy, and complete and durable responses have been observed with this type of treatment. Of 18 evaluable patients with cervical cancer, 5 patients had responses, including 2 complete responses. Another Phase I clinical trial (NCT02280811) using T cells genetically engineered with T cell receptors that recognize HLA-A*02:01-restricted HPV-16 oncoproteins showed responses in patients with HPV16-positive anal canal cancer, providing preliminary evidence of its efficacy.

[0031] In summary, due to the limitations and risks of currently available treatments and drugs, new treatment strategies are needed to provide more treatment options for patients with HPV-related cervical cancer.

[0032] 1.5 The role of T cells in HPV-related tumor immunotherapy and the basic principles of using TCR-T cells

[0033] T cells are immune cells that originate from the bone marrow and lymph nodes and mature in the thymus. They express T cell antigen receptors (TCRs) on their surface and play a crucial role in clearing infections and cancer cells in cell-mediated immunity. TCRs recognize and specifically bind to target antigen epitopes presented by major histocompatibility complex (MHC) molecules. Once a T cell recognizes its target, it kills the target cell through massive proliferation, cytokine release, and cytotoxicity.

[0034] When it comes to the scope of application of TCR-T therapy, people are currently trying to use T cell adoptive immunotherapy to treat human malignancies (such as leukemia) and viral diseases (such as hepatitis B virus (HBV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV). However, it is very difficult and time-consuming to isolate and amplify virus- or tumor-specific T cells from the patient's blood. Therefore, researchers have adopted a new treatment strategy by introducing T cell receptors (TCR) or TCRα / β heterodimers targeting specific antigens, allowing these gene-edited T lymphocytes to act on specific viruses or tumor antigens, giving T cells clear antigen specificity. The application of TCR-T in solid tumors, especially cervical cancer associated with HPV infection, has high clinical application value and significance.

[0035] TCR-T cell therapy relies on the presentation of the histocompatibility complex (MHC molecules) to identify targets and activate T cell function. It can recognize intracellular antigen fragments presented by MHC molecules. This feature determines that TCR-T cell therapy has a wider target range, including intracellular antigens, cell surface antigens, and new antigens produced after tumor cell mutations. It can overcome the differences in antigen expression of malignant tumor cells caused by tumor heterogeneity and embody higher therapeutic value.

[0036] Studies have shown that gene-edited T cells expressing HPV-specific T cell receptors (TCRs) can specifically recognize HPV16 E7-related cervical cancer cells, become activated and proliferate, and effectively eliminate HPV16 E7-positive tumor cells. Therefore, the HPV16 E7 protein is a therapeutic target for HPV-related cancers.

[0037] 1.6 T cell exhaustion and customer service strategies

[0038] T cell exhaustion is a persistent and often poorly understood cause of cell therapy efficacy in solid tumors. Common in malignant tumors, T cell exhaustion is a dysfunctional condition caused by prolonged exposure to persistent antigenic stimulation. This refers to the eventual loss of effector function after sustained T cell activity, manifested by defective production of tumor necrosis factor (TNF) and interferon-γ (IFN-γ), a mechanism by which malignant tumor cells evade the immune system.

[0039] Studies have found that the binding of programmed cell death-ligand 1 (PD-L1) expressed by tumor cells to programmed death receptor 1 (PD-1) on lymphocytes is an important signaling pathway leading to lymphocyte exhaustion. The exhausted T cell phenotype can be restored by antagonizing the PD-1 / PD-L1 interaction. This conclusion has important implications for resolving TCR-T exhaustion and improving the efficacy of solid tumors.

[0040] During tumor progression, tumor cells, mesenchymal fibroblasts, and other cells in the tumor microenvironment secrete large amounts of transforming growth factor-β (TGF-β). On the one hand, by secreting large amounts of TGF-β, tumor cells expose themselves to a high concentration of TGF-β, thereby establishing tolerance. On the other hand, TGF-β suppresses the anti-tumor activity of immune cells, allowing tumor cells to evade immune surveillance and further promote tumor progression. Most malignant tumors employ a wide range of active immune evasion strategies, suppressing the immune cell-mediated killing of tumor cells by producing TGF-β. Antagonizing TGF-β downstream signaling pathways has the potential to inhibit the immunosuppressive effects caused by the presence of TGF-β in the tumor microenvironment, preventing the weakening and exhaustion of T cell immune killing capacity, thereby further improving therapeutic efficacy. It also aims to prolong the survival of TCR-T cells in the body and promote T cell infiltration, addressing the pain points of TCR-T cell therapy, such as insufficient durability, suboptimal treatment efficacy, and the prevention of tumor recurrence, and better meet the needs of patients. Summary of the Invention

[0041] In view of the existing technical problems, the present application provides a modified immune cell that targets the specific HPV E7 antigen and can overcome the immune cell exhaustion caused by the immunosuppression of the tumor microenvironment. It can specifically kill cancer cells such as cervical cancer, oropharyngeal cancer, head and neck cancer, vaginal cancer, vulvar cancer, penile cancer, and anal cancer induced by HPV infection, thereby treating HPV-induced cervical cancer, oropharyngeal cancer, head and neck cancer, vaginal cancer, vulvar cancer, penile cancer, and anal cancer.

[0042] The first aspect of the present application provides a modified immune cell comprising a T cell receptor targeting HPV E7 antigen (HPV TCR) and a conversion stimulating molecule.

[0043] In some embodiments, the HPV TCR comprises a TCR α chain variable domain and a TCR β chain variable domain; wherein the amino acid sequence of the αCDR3 of the TCR α chain variable domain is as shown in SEQ ID NO: 3, or a variant thereof, in which one or two amino acids are replaced by other amino acids; and the amino acid sequence of the βCDR3 of the TCR β chain variable domain is as shown in SEQ ID NO: 6, or a variant thereof, in which one or two amino acids are replaced by other amino acids.

[0044] In some embodiments, the TCR α chain variable domain comprises complementarity determining regions αCDR1, αCDR2 and αCDR3, and the TCR β chain variable domain comprises complementarity determining regions βCDR1, βCDR2 and βCDR3, wherein: αCDR1, αCDR2 and αCDR3 are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; βCDR1, βCDR2 and βCDR3 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; or comprises the above-mentioned CDR variants, wherein one or two amino acids in one or more CDRs are replaced by other amino acids.

[0045] In some embodiments, the TCR α chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7; in some embodiments, the TCR β chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8.

[0046] In some embodiments, the HPV TCR comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the conversion stimulatory molecule comprises: an extracellular domain (ECD) of an immunosuppressive protein, wherein the ECD is fused to an intracellular domain (ICD) of a costimulatory molecule that mediates an immune cell activation signal; wherein the extracellular domain of the immunosuppressive protein binds to its ligand to generate an immune cell activation signal rather than an immune cell inactivation signal in the modified immune cell.

[0047] In some embodiments, the immunosuppressive protein is selected from any one or a combination of PD-1, CTLA4, BTLA, TIM3, TIGIT, TGFβ receptor and any other protein having immunosuppressive function or associated with an immunosuppressive signaling pathway, and the ECD sequence of the immunosuppressive protein may have at least one amino acid mutation.

[0048] In some embodiments, the ECD of the present application is TGFβ receptor II ECD; in some embodiments, the TGFβ receptor II ECD sequence may have at least one amino acid mutation; in some embodiments, the TGFβ receptor ECD amino acid sequence is as shown in SEQ ID NO:12.

[0049] In some embodiments, the ECD of the present application is PD-1 ECD; in some embodiments, the PD-1 ECD sequence may have at least one amino acid mutation; in some embodiments, the PD-1 ECD has an amino acid mutation, and the alanine at position 132 is mutated to leucine, which increases the affinity of PD-1 ECD to PDL1, and the ECD amino acid sequence is shown in SEQ ID NO:9.

[0050] In some embodiments, the costimulatory molecule comprises any one of CD28, 4-1BB, ICOS, CD27, IL-12R, CD3, and OX40 proteins, or a combination thereof, and the ICD sequence of the costimulatory molecule may have at least one amino acid mutation.

[0051] In some embodiments, the ICD of the present application is 4-1BB ICD; in some embodiments, the 4-1BB ICD sequence may have at least one amino acid mutation; in some embodiments, the 4-1BB ICD amino acid sequence is as shown in SEQ ID NO: 11.

[0052] In some embodiments, the TCR comprises a TCR alpha chain and a TCR beta chain, the TCR alpha chain variable domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; and / or

[0053] The TCR beta chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8.

[0054] In some embodiments, the ECD and the ICD are connected by a transmembrane region (TM) sequence; in some embodiments, the transmembrane region comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, and combinations thereof, and the transmembrane domains of the above proteins may have at least one amino acid mutation.

[0055] In some embodiments, the transmembrane region sequence is a CD8 transmembrane region sequence; in some embodiments, the CD8 transmembrane region sequence is shown in SEQ ID NO:10.

[0056] In some embodiments, the modified immune cell expresses the following exogenous polypeptides:

[0057] an AT cell receptor (TCR) that specifically targets the E7 antigen of HPV; and

[0058] B. A conversion stimulatory molecule, comprising: an extracellular domain (ECD) and an intracellular domain (ICD), wherein the ECD is selected from the ECD of an immunosuppressive molecule, and the ICD is selected from the ICD of a co-stimulatory molecule.

[0059] In one embodiment, the TCR is capable of binding to the HPV E7 antigen polypeptide presented by HLA-A*02, more preferably, the TCR is capable of binding to the HPV E7 antigen polypeptide presented by HLA-A*02:01; the surface antigen polypeptide comprises the amino acid sequences 11-19 of HPV16 E7 and HPV52 E7, and their corresponding amino acid sequences are YMLDLQPET or YILDLQPET, respectively.

[0060] In some embodiments, the conversion co-stimulatory molecule is PD1(ECD)-CD8(TM)-4-1BB(ICD)(PD1-BB); in some embodiments, the PD1-BB amino acid sequence is as shown in SEQ ID NO:14; in some embodiments, the conversion co-stimulatory molecule is TGFβ receptor (ECD)-CD8(TM)-4-1BB(ICD)(TGF-BB); in some embodiments, the TGFβ-BB amino acid sequence is as shown in SEQ ID NO:16.

[0061] In some embodiments, the modified immune cell comprises an HPV TCR and a conversion costimulatory molecule having a structure of PD1 (ECD) -CD8 (TM) -4-1BB (ICD) (PD1-BB); preferably, the modified immune cell comprises the amino acid sequence shown in SEQ ID NO: 15. Preferably, the modified immune cell comprises an HPV TCR and a conversion costimulatory molecule having a structure of TGFβ receptor (ECD) -CD8 (TM) -4-1BB (ICD) (TGF-BB); in some embodiments, the modified immune cell comprises the amino acid sequence shown in SEQ ID NO: 17.

[0062] In some embodiments, the immune cell is selected from lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, and mast cells; in some embodiments, the immune cell is a T cell.

[0063] The second aspect of the present application provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present application; and / or comprising a nucleic acid sequence encoding the conversion co-stimulatory molecule described in the first aspect of the present application.

[0064] In some embodiments, the HPV TCR is encoded by the nucleic acid sequence shown in SEQ ID NO:18.

[0065] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an HPV TCR molecule and a nucleic acid sequence encoding a switching co-stimulatory molecule of PD1-BB; in some embodiments, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 19.

[0066] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an HPV TCR molecule and a nucleic acid sequence encoding a switching co-stimulatory molecule having a structure of TGF-BB; in some embodiments, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 20.

[0067] The third aspect of the present application provides a vector, wherein the vector comprises the nucleic acid molecule described in the second aspect of the present application.

[0068] The fourth aspect of the present application provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the modified immune cells described in the first aspect of the present application, the nucleic acid molecules described in the second aspect of the present application, and the vector described in the third aspect of the present application.

[0069] The fifth aspect of the present application provides the use of the modified immune cells described in the first aspect of the present application, the nucleic acid molecules described in the second aspect of the present application, the vector described in the third aspect of the present application, or the pharmaceutical composition described in the fourth aspect of the present application in the preparation of a drug for preventing or treating related diseases caused by HPV infection; the HPV infection-related diseases include various malignant tumors caused by HPV, including one or more of cervical cancer, oropharyngeal cancer, head and neck cancer, vaginal cancer, vulvar cancer, penile cancer and anal cancer.

[0070] In a sixth aspect, the present application provides a T cell receptor or a fragment thereof targeting the HPV E7 antigen, wherein the T cell receptor comprises a TCR α chain variable domain and a TCR β chain variable domain selected from the following group:

[0071] (1) the TCR α chain variable domain comprises the following three CDRs: αCDR1 shown in SEQ ID NO: 1, αCDR2 shown in SEQ ID NO: 2, and αCDR3 shown in SEQ ID NO: 3, or CDR variants thereof in which one or two amino acids are replaced; and

[0072] (2) The TCR β chain variable domain comprises the following three CDRs: βCDR1 shown in SEQ ID NO: 4, βCDR2 shown in SEQ ID NO: 5, and βCDR3 shown in SEQ ID NO: 6, or the above CDR variants in which one or two amino acids are replaced.

[0073] In some embodiments, the TCR α chain variable domain comprises the following three CDRs: αCDR1 shown in SEQ ID NO: 1, αCDR2 shown in SEQ ID NO: 2, and αCDR3 shown in SEQ ID NO: 3; and

[0074] The TCR β chain variable domain comprises the following three CDRs: βCDR1 shown in SEQ ID NO: 4, βCDR2 shown in SEQ ID NO: 5, and βCDR3 shown in SEQ ID NO: 6.

[0075] In some embodiments, the TCR alpha chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7; and / or the TCR beta chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8.

[0076] In some embodiments, the T cell receptor or fragment thereof binds to an HPV E7 antigen polypeptide presented by HLA-A*02, more preferably, an HPV E7 antigen polypeptide presented by HLA-A*02:01. In some embodiments, the polypeptide comprises the amino acid sequence YMLDLQPET or YILDLQPET.

[0077] The seventh aspect of the present application provides a nucleic acid molecule, which comprises a T cell receptor encoding the HPV-targeted E7 antigen or a fragment thereof or a complementary sequence thereof as described in the sixth aspect of the present application.

[0078] The eighth aspect of the present application provides a vector, which comprises the nucleic acid molecule described in the seventh aspect of the present application, wherein the vector is selected from a plasmid, a binary vector, a DNA vector, an mRNA vector, a retroviral vector, a lentiviral vector, a transposon-based vector and an artificial chromosome.

[0079] The ninth aspect of the present application provides an isolated polypeptide, which is encoded by the nucleic acid molecule described in the seventh aspect of the present application or the vector described in the eighth aspect of the present application.

[0080] The tenth aspect of the present application provides an isolated cell, wherein the cell contains the T cell receptor or fragment thereof described in the sixth aspect of the present application, the nucleic acid molecule described in the seventh aspect of the present application, the vector of the eighth aspect of the present application, or the polypeptide described in the ninth aspect of the present application. Preferably, the cell contains HPV E7_SCG61 TCR, and the amino acid sequence of the HPV E7_SCG61 TCR is shown in SEQ ID NO: 13.

[0081] The eleventh aspect of the present application provides a pharmaceutical composition, characterized in that the composition contains a pharmaceutically acceptable carrier and the T cell receptor or fragment thereof described in the sixth aspect of the present application, the nucleic acid molecule described in the seventh aspect of the present application, the vector described in the eighth aspect of the present application, or the polypeptide described in the ninth aspect of the present application, or the cell described in the tenth aspect of the present application.

[0082] Use of the T cell receptor or fragment thereof described in the sixth aspect of the present application, the nucleic acid molecule described in the seventh aspect of the present application, the vector described in the eighth aspect of the present application, the polypeptide described in the ninth aspect of the present application, the cell described in the tenth aspect of the present application, or the pharmaceutical composition described in the eleventh aspect of the present application in the preparation of a medicament for preventing or treating related diseases caused by HPV infection. In some embodiments, the HPV infection-related disease comprises a variety of malignant tumors caused by human papillomavirus (HPV), including one or more of cervical cancer, oropharyngeal cancer, head and neck cancer, vaginal cancer, vulvar cancer, penile cancer, and anal cancer.

[0083] Beneficial effects of this application:

[0084] An anti-exhaustion TCR-T product targeting the E7 antigen of HPV-16 and HPV-52 related tumor cells. Structurally, HPV-targeted enhanced TCR-T cells can simultaneously express specific TCR and PD-1 or TGFβRII co-stimulatory fusion receptors. Among them, the TCR receptor is MHC restricted (only for HLA-A*02:01) and can specifically recognize the HPV-16 / 52E7 viral peptide presented by HLA-A*02:01, produce cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), etc., and play a role in cell lysis of tumor cells. HPV-targeted enhanced TCR-T cells contain PD-1 or TGFβRII co-stimulatory domain fusion receptors, and the intracellular activation signal domain replaces the intracellular inhibitory signal domain of the original inhibitory receptor. The purpose is to convert T cell inhibitory signals into activation signals to increase cell proliferation ability, cytokine release, etc., and increase the anti-exhaustion ability of TCR-T cells.

[0085] The use of the enhanced TCR-T cells targeting HPV-16 E7 of the present invention to treat HPV-related solid tumors has the following main advantages:

[0086] (a) Anti-tumor therapies targeting HPV E7 can overcome the protective effect of the tumor barrier on solid malignant tumor tissue. The tumor barrier is a self-protective mechanism of tumors that restricts the entry of immune effector cells into tumor tissue. Unlike TAA targets, the precancerous lesions caused by chronic HPV infection form an area of ​​high expression of HPV E7 targets between normal tissue and tumor tissue. These targets can guide the corresponding innate immune cells to infiltrate the tumor and exert a killing effect, thereby overcoming the tumor barrier.

[0087] (b) HPV E7 is a foreign antigen not expressed in normal tissues. TCR-T therapy has mild CRS and ICANS reactions and is well tolerated. Major adverse reactions are primarily due to the drug attacking normal tissue cells outside the tumor that express the corresponding target. Therefore, HPV-targeted enhanced TCR-T cells have a superior safety profile.

[0088] (c) HPV-targeted enhanced TCR-T cells can recognize HPV E7 peptides that meet the HLA-A*02:01 matching, regardless of whether the intact E7 antigen is expressed. Studies have shown that HPV-targeted enhanced TCR-T cells have a significant inhibitory effect on the growth of CDX cervical cancer and head and neck squamous cell carcinoma xenografts expressing HPV-16 E7 in immunodeficient mice, and this effect is dose-dependent.

[0089] (d) The present inventors successfully screened and obtained an all-natural HPV-specific TCR with ultra-high affinity and modified the TCR constant region to reduce the risk of mismatch with endogenous TCR chains.

[0090] (e) Enhanced TCR-T cells targeting HPV can simultaneously recognize HPV-16 and HPV-52 genotype-related cervical cancer, head and neck cancer and other HPV-related tumors, significantly improving the coverage of the patient population.

[0091] (f) Targeting the costimulatory molecule fusion receptor carried by HPV-enhanced TCR-T cells can also reverse T cell exhaustion mediated by signaling pathways, allowing TCR-T cells to continue to exert their anti-tumor effects. Antagonizing the downstream immunosuppressive signaling pathways prolongs TCR-T cell survival in vivo and promotes T cell infiltration, addressing the pain points of insufficient TCR-T cell therapy durability, poor therapeutic efficacy, and prevention of tumor recurrence, better meeting patient needs.

[0092] (g) Compared to chimeric switch receptor-enhanced CAR-T, enhanced TCR-T cells can recognize intracellular antigen fragments presented to the cell surface by MHC, which can overcome the differences in tumor heterogeneity in the expression of antigens on malignant tumor cells. In addition, TCR-T can recognize low-abundance tumor antigens. When paired with a chimeric switch receptor, while overcoming the inhibitory tumor microenvironment, it further enhances the proliferation and tumor-killing effects of CD8+ and CD4+ TCR-T cells, and promotes the long-term survival of memory T cells, ensuring the lasting and significant therapeutic effect of immune cell therapy, which is crucial for achieving breakthrough efficacy in solid tumors. In addition, the risk of cytokine release syndrome (CRS) in enhanced TCR-T cell therapy may be lower than that in enhanced CAR-T. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0094] Figure 1 is a schematic diagram of the SCG61 TCR structure;

[0095] Figure 2 is a TCR-T production flow chart;

[0096] Figure 3 shows the expression detection of SCG61 TCR-T cells infected with different MOIs;

[0097] Figure 4 shows the SCG61 TCR binding activity assay;

[0098] FIG5 shows the in vitro killing and cytokine secretion functions of SCG61 TCR on target cells;

[0099] Figure 6 shows the proliferation capacity of SCG61 TCR-T cells after multiple rounds of stimulation;

[0100] Figure 7 shows the recognition ability of SCG61 TCR-T for similar target epitopes;

[0101] Figure 8 shows the recognition ability of SCG61 TCR-T against peptides of different HPV subtypes;

[0102] Figure 9 shows the in vivo efficacy experiment of SCG61 TCR-T on CaSki xenograft tumor model in immunodeficient mice;

[0103] Figure 10 shows the sequence of SCG61-PD1-BB expressing HPV TCR and chimeric switch receptor structure;

[0104] Figure 11 shows the SCG61-PD1-BB TCR-T cell expression and phenotype detection;

[0105] Figure 12 shows SCG61-PD1-BB TCR-T cell proliferation;

[0106] FIG13 shows the in vitro killing function of SCG61-PD1-BB on target cells;

[0107] FIG14 shows the in vitro killing function of SCG61-PD1-BB against target cells of different HLA subtypes and HPV / PDL1±;

[0108] FIG15 shows the in vitro killing function of SCG61-PD1-BB against multiple rounds of tumor cell stimulation;

[0109] FIG16 shows the proliferation ability of SCG61-PD1-BB after multiple rounds of tumor cell stimulation;

[0110] FIG17 shows the in vivo efficacy study of SCG61-PD1-BB in a CaSki xenograft tumor model in immunodeficient mice;

[0111] FIG18 shows the in vivo efficacy study of SCG61-PD1-BB on the SCC-090 xenograft tumor model in immunodeficient mice;

[0112] Figure 19 shows the mismatch rate between SCG61-PD1-BB and endogenous TCR;

[0113] FIG20 shows the key amino acids recognized by SCG61-PD1-BB identified by alanine screening library technology;

[0114] FIG21 shows the functional affinity of SCG61-PD1-BB for T2 loaded with E7 polypeptide;

[0115] FIG22 shows the in vivo function of SCG61-PD1-BB in the CaSki xenograft tumor model in immunodeficient mice;

[0116] FIG23 shows the distribution of SCG61-PD1-BB in tumors, lungs, spleen and other tissues in mice;

[0117] FIG24 shows the sequence of SCG61-TGF-BB expressing HPV TCR and chimeric switch receptor structure;

[0118] Figure 25 shows the SCG61-TGF-BB TCR-T cell expression and phenotype detection;

[0119] Figure 26 shows the SCG61-TGF-BB TCR-T mismatch rate and memory exhaustion phenotype detection;

[0120] FIG27 shows the recognition ability of SCG61-TGF-BB TCR-T against different HPV subtype peptides;

[0121] Figure 28 shows the killing function of SCG61-TGF-BB TCR-T on target cells of different HPV subtypes;

[0122] Figure 29 shows the secretion of cytokines by SCG61-TGF-BB TCR-T cells against target cells of different HPV subtypes;

[0123] Figure 30 shows the inhibitory effect of SCG61-TGF-BB TCR-T on the pSMAD2 / 3 pathway;

[0124] Figure 31 is a functional validation of the affinity of SCG61-TGF-BB TCR-T for TGFβ1;

[0125] Figure 32 shows the target cell killing function of SCG61-TGF-BB TCR-T against TGFβ1;

[0126] FIG33 shows the in vitro killing function of SCG61-TGF-BB TCR-T cells after multiple rounds of tumor cell stimulation;

[0127] FIG34 shows the proliferation capacity of SCG61-TGF-BB TCR-T cells in response to multiple rounds of tumor cell stimulation;

[0128] FIG35 shows the cross-reactivity of SCG61-TGF-BB TCR-T to human polypeptide epitopes;

[0129] Figure 36 shows the in vitro killing function of the SCG61-TGF-BB TCR-T animal experimental batch on target cells;

[0130] FIG37 shows the in vivo killing function of SCG61-TGF-BB in the CaSki-E7 transplanted tumor model in immunodeficient mice;

[0131] FIG38 shows the in vivo expansion and persistence of SCG61-TGF-BB in the CaSki-E7 xenograft tumor model in immunodeficient mice;

[0132] FIG39 shows the in vivo killing function of SCG61-TGF-BB in the HepG2-E7 transplanted tumor model in immunodeficient mice;

[0133] FIG40 shows the in vivo expansion and persistence of SCG61-TGF-BB in the HepG2-E7 xenograft tumor model in immunodeficient mice. DETAILED DESCRIPTION

[0134] To facilitate understanding of the present invention, certain technical and scientific terms of the present invention are explained before describing the embodiments. Unless otherwise clearly defined elsewhere in the present application documents, all other technical and scientific terms used in the present invention have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0135] The term "HPV E7 antigen T cell receptor (TCR)" is defined herein as a TCR that binds to the HPV E7 surface antigen in the context of major histocompatibility complex (MHC) molecules to induce a helper or cytotoxic response in cells expressing the recombinant TCR. The TCR or fragment thereof of the present application is capable of recognizing a peptide corresponding to HPV-16 E7 that satisfies the HLA-A*02:01 matching, regardless of whether the intact E7 antigen is expressed.

[0136] The term "MHC molecule" refers to proteins in the immunoglobulin superfamily, either class I or class II. Therefore, they are specific for antigen presentation. Different individuals possess different MHCs, capable of presenting different short peptides from a protein antigen on their APC cell surfaces. The human MHC is often referred to as the HLA gene or HLA complex.

[0137] TCR is a glycoprotein on the surface of the cell membrane that exists in the form of a heterodimer of α chain / β chain or γ chain / δ chain. In 95% of T cells, TCR heterodimers are composed of α and β chains, while 5% of T cells have TCRs composed of γ and δ chains. Natural αβ heterodimeric TCRs have α chain and β chain, which constitute the subunits of αβ heterodimeric TCR. Each α and β chain contains a variable region and a constant region, and each variable region contains 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, embedded in the framework structure. The CDR regions of the α chain and β chain of the TCR of this application are delineated using the IMGT numbering rules. The CDR region determines the binding of the TCR to the pMHC complex. The sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT).

[0138] In this application, the terms "T cell receptor", "TCR", and "TCR molecule" are used interchangeably. TGFβRII and TGFβ receptor II have the same meaning and can be used interchangeably.

[0139] TCR molecules

[0140] The TCRs or fragments thereof disclosed herein recognize peptides corresponding to HPV-16 E7 that are HLA-A*02:01-matched. Approximately 50% of the population expresses the MHC class I molecule HLA-A*02. Therefore, HLA-A*02-restricted TCRs may have widespread therapeutic applications. Specifically, the TCRs disclosed herein can recognize products of multiple HLA-A*02 alleles, including HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0206, and HLA-A*0207. Although there can be significant differences in HLA gene subtypes between Caucasians and Asians; however, more than 95% of HLA-A2-positive Caucasians are HLA-A*0201; while HLA-A2-positive Chinese are composed of the following HLA-A2 subtypes: 23% HLA-A*0201; 45% HLA-A*0207; 8% HLA-A*0206; 23% HLA-A*0203.

[0141] In some embodiments, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, and the TCR alpha and beta chains each have three complementarity determining regions (CDRs).

[0142] In some embodiments, the amino acid sequence of the αCDR3 of the TCR α chain variable domain is as shown in SEQ ID NO: 3, or a variant thereof, in which one or two amino acids are replaced by other amino acids; and / or the amino acid sequence of the βCDR3 of the TCR β chain variable domain is as shown in SEQ ID NO: 6, or a variant thereof, in which one or two amino acids are replaced by other amino acids.

[0143] In some embodiments, the TCR α chain variable domain comprises complementarity determining regions αCDR1, αCDR2, and αCDR3, and the TCR β chain variable domain comprises complementarity determining regions βCDR1, βCDR2, and βCDR3, wherein:

[0144] αCDR1, αCDR2 and αCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and βCDR1, βCDR2 and βCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; or CDR variants in which one or two amino acids in one or more CDRs are replaced by other amino acids.

[0145] In some embodiments, the αCDR1, αCDR2 and αCDR3 are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and / or the βCDR1, βCDR2 and βCDR3 are shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0146] In some embodiments, the TCR comprises the amino acid sequence shown in SEQ ID NO:13.

[0147] The above-mentioned CDR region amino acid sequence of the present application can be embedded in any suitable framework structure to prepare a chimeric TCR. As long as the framework structure is compatible with the CDR region of the TCR of the present application, those skilled in the art can design or synthesize a TCR molecule with the corresponding function based on the CDR region disclosed in the present application. Therefore, the TCR molecule of the present application refers to a TCR molecule comprising the above-mentioned α and / or β chain CDR region sequence and any suitable framework structure. The TCR α chain variable domain of the present application is an amino acid sequence having at least 90%, preferably 95%, and more preferably 98% sequence identity with SEQ ID NO: 7; and / or the TCR β chain variable domain of the present application is an amino acid sequence having at least 90%, preferably 95%, and more preferably 98% sequence identity with SEQ ID NO: 8.

[0148] In some embodiments, the TCR is an αβ heterodimer comprising a TCRα chain constant domain and a TCRβ chain constant domain. In some embodiments, the constant domains of the TCR molecules of the present application are human constant domains. Those skilled in the art know or can obtain human constant domain amino acid sequences by consulting relevant books or the public database of IMGT (International Immunogenetics Information System). For example, the constant domain sequence of the α chain of the TCR molecule of the present invention can be "TRAC*01", and the constant domain sequence of the β chain of the TCR molecule can be "TRBC1*01" or "TRBC2*01". In some embodiments, the constant domain introduces an additional disulfide bond to improve stability and reduce mismatching between exogenously transferred TCR molecules and endogenous TCR molecules. The constant domains of the TCR molecules of the present application can also be mouse constant domains. Replacing TRAC and TRBC with mouse-derived constant domains simultaneously can avoid mismatching between exogenously transferred TCR molecules and endogenous TCR molecules, resulting in TCR targeting errors. This utility is similar to the purpose of exogenously introducing artificial disulfide bonds.

[0149] Converting co-stimulatory molecules

[0150] As used herein, the terms "switching stimulatory molecule" and "switching co-stimulatory molecule" are used interchangeably.

[0151] The conversion stimulatory molecule of the present application comprises: an extracellular domain (ECD) of an immunosuppressive protein, wherein the ECD is fused to an intracellular domain (ICD) of a co-stimulatory molecule that mediates immune cell activation signals; wherein the extracellular domain of the immunosuppressive protein binds to its ligand to produce an immune cell activation signal rather than an immune cell inactivation signal in the modified immune cells.

[0152] Proteins contained in immune cells that trigger immune cell inactivation signals after binding to their ligands include: immunosuppressive proteins including PD-1, CTLA4, BTLA, TIM3, TIGIT, TGFβ receptor and any other proteins with immunosuppressive functions or related to immunosuppressive signaling pathways, or any combination thereof, and the ECD of the above immunosuppressive proteins may have at least one amino acid mutation.

[0153] In some embodiments, the ECD of the present application is PD1 ECD; in some embodiments, the PD1 ECD sequence may have at least one amino acid mutation; in some embodiments, the PD-1 ECD has an amino acid mutation, and the alanine (A) at position 132 is mutated to leucine (L), which increases the affinity of PD-1 ECD to PDL1, and the ECD amino acid sequence is shown in SEQ ID NO:9.

[0154] In some embodiments, the ECD of the present application is a TGFβ receptor ECD; in some embodiments, the TGFβ receptor ECD sequence may have at least one amino acid mutation; in some embodiments, the TGFβ receptor ECD amino acid sequence is as shown in SEQ ID NO:12.

[0155] The costimulatory molecules of the present application include: any one of CD28, 4-1BB, ICOS, CD27, IL-12R, CD3, and OX40 proteins, or a combination thereof, and the ICD sequence of the above costimulatory molecules may have at least one amino acid mutation.

[0156] In some embodiments, the ICD of the present application is 4-1BB ICD; in some embodiments, the 4-1BB ICD sequence may have at least one amino acid mutation; in some embodiments, the 4-1BB ICD amino acid sequence is as shown in SEQ ID NO: 11.

[0157] In some embodiments, the TCR comprises a TCR alpha chain and a TCR beta chain, the TCR alpha chain variable domain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; and / or

[0158] The TCR beta chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8.

[0159] In some embodiments, the ECD and the ICD are connected by a transmembrane region (TM) sequence; the transmembrane region comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, and combinations thereof, and the transmembrane domains of the above proteins may have at least one amino acid mutation.

[0160] In some embodiments, the transmembrane region sequence is a CD8 transmembrane region sequence or a CD28 transmembrane region sequence; in some embodiments, the CD8 transmembrane region sequence is shown in SEQ ID NO:10.

[0161] In some embodiments, the conversion costimulatory molecule is PD1 (ECD) -CD8 (TM) -4-1BB (ICD) (PD1-BB); in some embodiments, the PD1-BB amino acid sequence is as shown in SEQ ID NO: 14; in some embodiments, the conversion costimulatory molecule is TGFβ (ECD) -CD8 (TM) -4-1BB (ICD) (TGF-BB); in some embodiments, the TGF-BB amino acid sequence is as shown in SEQ ID NO: 16.

[0162] Nucleic acid molecules

[0163] The nucleic acid molecule of the present application comprises a nucleic acid sequence encoding an HPV TCR molecule; and / or comprises a nucleic acid sequence encoding a conversion stimulating molecule.

[0164] The present application provides nucleic acid molecules encoding the TCR molecules described above or fragments thereof, wherein the fragments may be one or more CDRs, variable domains of α and / or β chains, and α and / or β chains.

[0165] In some embodiments, the nucleic acid encodes one or more structural features for increasing and / or stabilizing the association between the expressed TCR α and β chains. In some embodiments, the feature can be a specific amino acid or amino acid sequence. In some embodiments, the nucleic acid can encode one or more non-natural cysteine ​​residues for forming one or more disulfide bonds between the TCR α and β chains. In some embodiments, the nucleic acid can encode one or more non-natural cysteine ​​residues in the constant domains of the TCR α and β chains.

[0166] The nucleotide sequence of the nucleic acid molecule of the present application can be single-stranded or double-stranded, and the nucleic acid molecule can be RNA or DNA, and may or may not contain introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the present application does not contain introns but is capable of encoding the TCR of the present application and / or the conversion stimulating molecule of the present application.

[0167] The nucleotide sequence may be codon-optimized. Different cells utilize different codons, and expression can be increased by altering the codons in the sequence depending on the cell type. Codon usage tables for mammalian cells and various other organisms are well known to those skilled in the art.

[0168] In some embodiments, the coding sequence of the present application is a single chain, and the TCRβ chain coding sequence and the TCRα chain coding sequence are connected through the P2A coding sequence, and then connected to the costimulatory molecule coding sequence through the T2A coding sequence; and the single-chain coding nucleotides are in the same reading frame.

[0169] It should be understood that in gene cloning operations, it is often necessary to design appropriate restriction sites, which inevitably introduce one or more irrelevant residues at the end of the expressed amino acid sequence, but this does not affect the activity of the target sequence. In order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add certain amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein. For example, these include, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Therefore, the amino-terminus or carboxyl-terminus of the fusion protein of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used herein. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins.

[0170] In some embodiments, the HPV TCR is encoded by the nucleic acid sequence shown in SEQ ID NO: 18.

[0171] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an HPV TCR molecule and a nucleic acid sequence encoding a PD1-BB conversion co-stimulatory molecule; in some embodiments, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 19.

[0172] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an HPV TCR molecule and a nucleic acid sequence encoding a conversion co-stimulatory molecule having a structure of TGF-BB; in some embodiments, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 20.

[0173] carrier

[0174] The present invention also relates to vectors comprising the nucleic acid sequences described herein and one or more regulatory sequences operably linked to these sequences. The nucleic acid molecules of the present invention can be manipulated in a variety of ways to ensure expression of the fusion protein. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0175] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is usually operably linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, an untranslated region of an mRNA that is important for host cell translation. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in this application.

[0176] The nucleic acid molecules of the present application can be cloned into many types of vectors. For example, they can be cloned into plasmids, phagemids, phage derivatives, animal viruses and cosmids. Further, the vector is an expression vector. The expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include but are not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses.

[0177] Typically, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO 01 / 96584 and US Pat. No. 6,326,193).

[0178] For example, in certain embodiments, the present invention utilizes a lentiviral vector comprising an origin of replication, a 3' LTR, a 5' LTR, a polynucleotide sequence described herein, and optionally a selectable marker.

[0179] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence high-level expression that can be operably connected thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter and human gene promoter, such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, it is also possible to consider the use of inducible promoters. The use of inducible promoters provides a molecular switch that can open the expression of the polynucleotide sequence that can be operably connected to the inducible promoter when the deadline is expressed, and close expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0180] In order to assess the expression of the target gene, the expression vector introduced into the cell may also comprise any one or both of a selectable marker gene or a reporter gene so that from seeking to be transfected or infected cell mass by a viral vector, identification and selection of expressing cells can be performed. In other respects, selectable markers can be carried on a single section of DNA and used for cotransfection procedures. The flanks of selectable markers and reporter genes may all have suitable regulatory sequences so that expression in the host cell can be achieved. Useful selectable markers include, for example, antibiotic resistance genes, such as neo or the like.

[0181] The reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of the regulatory sequence. After DNA has been introduced into the recipient cells, the expression of the reporter gene is measured at the appropriate time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein genes. Suitable expression systems are known and can utilize known technology to prepare or commercially obtain.

[0182] Methods for introducing genes into cells and expressing genes in cells are known in the art. Vectors can be easily introduced into host cells, for example, mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0183] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0184] Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly lentiviral vectors, which have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0185] immune cells

[0186] The immune cells of the present application are selected from lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells. In some embodiments, the immune cells are lymphocytes; in some embodiments, the immune cells are NK cells; in some embodiments, the immune cells are B cells; in some embodiments, the immune cells are TIL cells. In some embodiments, the immune cells are T cells, which can be derived from T cells separated from the subject, or can be a part of a mixed cell population separated from the subject, such as a peripheral blood lymphocyte (PBL) group. For example, the cell can be separated from peripheral blood mononuclear cells (PBMC), can be CD4+ helper T cells or CD8+ cytotoxic T cells. The cell can be in a mixed group of CD4+ helper T cells / CD8+ cytotoxic T cells. Generally, the cell can be activated with antibodies (such as, anti-CD3 antibodies) to enable them to be more easily transfected.

[0187] The modified immune cells of the present application are immune cells comprising the above-mentioned TCR receptor molecules and the above-mentioned immune co-stimulatory molecules; in some embodiments, the modified immune cells are constructed by introducing the coding sequences encoding the above-mentioned TCR receptor molecules and the above-mentioned immune co-stimulatory molecules or vectors comprising the above-mentioned coding sequences into isolated immune cells; in some embodiments, the modified immune cells are constructed by introducing the coding sequences encoding the above-mentioned TCR receptor molecules and the above-mentioned immune co-stimulatory molecules or vectors comprising the above-mentioned coding sequences into immune cells in the body; in some embodiments, the coding sequences of the TCR receptor molecules and the immune co-stimulatory molecules are expressed in tandem and are in the same reading frame.

[0188] In some embodiments, the modified immune cells comprise HPV TCR and a conversion co-stimulatory molecule having a structure of PD1(ECD)-CD8(TM)-4-1BB(ICD)(PD1-BB); in some embodiments, the modified immune cells comprise the amino acid sequence shown in SEQ ID NO: 15.

[0189] In some embodiments, the modified immune cells comprise HPV TCR and a conversion co-stimulatory molecule having the structure of TGFβ receptor (ECD)-CD8(TM)-4-1BB(ICD)(TGF-BB); in some embodiments, the modified immune cells comprise the amino acid sequence shown in SEQ ID NO: 17.

[0190] Composition

[0191] The present application also provides compositions comprising the modified immune cells, nucleic acids, or vectors of the present invention. In some embodiments, the compositions are pharmaceutical compositions. In some embodiments, the compositions are compositions suitable for research, treatment, prevention, and / or diagnosis.

[0192] In some embodiments, the modified immune cells, nucleic acids or vectors of the present application are preferably formulated into a medicament or drug together with one or more other pharmaceutically acceptable ingredients known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings and sweeteners. The term "pharmaceutically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, etc., which are suitable for contacting the tissues of the subject in question (e.g., humans) without excessive toxicity, irritation, allergic reactions or other problems or complications, and are commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. Each carrier, adjuvant, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical textbooks, for example, Remington's Pharmaceutical Sciences; and Handbook of Pharmaceutical Excipients.

[0193] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease.

[0194] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered at a dose of E4 to E9 cells / kg body weight, preferably E5 to E7 cells / kg body weight. The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0195] Administration of the subject composition can be carried out in any convenient manner, including by nebulization, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally.

[0196] Medical uses

[0197] In another aspect, provided is a use of the modified immune cell, nucleic acid, vector or pharmaceutical composition of the present application in the preparation of a medicament for treating or preventing a disease or disorder.

[0198] In some embodiments, the modified immune cells, nucleic acids, vectors, or pharmaceutical compositions of the present application can be used to prevent or treat diseases caused by HPV infection. Diseases caused by HPV infection include one or more of cervical cancer, oropharyngeal cancer, head and neck cancer, vaginal cancer, vulvar cancer, and penile cancer.

[0199] Treatment and prevention methods

[0200] Treatment can be performed by isolating T cells from patients or volunteers suffering from HPV-related diseases, introducing the nucleic acid molecules or vectors of the present application into the above-mentioned T cells, and then returning these genetically engineered cells to the patient's body. Therefore, the present application provides a method for treating HPV-related diseases, comprising: returning separated T cells expressing the TCR of the present application, preferably, the T cells are derived from the patient himself, to the patient's body. Generally, it comprises (1) isolating the patient's T cells, (2) transducing the T cells in vitro with the nucleic acid molecules or vectors of the present application, and (3) returning the genetically engineered T cells to the patient's body. The number of cells separated, transfected, and returned can be determined by the physician.

[0201] In some embodiments of the present invention, the modified immune cells, nucleic acids, vectors, or pharmaceutical compositions of the present invention may be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiotherapy, immunosuppressants, and viral inhibitors. For example, treatment may be combined with nucleotide analogs or interferons known in the art for treating HPV-induced diseases.

[0202] "Patient," "subject," "individual," and the like are used interchangeably herein to refer to a living organism, such as a mammal, in which an immune response can be elicited. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

[0203] The technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application. It should be noted that the description order of the following embodiments does not limit the preferred order of the embodiments.

[0204] The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions such as those described in Sambrook et al. in Molecular cloning: A Laboratory manual (Molecular cloning-A Laboratory manual. 3rd edition. 2001), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise stated, the reagents and materials involved in the text are commercially available, or can be prepared by those skilled in the art based on common knowledge. Any methods and materials similar or equivalent to those described herein can be applied to this application. The preferred embodiments and materials herein are for demonstration purposes only and do not limit the content of this application.

[0205] Example 1: Construction of viral vector expressing SCG61 TCR

[0206] Through large-scale screening, the present inventors unexpectedly obtained a TCR that recognizes the corresponding HPV-16 E7 peptide segment with the HLA-A*02:01 matching. The TCR, named SCG61 TCR, comprises a TCR α chain variable domain and a TCR β chain variable domain, and the TCR α and β chains each have three complementarity determining regions (CDRs): αCDR1, αCDR2, and αCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; the βCDR1, βCDR2, and βCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0207] A schematic diagram of a preferred SCG61 TCR structure is shown in Figure 1. The coding sequence for the HPV TCR fragment was synthesized using a full-length gene. The synthesized gene was then double-digested and inserted into a lentiviral vector to construct a plasmid. The resulting vector was then packaged using lentiviral technology. The SCG61 TCR consists of the HPV E7 T cell receptor β chain variable region (E7 TCR Vβ), the T cell receptor β chain constant region (TCR Cβ), a 2A self-cleaving peptide, the HPV E7 T cell receptor α chain variable region (E7 TCR Vα), and the T cell receptor α chain constant region (TCR Cα). The TCR constant region has been sequence-modified to reduce endogenous TCR mispairing.

[0208] Example 2: Preparation of SCG61 TCR-T

[0209] Preparation of complete T cell culture medium: CTS TM OpTmizer TM +Supplement(Gibco,A379040-01)、5%CTS TMImmune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), 400 IU / mL IL-2 for injection (Shandong Quangang Pharmaceutical Co., Ltd., 08-102) were mixed by inversion and placed in a 4°C refrigerator for later use.

[0210] Preparation of T cell freezing solution: 75% CS10 (ThermoFisher, A2596101) + 25% HSA (FLEXBUMIN, S20181007).

[0211] The production process is shown in Figure 2;

[0212] Day 0: Pure CD3+ T cells were isolated from single blood samples and the cell concentration was adjusted to 1×10 6 / mL, according to Transact (CD3 / CD28 microspheres) (Macs, 6201000014): cell suspension = 1:30, add activator, gently mix thoroughly, stimulate culture for 24h, and then infect with different MOI viruses;

[0213] Day 1: Count cells and adjust the density of T cells to 5×10 5 / mL, add virus solution;

[0214] Day 2-11: After cell infection, the cell status was observed every day and T cell complete culture medium was added in time to maintain the T cell density at 5×10 5 / mL, which allows cells to expand;

[0215] Day 12: Harvest cells by centrifugation at 300 g for 5 minutes, wash the cells with physiological saline solution containing 5% human albumin, freeze them at an appropriate density using T cell freezing solution, freeze them in a programmed cooling device, and store them in liquid nitrogen.

[0216] Example 3: Detection of TCR-T cell expression at different MOIs of SCG61 infection

[0217] Flow cytometry buffer was prepared as follows: DPBS (Gibco, 14190250), 2% FBS (Gibco, 10099141), and stored in a 4°C refrigerator until ready for use.

[0218] SCG61 TCR-T cells and UT cells (control group) were washed once with flow cytometry buffer and the supernatant was discarded. PE HLA-A*02:01HPV E7 Tetramer (MBL, TB-0031-1) was added and incubated at 4°C in the dark for 60 min. The cells were then washed with flow cytometry buffer, resuspended, and detected by flow cytometry (Beckman CytoFLEX).

[0219] The results showed that at MOI = 0.3-8, the SCG61 TCR positivity rate ranged from 17% to 96%, and PD1 expression did not increase significantly ( Figure 3 ), indicating that SCG61 TCR expression was positively correlated with MOI and did not upregulate the expression of PD1 in the exhausted phenotype.

[0220] Example 4: SCG61 TCR binding activity detection

[0221] TCR affinity measures the strength of TCR-pMHC binding, while also accounting for the influence of other molecules (such as TCR co-receptors) in the interaction. Using mTCR to label TCR-positive cells, we then circled the TCR-positive cell population. These selected positive cells were then stained with E7 11-19pMHC tetramer at varying concentrations. The binding capacity of positive SCG142 cells to the E7 11-19peptide-MHC tetramer at these concentrations was examined. A clear positive correlation between the SCG61 TCR and tetramer concentrations was observed (Figure 4).

[0222] Example 5: In vitro killing of target cells and cytokine secretion by SCG61 TCR

[0223] The experiment used real-time cell analysis (RTCA) technology to evaluate the killing effect of SCG61 TCR on target cells. Human cervical cancer Caski cells (HPV-16 E7+) were cultured as target cells in 96-well RTCA plates. After approximately 16 hours of incubation, SCG61 TCR-T and UT were added for co-culture. The cells were then observed for 36 hours. The survival curves of the target cells were plotted as a continuous graph for comparison, and the co-culture supernatant was collected for cytokine analysis.

[0224] M10 cell complete culture medium was prepared as follows: DMEM (Gibco, 11965-092), 10% FBS (Gibco, 10099141), 1% Sodium Pyruvate (Gibco, 11360070), 1% HEPES (Gibco, 15630080), and 1% NEAA (Gibco, 11140-050). The culture medium was mixed by inversion and placed in a 4°C refrigerator until ready for use.

[0225] Complete culture medium for R10 cells was prepared as follows: RPMI1640 (Gibco, 22400-089), 10% FBS (Gibco, 10099141), mixed by inversion, and placed in a 4°C refrigerator for later use.

[0226] T cell culture medium preparation: CTS TM OpTmizer TM+Supplement(Gibco,A379040-01)、5%CTS TM Immune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), mix thoroughly by inversion, and place in a 4°C refrigerator until use.

[0227] SCG61 TCR-T cell killing (RTCA)

[0228] Day 0: Take well-growing human cervical cancer CaSki cells (R10 complete cell culture medium), digest and adjust the cell density to 4×10 5 / ml for standby use, take a 96-well plate with good collagen coating, add 50ul / well of corresponding culture medium for instrument baseline measurement, and then add 4×10 5 50ul of target cells / ml were placed in a static state for about 5 minutes before loading onto the instrument and the growth curve was continuously monitored for about 16 hours;

[0229] Day 1: Take SCG61 TCR-T and UT cells with measured positive rates and cell viability, calculate the number of effector cells based on the positive rates, add 50 μl of effector cells per well at effector-target ratios of 1:1 and 1:3, and continuously monitor the killing curve on the instrument. After 24 hours, collect the supernatant and measure cytokines.

[0230] Cytokine detection:

[0231] Take the cytokine kit Human Th1 / Th2 Cytokine Cytometric Bead Array Kit II (BD, 551809) and equilibrate to room temperature;

[0232] Pipette 2 ml of Assay Diluent to reconstitute the standard to a concentration of 5000 pg / ml and equilibrate at room temperature for 30 minutes;

[0233] Preparation of standard: Take the standard, marked as S1, and dilute the standards S2-S9 by 2 times in sequence, with S10 as blank;

[0234] Prepare Human Th1 / Th2 Cytokine Capture Beads mixture: Take microsphere solutions A1-A6, shake well, and mix in equal proportions.

[0235] Take a 96-well U-bottom plate and add 50ul of Human Th1 / Th2 Cytokine Capture Beads mixture to each sample well;

[0236] Add test samples and calibration curve samples S1-S10 at 50 μl / well;

[0237] Add 50ul of Human Th1 / Th2 PE Detection Reagent;

[0238] Incubate in the dark at room temperature for 180 min;

[0239] Add 100 μl of wash buffer, centrifuge at 300 g for 5 min, and discard the supernatant;

[0240] Resuspend with 100ul Wash buffer, load on flow cytometry, and analyze the results using FCAP Array v3 software.

[0241] The results showed that SCG61 TCR-T cells significantly cytotoxicized CaSki expressing HPV-16 E7+, with the cytotoxicity rate positively correlated with the effector-target ratio (Figure 5A). After 36 hours of co-culture, the E:T = 1:1 and E:T = 1:3 groups achieved nearly 100% cytotoxicity against positive target cells, and the IFN-γ release was significantly higher than that of the mock T group (Figure 5B).

[0242] Example 6: Proliferation ability of SCG61 TCR-T cells after multiple rounds of stimulation

[0243] The proliferation capacity of SCG61 TCR-T was studied through multiple rounds of tumor cell stimulation experiments.

[0244] CaSki (HPV-16 E7+) target cells were added to SCG61 TCR-T and UT cell culture dishes at a 1:1 ratio for stimulation, and the cells were counted every 2-3 days. After 5-7 days of stimulation, the cell weight of SCG61 TCR-T and UT was adjusted, and an equal amount of tumor cells was added. The above steps were repeated for a total of three rounds of stimulation.

[0245] Experimental data showed that after each round of tumor cell stimulation, the proliferation rate of SCG61 TCR-T cells was significantly higher than that of UT cells (Figure 6). SCG61 TCR-T cells were able to maintain sustained proliferation activity after multiple rounds of tumor stimulation.

[0246] Example 7: SCG61 TCR-T recognition ability of similar target epitopes

[0247] The experiment used T2 cells loaded with nonapeptides (HPV E7 11-19), (HPV E7 12-20) and decapeptides (HPV E7 11-20) and then co-incubated with SCG61 TCR-T. The IFN-γ content in the supernatant was detected to determine the recognition epitope of SCG61 TCR-T, providing support and basis for non-clinical safety studies.

[0248] Target cell preparation: Take T2 cells that have been passaged several times and are in good growth condition, centrifuge at 500g for 5 minutes, remove the supernatant, resuspend the cells in R10 medium, count the cells, take a 96-well U-bottom plate, add T2 cells at 100μl / well, and add 2×10 4 / well; add 11ul of the corresponding peptide solution at 10uM and 1uM respectively to make the final concentrations: 1uM and 100nM; place the 96-well U-bottom plate in a 37°C incubator and incubate for 2 hours, then add the effector cells.

[0249] Effector cell preparation: SCG61 TCR-T cells were homogenized and counted, and the cell density was adjusted to 4.0×10 5 / ml, add effector cells at 50μl / well, blow evenly, and place in a 37℃ incubator. After incubation for about 24 hours, the cells were collected and cytokine levels were analyzed by flow cytometry.

[0250] The results showed that using two peptide concentration gradients (1uM and 100nM), SCG61 TCR-T significantly secreted the cytokine IFN-γ to T2 cells loaded with E7 11-19 peptide, but had relatively weak recognition ability for E7 11-20 peptide. At the same time, no cytokine secretion was detected for the negative control group E629-38 peptide and E7 12-20 peptide (Figure 7), indicating that the recognition epitope of SCG61 TCR-T is HPV E7 11-19 nonapeptide.

[0251] Example 8: Recognition ability of SCG61 TCR-T for different HPV subtype peptides

[0252] HPV is a non-enveloped, double-stranded, circular DNA virus with high host specificity and affinity. This study focused on the globally high-risk subtypes HPV16, HPV31, and HPV52. T2 cells were loaded with varying concentrations of HPV16-E7, HPV31-E7, and HPV52-E7 peptides, and cytokine secretion was measured in the co-cultured cells to investigate whether the SCG61 TCR-T cell receptor (TCR) could recognize different HPV subtypes.

[0253] Co-incubation experiment of SCG61 TCR-T cells with T2 loaded with different subtypes of HPV-E7 peptides

[0254] Complete culture medium for R10 cells was prepared as follows: RPMI1640 (Gibco, 22400-089), 10% FBS (Gibco, 10099141), mixed by inversion, and placed in a 4°C refrigerator for later use.

[0255] T cell culture medium preparation: CTS TM OpTmizer TM +Supplement(Gibco,A379040-01)、5%CTS TM Immune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), mix thoroughly by inversion, and place in a 4°C refrigerator until use.

[0256] Target cell preparation: Take T2 cells that have been passaged several times and are in good growth condition, centrifuge at 500g for 5 minutes, remove the supernatant, resuspend the cells in R10 complete cell culture medium, count the cells, take a 96-well U-bottom plate, add T2 cells at 100μl / well, and add 2×10 4 / well, 100ul TCM culture medium was added to each well of the negative control group; the corresponding HPV16-E7, HPV31-E7, HPV52-E7, and HPV16-E6 peptide solutions were added according to the concentration gradient to make the final concentration: 10 -5 M~10 -12 M.

[0257] Effector cell preparation: SCG61 TCR-T cells were homogenized by centrifugation at 500 g for 5 min, and the supernatant was removed. The cells were resuspended in T cell culture medium and the cell density of SCG61 TCR-T positive cells was adjusted to 4.0 × 10 5 / ml, add effector cells at 50μl / well, blow evenly, and place in a 37℃ incubator. After incubation for 24 hours, collect the cell supernatant and analyze the cytokine production level by flow cytometry.

[0258] The results showed that the EC50 values ​​of SCG61 TCR-T for recognition of HPV16-E7, HPV31-E7, and HPV52-E7 peptides loaded on T2 cells at gradient concentrations of 10uM to 1pM were 2.4×10 -9 M, 1.8×10 -8 M and 6.8×10 -9 M (Figure 8). This indicates that SCG61 TCR-T has similar recognition abilities for high-risk HPV16 and HPV52, and better recognition than HPV31.

[0259] Example 9: In vivo efficacy study of SCG61 TCR-T on CaSki transplanted tumor model in immunodeficient mice

[0260] In order to understand the killing effect of SCG61 TCR-T on tumor cells in vivo, 25 NPG immunodeficient female mice were used in the experiment. The mice were inoculated with 5×10 6 Ten days later, the mice were randomly divided into five groups, three of which were SCG61TCR-T low-dose, medium-dose, and high-dose groups, and each group was injected with SCG61TCR-T via the tail vein at a dose of 2×10 6 , 6×10 6 , 2×10 7 Positive T cells / rat; negative control group received a single injection of 2×10 UT cells via tail vein 7 T cells / mouse; at the same time, mice in the model control group were injected once with an equal amount of the test substance vehicle via the tail vein. After administration, the mice were observed for 24 days. The long and short diameters of the tumors were measured twice a week, and the tumor volume was calculated.

[0261] On Day 24, compared with the model control group and the negative control group, the tumor volume of the SCG61 TCR-T low-dose group, the medium-dose group, and the high-dose group was significantly reduced, and the tumor inhibition rate was positively correlated with the administration dose (Figure 9).

[0262] The above results showed that SCG61 TCR-T could significantly inhibit the growth of subcutaneous transplanted tumors of human cervical cancer CaSki cells, especially at a high dose of 2×10 7 T cells / SCG61 TCR-T has the most significant effect in inhibiting tumors.

[0263] Example 10: Construction of viral vector expressing SCG61-PD1-BB

[0264] The structure of SCG61-PD1-BB is shown in Figure 10. The coding sequence of the HPV TCR-PD1-BB fragment was fully synthesized. The synthesized gene was double-digested and inserted into a lentiviral vector to construct a plasmid. The resulting vector was constructed and packaged using lentiviral technology. SCG61-PD1-BB consists of the HPV E7 T cell receptor β chain variable region (E7 TCR Vβ), the T cell receptor β chain constant region (TCR Cβ), a 2A self-cleavage peptide, the HPV E7 T cell receptor α chain variable region (E7 TCR Vα), the T cell receptor α chain constant region (TCR Cα), and the 2A self-cleavage peptide, the PD-1 extracellular domain, the CD8 transmembrane domain, and the 4-1BB intracellular domain.

[0265] Example 11: Flow cytometry detection of SCG61-PD1-BB TCR-T and PD-1 specific expression

[0266] Flow cytometry buffer was prepared as follows: DPBS (Gibco, 14190250), 2% FBS (Gibco, 10099141), and stored in a 4°C refrigerator until ready for use.

[0267] SCG61-PD1-BB TCR-T cells and mock T cells (control group) were obtained and washed once with flow cytometry buffer. The supernatant was discarded and PE HLA-A*02:01HPV E7 Tetramer (MBL, TB-0031-1), PE-Cy7 anti-human CD4 (BIOLEGEND, 300512) and PerCP / Cy5.5 anti-human CD8a (BIOLEGEND, 301032) were added. The cells were incubated in the dark at 4°C for 60 min, washed with flow cytometry buffer, resuspended, and finally detected by flow cytometry (Beckman CytoFLEX).

[0268] The results showed that the SCG61-PD1-BB TCR positivity rate was above 60%, and the proportions of CD4+ and CD8+ T cells were basically maintained at around 50%, with no difference compared with Mock T (Figure 11), indicating that SCG61-PD1-BB TCR can be stably expressed, and the newly added TCR and PD-1 sequences did not significantly affect the proportions of CD4+ and CD8+ T cells.

[0269] In addition, the in vitro expansion capacity showed that SCG61-PD1-BB could expand more than 100-fold around day 10 ( FIG12 ), indicating that TCR and PD-1 transfection did not affect the proliferation and viability of T cells.

[0270] Example 12: In vitro tumor cell killing ability of SCG61-PD1-BB

[0271] The experiment used real-time cell analysis (RTCA) technology to evaluate the killing effect of SCG61-PD1-BB on target cells. Human cervical cancer Caski cells (HPV-16 E7+ / PD-L1+), human head and neck squamous cell carcinoma SCC-090 cells (HPV-16 E7+ / PD-L1–), and human liver cancer HepG2 cells (HPV-16 E7– / PD-L1–) were cultured as target cells on 96-well RTCA plates. After incubation for approximately 16 hours, SCG61-PD1-BB and Mock T were added for co-culture. The cells were observed for 60 hours, and the survival curves of the target cells were plotted as continuous graphs for comparison. After the killing was completed, the co-culture supernatant was collected for cytokine detection.

[0272] SCG61-PD1-BB kills tumor cells (RTCA)

[0273] Day 0: Take well-grown human cervical cancer CaSki-Luci-GFP cells (R10 complete cell culture medium), human head and neck squamous cell carcinoma SCC-090 cells (M10 complete cell culture medium), and human liver cancer HepG2 cells (M10 complete cell culture medium), and adjust the cell density to 4×10 5 / ml for standby use, take a 96-well plate with good collagen coating, add 50ul / well of corresponding culture medium for instrument baseline measurement, and then add 4×10 5 50ul of target cells / ml were placed in a static state for about 5 minutes before loading onto the instrument and the growth curve was continuously monitored for about 16 hours;

[0274] Day 1: Take the SCG61-PD1-BB TCR-T and Mock T cells whose positive rate and cell viability have been measured, calculate the number of effector cells according to the positive rate, add 50ul / well of effector cells at the effector-target ratio of 5:1 and 1:1, and continuously monitor the killing curve on the instrument;

[0275] Day 3: Copy the RTCA growth and killing curves, stop the culture, and collect the co-culture supernatant for cytokine detection.

[0276] The results showed that SCG61-PD1-BB had a significant specific killing effect on both CaSki-Luci-GFP and HPV-16 E7+ / PD-L1+-expressing HPV-16 E7+ / PD-L1–SCC-090, and the killing rate was positively correlated with the effector-target ratio (Figure 13A). The half-killing time at an effector-target ratio of 5:1 was significantly shorter than that at an effector-target ratio of 1:1. After 40 hours of co-culture, the killing rate of positive target cells in the E:T = 5:1 and E:T = 1:1 groups was close to 100% (Figure 13B). The levels of released IFN-γ and TNF were significantly higher than those in the Mock T group (Figures 13C, D). At the same effector-target ratio, the half-life of SCG61-PD1-BB against CaSki cells that overexpress PD-L1 was significantly shorter than that against PD-L1-negative SCC-090 cells, and the concentration of released cytokines was positively correlated with the killing rate. These results suggest that SCG61-PD1-BB may have a stronger anti-tumor effect against HPV-16 E7+ tumor cells that overexpress PD-L1.

[0277] Example 13: The anti-tumor effect of SCG61-PD1-BB is HLA-A*02:01 and HPV specific

[0278] To verify whether SCG61-PD1-BB has different killing abilities in response to differences in tumor cell expression of HLA-A*02:01 and HPV-16 E7, four target cell lines, including HLA-A*02:01+ / HPV-16 E7+ / PD L1+ (CaSki cells), HLA-A*02:01+ / HPV-16 E7+ / PD L1– (SCC-090 cells), HLA-A*02:01+ / HPV-16 E7– / PD L1– (human cervical cancer C-33A cells), and HLA-A*02:01– / HPV-16 E7+ / PD L1– (human cervical cancer SiHa cells), were prepared for functional experimental detection ( Figure 14A ). First, SCG61-PD1-BB and Mock T cells were cultured with the above four target cells, and the killing effect of the target cells was observed using RTCA technology. After the killing was completed, the co-culture supernatant was collected for cytokine detection.

[0279] SCG61-PD1-BB kills tumor cells (RTCA)

[0280] Day 0: Take CaSki cells (R10 complete cell culture medium), SCC-090 cells (M10 complete cell culture medium), C-33A cells (M10 complete cell culture medium), and SiHa cells (M10 complete cell culture medium) in good growth state, and adjust the cell density to 4×10 5 / ml for standby use, take a 96-well plate for RTCA, add 50ul / well of the corresponding culture medium for instrument baseline measurement, and then add a density of 4×10 5 50ul of target cells / ml were placed in a static state for about 5 minutes before loading onto the instrument and the growth curve was continuously monitored for about 16 hours;

[0281] Day 1: Take the SCG61-PD1-BB TCR-T and Mock T cells whose positive rate and cell viability have been measured, calculate the number of effector cells according to the positive rate, add 50ul of effector cells per well at an effector-target ratio of 1:2, and continuously monitor the killing curve on the instrument;

[0282] Day 3: Copy the RTCA growth and killing curves, stop the culture, and collect the co-culture supernatant for cytokine detection.

[0283] The results showed that at an effector-target ratio of 1:2, SCG61-PD1-BB specifically recognized HLA-A*02:01+ / HPV-16 E7+ target cells, achieving a 24-hour killing rate exceeding 70%. Furthermore, SCG61-PD1-BB showed stronger killing effects against HPV-16 E7+ / PD L1+ cells than against HPV-16 E7+ / PD L1- cells (Figure 14B). However, SCG61-PD1-BB showed no significant killing effects against target cells expressing HLA-A*02:01+ / HPV-16 E7– and HLA-A*02:01– / HPV-16 E7+ (Figure 14B). Cytokine expression was highly consistent with the killing results (Figure 14C). This suggests that the anti-tumor effect of SCG61-PD1-BB is specific for HLA-A*02:01 and HPV-16 E7 expression.

[0284] SCG61-PD1-BB can specifically target and kill tumor cells expressing HLA-A*02:01 and HPV-16 E7 and release effector cytokines, but does not recognize and kill tumor cells that only express HLA-A*02:01 or HPV-16 E7; and because SCG61-PD1-BB can convert the inhibitory signal of PD-1 into an activation stimulation signal, the anti-tumor effect of SCG61-PD1-BB is more prominent for HPV-16 E7 cancer cells that highly express PD-L1.

[0285] Example 14: SCG61-PD1-BB retains killing and proliferation capabilities after multiple rounds of target cell stimulation

[0286] The ability of SCG61-PD1-BB to kill tumor cells against exhaustion was investigated through multiple rounds of tumor cell stimulation. RTCA technology was used to observe the killing effect on target cells. HPV-16 E7+ / PD-L1+ (CaSki) and HPV-16 E7+ / PD-L1– (SCC-090) target cells were plated on RTCA plates. After 24 hours of culture, SCG61-PD1-BB cells and mock T cells were added at an effector-target ratio (E:T) of 2:1. Four days after the killing, T cells were harvested and added to RTCA plates pre-plated with target cells for 24 hours for multiple rounds of killing. This procedure was repeated for a total of four rounds.

[0287] Experimental data showed that SCG61-PD1-BB still had a significant killing effect on CaSki and SCC-090 tumor cells after four rounds of tumor cell stimulation ( Figure 15 ).

[0288] CaSki (HPV-16 E7+ / PD-L1+) target cells were added to SCG61-PD1-BB and Mock T cell culture dishes at a 1:1 ratio for stimulation, and the cells were counted every 2-3 days. After 5-7 days of stimulation, the cell weights of SCG61-PD1-BB and Mock T cells were adjusted, and an equal amount of tumor cells were added. The above steps were repeated for a total of three rounds of stimulation.

[0289] Experimental data showed that after each round of tumor cell stimulation, the proliferation rate of SCG61-PD1-BB was significantly higher than that of Mock T ( Figure 16 ).

[0290] SCG61-PD1-BB incorporates a PD-1 co-stimulatory fusion receptor (PD-1 auxiliary sequence) to convert the braking signal (PD-L1) on the surface of tumor cell lines into a stimulatory signal, thereby promoting cell proliferation and preventing cells from prematurely entering a state of exhaustion. Results demonstrated that SCG61-PD1-BB can indeed maintain sustained anti-tumor effects and proliferation activity after multiple rounds of tumor stimulation (simulating an exhaustive environment for T cells).

[0291] Example 15: Efficacy of SCG61-PD1-BB on subcutaneous xenografts of human cervical cancer CaSki cells in NPG mice

[0292] To understand the killing effect of SCG61-PD1-BB on tumor cells in vivo, the experiment used 30 NPG immunodeficient female mice. On Day 7, the mice were inoculated with CaSki cells. The mice were randomly divided into five groups with tumor volumes ranging from 99.9 to 133.9 mm. 3 The three groups were SCG61-PD1-BB low, medium and high dose groups, and each group was injected with SCG61-PD1-BB via tail vein at a dose of 2×10 6 , 6×10 6 , 2×10 7 Positive T cells / mouse; negative control group received a single injection of 2×10 Mock T cells via tail vein 7 T cells / mouse; at the same time, mice in the model control group were injected once with an equal amount of the test substance vehicle via tail vein injection. After administration, the mice were observed for 4 weeks. The long and short diameters of the tumors were measured twice a week, and the tumor volume was calculated.

[0293] On Day 29, the tumor sizes of the model control group, negative control group, and SCG61-PD1-BB low-, medium-, and high-dose groups were 761±127 mm, respectively. 3 、674±101mm 3 、444±80mm 3 、466±69mm 3 and 206±119mm3 (Figure 17). From Day 8 to Day 29, although the tumor volume of animals in the negative control group was lower than that in the model control group, there was no statistical difference (P>0.05). Compared with the model control group, the tumor volume of the SCG61-PD1-BB low-dose group was significantly reduced from Day 15 to Day 25 (P<0.05). Compared with the model control group, the tumor volume of the SCG61-PD1-BB medium-dose group was significantly reduced from Day 22 and Day 25 (P<0.05). Compared with the model control group, the tumor volume of the SCG61-PD1-BB high-dose group was significantly reduced from Day 8 to Day 29 (P<0.01).

[0294] The above results showed that SCG61-PD1-BB could significantly inhibit the volume of subcutaneous transplanted tumors of human cervical cancer CaSki cells, especially at a high dose of 2×10 7 T cells / SCG61-PD1-BB had the most significant tumor inhibition effect.

[0295] Grouping of SCG61-PD1-BB efficacy test on CaSki cell NPG mouse subcutaneous xenograft tumors

[0296] “NA” means not applicable.

[0297] Example 16: Efficacy of SCG61-PD1-BB on subcutaneous xenografts of human head and neck squamous cell carcinoma SCC-090 cells in NCG mice

[0298] To understand the killing effect of SCG61-PD1-BB on tumor cells in vivo, 30 NCG immune-deficient female mice were used in the experiment. On Day 14, the right side of the mice were inoculated with 5×10 6 Human head and neck squamous cell carcinoma SCC-090 cells. Mice were randomly divided into five groups, and the tumor size was approximately 120 mm. 3 Three groups received different doses of SCG61-PD1-BB (2×10 6 cells / mouse, 6×10 6 cells / cell, 1.5×10 6 The remaining two groups served as control groups and received vehicle and Mock T, respectively. During the experiment, tumor volume and body weight were measured twice a week.

[0299] On Day 32 after administration, the tumor sizes of the model control group, negative control group, SCG61-PD1-BB low-dose, medium-dose, and high-dose groups were 1585±72mm, respectively. 3 , 555±72mm 3 , 98±34mm 3, 9±6mm 3 , 9±8mm 3 (Figure 18) The mean tumor volumes in the negative control group and the SCG61-PD1-BB low-, medium-, and high-dose groups were significantly lower than those in the vehicle control group (P < 0.01). The mean tumor volumes in the SCG61-PD1-BB low-, medium-, and high-dose groups were significantly lower than those in the negative control group (P < 0.01).

[0300] In summary, NCG mice bearing human head and neck squamous cell carcinoma SCC-090 cells were injected intravenously once with a dose of 0.2×10 7 ~1.5×10 7 Cells / SCG61-PD1-BB showed significant tumor-suppressing effects on mouse transplanted tumors.

[0301] Grouping of SCG61-PD1-BB efficacy test on SCC-090 cell subcutaneous xenograft tumors in NCG mice

[0302] “NA” means not applicable.

[0303] Example 17: Mismatch Rate of SCG61-PD1-BB Endogenous TCR

[0304] The antibody APC Hamster Anti-mTCRβ (BD, 553174) can detect all TCR chains expressed in the SCG61-PD1-BB cell product, while the PE HLA-A*02:01 HPV E7 Tetramer (MBL, TB-0031-1) detects the specific TCR in the SCG61-PD1-BB cell product. Therefore, these two antibodies were used to detect the SCG61-PD1-BB cell product, and the TCR mismatch rate result was calculated. SCG61-PD1-BB mismatch rate = [1-(E7 tetramer+ / mTCRβ+)] × 100%

[0305] The results showed that TCR expression was positively correlated with MOI (Multiplicity of Infection, which refers to the ratio of lentiviral vector to target T cells). This was demonstrated by the fact that at an MOI of 0.2, the TCR positivity rate of SCG61-PD1-BB was only 22.1%, while at an MOI of 5, the TCR positivity rate reached 79.2% (Figure 19). Furthermore, the TCR mismatch rate remained below 10% at MOIs of 0.2 to 5.

[0306] In summary, the newly added TCR and PD-1 sequences will basically not cause mismatches and lead to potential safety risks.

[0307] Example 18: Key amino acid recognition and affinity of SCG61-PD1-BB HPV-16 E7 epitope peptide

[0308] This study aimed to mutate each amino acid residue in the HPV-16 E7 epitope peptide to A (alanine) using an alanine screening library technique to identify the effect of a specific amino acid on TCR affinity and activity, thereby clarifying the recognition site of SCG61-PD1-BB within the target antigen peptide. T2 cells are HLA-A*02:01-positive T and B lymphocyte hybridoma cells. T2 cells were loaded with the mutated peptides, incubated at 37°C, and then SCG61-PD1-BB was added. After 24 hours, the supernatant was assayed for IFN-γ secretion.

[0309] The binding functional activity of SCG61-PD1-BB with the HPV-16 E7 epitope peptide-MHC complex in vitro can be evaluated by studying the ability of SCG61-PD1-BB to secrete cytokines after specifically recognizing T2 loaded with HPV-16 E7 epitope peptides of different concentration gradients.

[0310] Complete culture medium for R10 cells was prepared as follows: RPMI1640 (Gibco, 22400-089), 10% FBS (Gibco, 10099141), mixed by inversion, and placed in a 4°C refrigerator for later use.

[0311] T cell culture medium preparation: CTS TM OpTmizer TM +Supplement(Gibco,A379040-01)、5%CTS TM Immune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), mix thoroughly by inversion, and place in a 4°C refrigerator until use.

[0312] Peptide preparation: Dissolve 2 mg of each peptide in 190 μl of DMSO to a final concentration of 10 mM. Dilute 3 μl of the 10 mM solution to 100 μM using 297 μl of TCM. Repeat this process with a 10-fold serial dilution to 1 nM. See the table below for details.

[0313] Target cell preparation: Take T2 cells that have been passaged for several times and are in good growth condition, centrifuge at 500g / 5min, remove the supernatant, resuspend the cells in R10 complete cell culture medium, count the cells, and take 2.4×10 6 Resuspend the T2 cells in 12 ml TCM medium, take a 96-well U-bottom plate, add T2 cells at 100 μl / well, and 2×10 4 / well; add the corresponding peptide solution according to the concentration gradient to make the final concentration: 10 -5 M~10 -12 M; Place the 96-well U-bottom plate in a 37-degree incubator and incubate for 2 hours before adding the effector cells.

[0314] Effector cell preparation: SCG61-PD1-BB was homogenized and counted to 3.6×10 6 The cells were resuspended in 6 ml of T cell culture medium to adjust the cell density of SCG61-PD1-BB positive cells to 4 × 10 5 / ml, add effector cells at 50μl / well, blow evenly and place in a 37℃ incubator. After incubation for 24h, collect the co-culture supernatant and detect cytokine secretion by flow cytometry.

[0315] The results showed that cytokine secretion was significantly reduced after incubation of SCG61-PD1-BB with HPV-16 E7 peptides with amino acid mutations at positions 2 / 4 / 5 / 6 (Figure 20), confirming that the 2 / 4 / 5 / 6 recognition sites are the four key amino acids. These four key amino acid residues are believed to play a key role in the direct binding of TCR to HPV-16 E7 epitope peptides or in controlling the spatial structure of HPV-16 E7 epitope peptides.

[0316] T2 cell load 10 -5 M~10 -12 After being co-incubated with SCG61-PD1-BB in a concentration range of HPV-16 E7 epitope peptide, the IFN-γ secretion was positively correlated with the concentration of HPV-16 E7 epitope peptide loaded. However, Mock T cells could not recognize the HPV-16 E7 epitope peptide loaded on T2 cells. The half-maximal effect concentration (EC50) of SCG61-PD1-BB for recognizing the HPV-16 E7 epitope peptide was 6.76×10 -10 M (Figure 21). The results showed that SCG61-PD1-BB could recognize the key binding site of HPV-16 E7 epitope peptide and had strong binding functional activity.

[0317] Example 19: Expansion of SCG61-PD1-BB cells in CaSki cell-bearing NPG mice

[0318] In order to understand the data on the continuous expansion of SCG61-PD1-BB in the in vivo environment, the experiment constructed a CaSki cervical cancer cell NPG immunodeficient mouse model to simulate the in vivo expansion of SCG61-PD1-BB cells after receiving lymphoproliferative chemotherapy.

[0319] On Day 0, 18 mice were divided into three groups and received different doses of SCG61-PD1-BB (2×10 6 cells / mouse, 6×10 6 cells / cell, 2×10 7 The cells were injected into the tail vein of the mice, and the peripheral blood of the mice was collected every 7 days. The viral vector copy number (VCN) was detected by fluorescent quantitative PCR, and the data were collected and plotted into a graph showing the changes in the SCG61-PD1-BB content in the blood.

[0320] The experimental results showed that different doses of SCG61-PD1-BB cells could be significantly expanded in the animals. The values ​​of VCN in the blood of the three groups of mice on the 21st day were 40.35±37.88, 44.53±44.10, and 397.50±65.78 copies / μg gDNA, respectively (Figure 22). 7 The VCN content in the blood of mice with SCG61-PD1-BB cells was significantly higher than that in the other two dose groups.

[0321] The above data show that SCG61-PD1-BB can complete immune reconstitution in mice, persist and expand in the body for a long time, and the expansion of SCG61-PD1-BB in the body is dose-dependent.

[0322] Example 20: Tissue distribution of SCG61-PD1-BB cells in CaSki cell-bearing NPG mice

[0323] The experiment used CaSki cell-bearing NPG immunodeficient female mice. On Day 0, 2×10 SCG61-PD1-BB were injected into the tail vein of each mouse. 7 Whole blood and tissues were collected before administration and on Day 1, Day 7, Day 14, Day 21 and Day 28 after administration. The tissues included heart, liver, spleen, lung, kidney, tumor, brain, eyeball and optic nerve, ovary, uterus, bladder, stomach, skeletal muscle and tail vein injection site. Tissue distribution was studied by fluorescence quantitative PCR.

[0324] The results showed that, except for tumor tissue, the VCN of other tissues gradually decreased over time after transfusion; the VCN in tumor tissue was maintained at a relatively high level, reaching a peak of 8.22×10 4 copies / μg gDNA, then decreased, and began to increase gradually on Day 28, reaching 2.25×10 4SCG61-PD1-BB cells were exposed to a high concentration of 6.98 × 10 copies / μg gDNA in tumor tissue, lung, spleen, whole blood, injection site, and liver. The order from largest to smallest was tumor, lung, spleen, tail vein injection site, whole blood, liver, heart, left eyeball and optic nerve, and uterus. The VCNs were 6.98 × 10 5 , 5.04×10 5 , 2.87×10 5 , 9.84×10 4 , 6.78×10 4 , 2.26×10 4 , 9.32×10 3 , 2.81×10 3 , 2.12×10 3 days*(copies / μg gDNA) (Figure 23). Because the lungs are the first organ reached after tail vein injection in mice, the exposure level is higher in the lungs. The spleen is a hematopoietic organ, and SCG61-PD1-BB colonizes and expands in vivo after transfusion, resulting in higher exposure in the spleen. There is no distribution in the bladder, stomach, skeletal muscle, kidney, brain, or ovaries.

[0325] The above data show that SCG61-PD1-BB can specifically accumulate in tumor tissue after being infused back into the body; the lungs and spleen have higher exposure due to abundant blood flow; the exposure of the study drug in other organs is weak and transient.

[0326] Example 21: Construction of viral vector expressing SCG61-TGF-BB

[0327] The structural principle of SCG61-TGF-BB is shown in Figure 24. The coding sequence of the HPV TCR-TGF-BB fragment is fully synthesized. The synthesized gene is double-enzyme digested and inserted into a lentiviral vector to construct a plasmid. The resulting vector is then packaged using lentiviral technology. SCG61-TGF-BB consists of the HPV E7 T cell receptor β chain variable region (E7 TCR Vβ), the T cell receptor β chain constant region (TCR Cβ), a 2A self-cleavage peptide, the HPV E7 T cell receptor α chain variable region (E7 TCR Vα), the T cell receptor α chain constant region (TCR Cα), and a 2A self-cleavage peptide, the TGFβRII extracellular region, the CD8 transmembrane region, and the 4-1BB intracellular region. The TCR constant region has been sequence-modified to reduce endogenous TCR mispairing.

[0328] Example 22: Flow cytometry detection of SCG61-TGF-BB TCR-T specific expression and phenotypic detection

[0329] Flow cytometry buffer was prepared as follows: DPBS (Gibco, 14190250), 2% FBS (Gibco, 10099141), and stored in a 4°C refrigerator until ready for use.

[0330] SCG61-TGF-BB TCR-T, SCG61 TCR-T and Mock T cells (control group) were obtained and washed once with flow cytometry buffer, and the supernatant was discarded. PE HLA-A*02:01HPV E7 Tetramer (MBL, TB-0031-1), APC mTCRβ (BD, 553174), APC Anti-hTGF-βRII (Biolegend, 399706) and BV421 Mouse anti-hCD8a (BD, 743064) were added. After incubation at 4°C in the dark for 60 min, the cells were washed with flow cytometry buffer, resuspended, and finally detected by flow cytometry (Beckman CytoFLEX).

[0331] The results showed that the E7 Tetramer positivity rates of SCG61 TCR and SCG61-TGF-BB TCR were over 90%, and SCG61-TGF-BB could specifically express TGF-βRII. There was no significant difference in the proportion of CD8+ T cells between the two (Figure 25). This indicates that both SCG61 and SCG61-TGF-BB can be stably expressed, and the newly added TCR sequence and 41-BB sequence did not significantly affect the proportion of CD8+ T cells.

[0332] SCG61-TGF-BB TCR-T and Mock T cells (control group) were taken, washed once with flow cytometry buffer and the supernatant was discarded. Antibodies were added as shown in the table below, incubated at 4°C in the dark for 60 minutes, washed with flow cytometry buffer, resuspended, and finally detected by flow cytometry (Beckman CytoFLEX).

[0333] The results showed that SCG61-TGF-BB TCR-T cells maintained high CD8 and memory subset Tscm+Tcm ratios, as well as low PD-1 and Treg ratios ( FIG26 ).

[0334] Example 23: Functional evaluation of SCG61-TGF-BB against different HPV subtypes

[0335] HPV is a non-enveloped, double-stranded, circular DNA virus with high host specificity and affinity. This experiment primarily targets the subtypes with the highest global infection rates: high-risk HPV16 and HPV52. The corresponding HPV-E7 is a major oncogenic protein, the sequence of which is shown in Figure 27. The experiment involved loading T2 cells with varying concentrations of HPV16-E7 and HPV52-E7 peptides to detect cytokine secretion in co-cultured cells. Furthermore, HepG2 cell lines expressing HPV16-E7 and HPV52-E7 were constructed to investigate whether SCG61-TGF-BB could recognize HPV16-E7 and HPV52-E7 epitope peptides endogenously processed and presented by target cells, thereby evaluating its ability to target different HPV subtypes.

[0336] Co-incubation experiment of SCG61-TGF-BB on T2 loaded with different subtypes of HPV-E7 peptides

[0337] Complete culture medium for R10 cells was prepared as follows: RPMI1640 (Gibco, 22400-089), 10% FBS (Gibco, 10099141), mixed by inversion, and placed in a 4°C refrigerator for later use.

[0338] T cell culture medium preparation: CTS TM OpTmizer TM +Supplement(Gibco,A379040-01)、5%CTS TM Immune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), mix thoroughly by inversion, and place in a 4°C refrigerator until use.

[0339] Target cell preparation: Take T2 cells that have been passaged several times and are in good growth condition, centrifuge at 500g for 5 minutes, remove the supernatant, resuspend the cells in R10 complete cell culture medium, count the cells, take a 96-well U-bottom plate, add T2 cells at 100μl / well, and add 2×10 4 / well, 100ul TCM culture medium was added to each well of the negative control group; the corresponding HPV16-E7 and HPV52-E7 peptide solutions were added according to the concentration gradient to make the final concentration: 10 -5 M~10 -12 M.

[0340] Effector cell preparation: SCG61-TGF-BB and Mock T cells were mixed and centrifuged at 500g for 5 min. The supernatant was removed and the cells were resuspended in T cell culture medium. After counting, the density of SCG61-TGF-BB positive cells was adjusted to 4.0×10 5 / ml, add effector cells at 50μl / well, blow evenly, and place in a 37℃ incubator. After incubation for 24 hours, collect the cell supernatant and analyze the cytokine production level by flow cytometry.

[0341] SCG61-TGF-BB kills HepG2 cells expressing different subtypes of HPV-E7 (RTCA)

[0342] M10 cell complete culture medium was prepared as follows: DMEM (Gibco, 11965-092), 10% FBS (Gibco, 10099141), 1% Sodium Pyruvate (Gibco, 11360070), 1% HEPES (Gibco, 15630080), and 1% NEAA (Gibco, 11140-050). The culture medium was mixed by inversion and placed in a 4°C refrigerator until ready for use.

[0343] T cell culture medium preparation: CTS TM OpTmizer TM +Supplement(Gibco,A379040-01)、5%CTS TM Immune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), mix thoroughly by inversion, and place in a 4°C refrigerator until use.

[0344] Target cell plating: On Day 0, take the coated 96-well RTCA plate, add 50 μl of M10 medium, and place it in a 37°C incubator to detect the baseline. Take the target cells (HepG2, HepG2-HPV16-E7-LG, HepG2-HPV52-E7-LG), digest them, centrifuge and discard the supernatant, resuspend them in M10 complete cell culture medium, count them, take the RTCA plate, add each target cell at 50 μl / well, and add 4×10 4 / well, add 50μl T cell culture medium to the blank well, shake well and place in a 37℃ incubator for RTCA.

[0345] Add effector cells: On Day 0, resuscitate SCG61-TGF-BB and mock T cells, shake well, and place in a 37°C incubator. On Day 1, blow the cells evenly, count them, and add effector cells at effector-target ratios of 2:1, 1:2, and 1:8 based on the positive rate, at 50μl / well. Place in a 37°C incubator, load the RTCA machine, and start the experiment. On Day 2, remove the RTCA plate and carefully aspirate the 24-hour culture supernatant at 20uL / well to detect cytokines. Return the RTCA plate to the 37°C incubator and continue the experiment. On Day 3-Day 4, end the experiment based on the cell killing situation and remove the RTCA plate.

[0346] The results showed that SCG61-TGF-BB had similar affinity for T2 cells loaded with HPV16-E7 and HPV52-E7 peptides at gradient concentrations of 10uM to 1pM, with the EC50 values ​​of 8.39×10 -9 and 8.82×10 -9 (Figure 27); and SCG61-TGF-BB was able to completely eliminate HPV16-E7 and HPV52-E7 positive target cells at effector-target ratios of 2:1 and 1:2, with the killing rate exceeding 90% at 72 hours; at an effector-target ratio of 1:8, the killing rate of HPV16-E7 positive target cells was still over 60%, and SCG61-TGF-BB had no killing effect on negative target cells (Figure 28). The detection results of cytokines IFN-γ and TNF were highly consistent with the killing results (Figure 29); Mock T had no function on target cells.

[0347] In summary, SCG61-TGF-BB has significant antitumor activity against high-risk HPV16 and HPV52, regardless of whether they are T2 cell-loaded peptides or epitope peptides endogenously expressed in the cell lines. Therefore, including HPV genotypes 16 and 52 as inclusion criteria for the target population may benefit a wider range of patients.

[0348] Example 24: Mechanism Verification of SCG61-TGF-BB Inhibitory Effect on the pSMAD2 / 3 Pathway

[0349] There are many immunosuppressive mechanisms in the tumor microenvironment that may hinder the efficacy of adoptively transferred T cells. One of these mechanisms is mediated by TGF-β, a cytokine secreted by tumor cells and infiltrating suppressor immune cells that directly inhibits the activity of effector T cells. Effector T cells express the TGF-β receptors TGFBRI and TGFBRII. T cells exposed to TGF-β induce heterodimerization of these receptors and phosphorylation of the main TGF-β signaling mediators SMAD2 and SMAD3. Phosphorylated SMAD proteins induce an inhibitory transcriptional program, ultimately leading to reduced cytokine production, reduced cytotoxicity, and failure to proliferate under antigen stimulation. In this experiment, the inhibitory effect of SCG61-TGF-BB on the TGF signaling pathway was verified by detecting the degree of intracellular SMAD2 / 3 phosphorylation after incubation with TGF-β.

[0350] T cell culture medium preparation: CTS TM OpTmizer TM +Supplement(Gibco,A379040-01)、5%CTS TMImmune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), mix thoroughly by inversion, and place in a 4°C refrigerator until use.

[0351] Flow cytometry buffer was prepared as follows: DPBS (Gibco, 14190250), 2% FBS (Gibco, 10099141), and stored in a 4°C refrigerator until ready for use.

[0352] Preparation of TCR-T cells: SCG61-TGF-BB and Mock T were mixed by vortexing, centrifuged at 500 g for 5 min, and the supernatant was removed. The cells were resuspended in T cell culture medium and counted, and the cell density was adjusted to 1.0 × 10 6 / ml, set up two sets of replicate wells in a 24-well plate, adding 1ml / well. Add 10ng / ml TGFβ to one set, and the other set serves as a control. After agitation, place the cells in a 37°C incubator. After incubation for 1 hour, harvest the cells and perform flow cytometry analysis.

[0353] SCG61 TCR-T and SCG61-TGF-BB TCR-T cells were taken and washed once with flow cytometry buffer, after which the supernatant was discarded. PE Hamster Anti-mTCRβ (BD, 553172), BV421 Mouse Anti-hCD8 (BD, 743064), and Ghost Dye Red 710 (TONBO, 13-0871-T100) were added and incubated at 4°C in the dark for 60 min. The cells were washed with flow cytometry buffer, centrifuged, and preheated 37°C BD PhosFlow Fix Buffer I (BD, 557870) was added. The cells were fixed at 37°C in the dark for 15 min, washed with flow cytometry buffer, centrifuged, and precooled 4°C BD PhosFlow Perm Buffer III (BD, 558050) was added. The membrane was permeabilized at 4°C in the dark for 60 min. The cells were washed with flow cytometry buffer, centrifuged, and added with AF647 Mouse Antibody. Anti-pSmad2 / 3 (BD, 562696) was incubated at 4°C in the dark for 60 min, washed with flow cytometry buffer, resuspended by centrifugation, and finally detected by flow cytometry (Beckman CytoFLEX).

[0354] The results showed that compared to SCG61 TCR, SMAD2 / 3 phosphorylation was barely upregulated in SCG61-TGF-BB-positive cells, and the proportions of activated and inactivated pSMAD2 / 3 in CD4+ / CD8+ subsets were roughly the same (Figure 30). This suggests that SCG61-TGF-BB protects T cells from the effects of TGF-β by blocking TGF-β's ability to induce SMAD2 / 3 phosphorylation.

[0355] Example 25: Functional Verification of SCG61-TGF-BB on T2-loaded Peptide under TGFβ1 Conditions

[0356] The experiment used T2 cells, loaded with HPV-16 E7 epitope peptides at different concentration gradients, and divided them into two groups. One group was added with 10 ng / ml TGF-β1. After incubation for 2 hours, effector cells were added and incubated at 37°C for 24 hours. The supernatant was collected to detect the secretion of cytokine IFN-γ to verify the effect of TGF-β1 on the killing function.

[0357] Complete culture medium for R10 cells was prepared as follows: RPMI1640 (Gibco, 22400-089), 10% FBS (Gibco, 10099141), mixed by inversion, and placed in a 4°C refrigerator for later use.

[0358] T cell culture medium preparation: CTS TM OpTmizer TM +Supplement(Gibco,A379040-01)、5%CTS TM Immune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), mix thoroughly by inversion, and place in a 4°C refrigerator until use.

[0359] Target cell preparation: Take T2 cells that have been passaged for several times and are in good growth condition, centrifuge at 500g / 5min, remove the supernatant, resuspend the cells in R10 complete cell culture medium, count the cells, and take 2.4×10 6 Resuspend the T2 cells in 12 ml TCM medium, take a 96-well U-bottom plate, add T2 cells at 100 μl / well, and 2×10 4 / well; add the corresponding polypeptide solution according to the concentration gradient to make the final concentrations: 100nM and 10nM; divide into 2 groups, one group is added with TGF-β1, and the other group is used as a control. Place the 96-well U-bottom plate in a 37-degree incubator and incubate for 2 hours, then add the effector cells.

[0360] Effector cell preparation: SCG61 TCR-T and SCG61-TGF-BB TCR-T were homogenized and counted to 3.6 × 10 6 The cells were resuspended in 6 ml of T cell culture medium to adjust the cell density of SCG61-TGF-BB positive cells to 4 × 10 5 / ml, add effector cells at 50μl / well, blow evenly and place in a 37℃ incubator. After incubation for 24h, collect the co-culture supernatant and detect cytokine secretion by flow cytometry.

[0361] The results showed that when TGF-β1 was not added, the killing functions of SCG61 TCR-T and SCG61-TGF-BB TCR-T on T2 cells loaded with 100nM and 10nM HPV-16 E7 epitope peptides were basically the same; when TGF-β1 was added, the function of SCG61 TCR-T was inhibited, while the cytokine secretion ability of SCG61-TGF-BB was stronger than when it was not added (Figure 31). This is because SCG61-TGF-BB uses a lentiviral vector to genetically modify T cells. The lentiviral vector encodes a TCR that recognizes the HPV E7 peptide presented by HLA-A*02:01 on tumor cells and a switch receptor that is a fusion of the TGF-β receptor II external domain and the 4-1BB internal domain, converting the binding of the immunosuppressive cytokine TGF-β into a co-stimulatory signal. In the high TGF environment in the simulated tumor microenvironment, the function of SCG61-TGF-BB was not only not inhibited, but also showed a stronger anti-tumor effect.

[0362] Example 26: Cytotoxicity of SCG61-TGF-BB on Tumor Cells in Vitro Under TGFβ1 Conditions

[0363] The experiment used real-time cell analysis (RTCA) technology to evaluate the cytotoxicity of SCG61-TGF-BB against target cells in the presence of TGFβ1. Human cervical cancer Caski cells were cultured as target cells in 96-well RTCA plates and divided into two groups. One group was treated with 10ng / ml TGF-β1. After approximately 16 hours of incubation, effector cells were added to the culture and co-cultured. The cells were then observed for approximately 48 hours, and the survival curves of the target cells were plotted as a continuous graph for comparison. After the cytotoxicity was complete, the co-culture supernatant was collected for cytokine analysis.

[0364] Complete culture medium for R10 cells was prepared as follows: RPMI1640 (Gibco, 22400-089), 10% FBS (Gibco, 10099141), mixed by inversion, and placed in a 4°C refrigerator for later use.

[0365] T cell culture medium preparation: CTS TM OpTmizer TM +Supplement(Gibco,A379040-01)、5%CTS TM Immune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), mix thoroughly by inversion, and place in a 4°C refrigerator until use.

[0366] Day 0: Take well-growing human cervical cancer CaSki cells (R10 complete cell culture medium), digest and adjust the cell density to 4×10 5 / ml for standby use. Take a 96-well RTCA plate and add 50ul / well of the corresponding culture medium for instrument baseline measurement. Then add 4×10 5 50ul of target cells / ml were placed in a static state for about 5 minutes before loading onto the instrument and the growth curve was continuously monitored for about 16 hours;

[0367] Day 1: Take SCG61 TCR-T, SCG61-TGF-BB TCR-T, and Mock T cells, whose positive rates and cell viability have been measured, calculate the number of effector cells according to the positive rate, add 50 μl of effector cells per well at effector-target ratios of 2.5:1 and 1:2, and continuously monitor the killing curve on the instrument;

[0368] Day 2: Collect 24-hour co-culture supernatant for cytokine detection;

[0369] Day 3: Stop the machine and copy the growth and kill curves of RTCA.

[0370] The results showed that after 48 hours of co-culture, SCG61-TGF-BB had a significant specific killing effect on tumor cells under the condition of adding 10ng / ml TGF-β1, and the killing ratio was positively correlated with the effector-target ratio; while SCG61 had a significant inhibitory effect on tumor cells at a high effector-target ratio (2.5:1); the cytokine release of SCG61-TGF-BB at both effector-target ratios was significantly higher than that of the SCG61 group (Figure 32), indicating that SCG61-TGF-BB has a stronger anti-tumor effect under the condition of TGFβ1.

[0371] Example 27: Multiple rounds of target cell stimulation assay for tumor depletion using SCG61-TGF-BB under TGFβ1 conditions

[0372] The experiment simulated the immune cell inhibition caused by TGFβ1 in the tumor microenvironment. Through multiple rounds of tumor cell stimulation experiments to simulate the condition of T cell exhaustion in vivo, the ability of SCG61 and SCG61-TGF-BB to kill tumor cells was compared to verify the role of the TGF-BB auxiliary sequence.

[0373] Complete culture medium for R10 cells was prepared as follows: RPMI1640 (Gibco, 22400-089), 10% FBS (Gibco, 10099141), mixed by inversion, and placed in a 4°C refrigerator for later use.

[0374] T cell culture medium preparation: CTS TM OpTmizer TM +Supplement(Gibco,A379040-01)、5%CTS TMImmune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), mix thoroughly by inversion, and place in a 4°C refrigerator until use.

[0375] Day 0: Take well-growing human cervical cancer CaSki cells (R10 complete cell culture medium), digest and adjust the cell density to 4×10 5 / ml for standby use, take a RTCA96-well plate, add 50ul / well of the corresponding culture medium for instrument baseline measurement, and then add a density of 4×10 5 50ul of target cells / ml were placed in a static state for about 5 minutes before loading onto the instrument and the growth curve was continuously monitored for about 16 hours;

[0376] Day 1: Take SCG61 TCR-T, SCG61-TGF-BB TCR-T, and Mock T cells, whose positive rates and cell viability have been measured, calculate the number of effector cells according to the positive rate, add 50 μl of effector cells per well at an effector-target ratio of 1:2, and continuously monitor the killing curve on the instrument;

[0377] Day 3: Follow the same procedures as Day 0, plate the target cells on the second RTCA plate, and incubate at 37°C for approximately 16 hours.

[0378] Day 4: Remove the first RTCA plate and transfer the effector cells to the same position on the second RTCA plate. Continuously monitor the killing curve on the machine.

[0379] Day 7: Stop the machine and copy the killing curve of RTCA, and collect the 24-hour co-culture supernatant for cytokine detection.

[0380] The results showed that, when 10 ng / ml TGF-β1 was added, SCG61-TGF-BB had a significant specific killing effect on tumor cells stimulated for the first time, and SCG61 had a significant inhibitory effect on tumor cells. During the second round of stimulation, the half-life of SCG61-TGF-BB killing of tumor cells was significantly shortened, while the killing effect of SCG61 was significantly weakened. Cytokine release by SCG61-TGF-BB after multiple rounds of stimulation was significantly higher than that of the SCG61 group, both with and without TGF-β1 (Figure 33), indicating that SCG61-TGF-BB has a stronger anti-tumor effect under the condition of TGFβ1. SCG61-TGF-BB is genetically modified using a lentiviral vector encoding a TCR (HLA-A*02:01) that recognizes HPV16 and HPV52 E7 (epitopes 11-19) and a switch receptor. By fusing the TGF-β receptor II (TGFBRII) ectodomain with the 4-1BB endodomain, the immunosuppressive factor TGFβ is converted into a co-stimulatory signal, thereby preventing T cell apoptosis, improving exhaustion, and enhancing persistence. Results showed that SCG61-TGF-BB can maintain a lasting anti-tumor effect even after multiple rounds of tumor stimulation (simulating a T cell exhaustion environment).

[0381] Example 28: Detection of the proliferation ability of target cells after multiple rounds of stimulation with SCG61-TGF-BB under TGFβ1 conditions

[0382] The ability of SCG61-TGF-BB to kill tumor cells was studied through multiple rounds of tumor cell stimulation experiments. SCG61 TCR-T, SCG61-TGF-BB TCR-T and Mock T were counted and resuspended in medium without IL-2. 6 / ml density, 1ml / well was added to a 24-well plate, 10ng / ml TGF-β1 was added, and target cells HepG2-E7 were added at an effector-target ratio of 1:1; after 5 days of stimulation, the cells were counted, the number of effector cells was adjusted, and an equal amount of target cells were added for stimulation again; during this period, the cells were counted every 2-3 days and their proliferation rate was calculated.

[0383] Experimental data showed that after each round of tumor cell stimulation, the proliferation fold of SCG61-TGF-BB was significantly higher than that of SCG61TCR-T and Mock T ( Figure 34 ).

[0384] Example 29: Cross-reactivity of SCG61-TGF-BB to human polypeptide library

[0385] The human TCR repertoire displays inherent cross-reactivity, with up to 108 unique TCRs providing over 10 15Identification of potential peptides. In clinical trials, TCR cross-reactivity can lead to unintended targeting of healthy human tissues, resulting in severe toxicity. The key amino acid positions of the HPV16 E711-19 epitope peptide recognized by SCG61-TGF-BB are known to be 2 / 4 / 5 / 6. Bioinformatics predictions and BLAST sequence alignment of a human peptide library revealed 18 peptide sequences containing six amino acid residues identical to the E7 epitope peptide (five amino acid residues plus one conserved amino acid residue). Analysis of four key fragments (aa 2 / 4 / 5 / 6) of the fixed sequence using Expitope 2.0 revealed two fragments with four mismatches and 14 fragments with five mismatches. T2 cells loaded with these peptides were co-incubated with SCG61-TGF-BB, and IFN-γ levels in the supernatant were measured to investigate cross-reactivity between SCG61-TGF-BB and these peptides and to determine potential off-target effects, providing support and evidence for nonclinical safety studies.

[0386] Complete culture medium for R10 cells was prepared as follows: RPMI1640 (Gibco, 22400-089), 10% FBS (Gibco, 10099141), mixed by inversion, and placed in a 4°C refrigerator for later use.

[0387] T cell culture medium preparation: CTS TM OpTmizer TM +Supplement(Gibco,A379040-01)、5%CTS TM Immune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), mix thoroughly by inversion, and place in a 4°C refrigerator until use.

[0388] Peptide preparation: Take 2 mg of each of the 36 peptides, No. 1-36, and dissolve them in 150 μl to 200 μl of DMSO to a final concentration of 10 mM. See the table below for details.

[0389] Target cell preparation: Take T2 cells that have been passaged several times and are in good growth condition, centrifuge at 500g for 5 minutes, remove the supernatant, resuspend the cells in R10 medium, count the cells, take a 96-well U-bottom plate, add T2 cells at 100μl / well, and add 2×10 4 / well; add 11ul of the corresponding peptide solutions No. 1 to 36 at 10uM and 1uM respectively, so that the final concentrations are: 1uM and 100nM; place the 96-well U-bottom plate in a 37°C incubator and incubate for 2 hours, then add the effector cells.

[0390] Effector cell preparation: SCG61-TGF-BB was evenly distributed and the cell density was adjusted to 4.0×10 cells according to the positive rate after counting. 5 / ml, add effector cells at 50μl / well, blow evenly, and place in a 37℃ incubator. After incubation for about 24 hours, the cells were collected and cytokine levels were analyzed by flow cytometry.

[0391] The results showed that using two peptide concentration gradients (1uM and 100nM), SCG61-TGF-BB was able to stimulate T2 cells loaded with E711-19 peptide to significantly secrete the cytokine IFN-γ. However, no cytokine secretion was detected for the negative control group S20-28 peptide and the 34 experimental group peptides. This indicates that SCG61-TGF-BB has no cross-reactivity with the peptides with the highest consistency with E711-19 (six identical amino acids or five amino acids plus one conserved amino acid) in the human autoantigen peptide library (Figure 35), and has no cross-reactivity with peptides containing four key amino acid motifs in the human autoantigen peptide library. This indicates that the potential off-target toxicity risk of SCG61-TGF-BB against endogenous human antigen peptides is very low.

[0392] Example 30: In vitro killing function of SCG61-TGF-BB animal experimental batch on target cells

[0393] The experiment used real-time cell analysis (RTCA) technology to evaluate the in vitro killing ability of the SCG61-TGF-BB animal experimental batch on target cells. Caski-E7 and HepG2-E7 cells were cultured as target cells in 96-well RTCA plates. After approximately 16 hours of incubation, effector cells were added to the culture. The supernatant of the co-culture was collected for 24 hours for cytokine analysis. The cells were then observed continuously for 48-60 hours, and the survival curves of the target cells were plotted as a continuous graph for comparison.

[0394] M10 cell complete culture medium was prepared as follows: DMEM (Gibco, 11965-092), 10% FBS (Gibco, 10099141), 1% Sodium Pyruvate (Gibco, 11360070), 1% HEPES (Gibco, 15630080), and 1% NEAA (Gibco, 11140-050). The culture medium was mixed by inversion and placed in a 4°C refrigerator until ready for use.

[0395] Complete culture medium for R10 cells was prepared as follows: RPMI1640 (Gibco, 22400-089), 10% FBS (Gibco, 10099141), mixed by inversion, and placed in a 4°C refrigerator for later use.

[0396] T cell culture medium preparation: CTS TM OpTmizer TM+Supplement(Gibco,A379040-01)、5%CTS TM Immune Cell SR (Gibco, A25961-01) + 2% Glutamax (Gibco, A12860-01), mix thoroughly by inversion, and place in a 4°C refrigerator until use.

[0397] Day 0: Take well-growing human cervical cancer CaSki-E7 cells (R10 complete cell culture medium) and HepG2-E7 cells (M10 complete cell culture medium), digest them and adjust the cell density to 4×10 5 / ml for standby use, take a coated RTCA96-well plate, add 50ul / well of the corresponding culture medium for instrument baseline measurement, and then add a density of 4×10 5 50ul of target cells / ml were placed in a static state for about 5 minutes before loading onto the instrument and the growth curve was continuously monitored for about 16 hours;

[0398] Day 1: Take the SCG61-TGF-BB TCR-T and Mock T cells whose positive rate and cell viability have been measured, calculate the number of effector cells according to the positive rate, add 50ul of effector cells per well at effector-target ratios of 2:1, 1:2, and 1:8, and continuously monitor the killing curve on the instrument;

[0399] Day 2: Collect 24-hour co-culture supernatant for cytokine detection;

[0400] Day 3: Stop the machine and copy the growth and kill curves of RTCA.

[0401] The results showed that SCG61-TGF-BB had a significant specific killing effect on Caski-E7 at effector-target ratios of 2:1 and 1:2, and the killing ratio was positively correlated with the effector-target ratio; SCG61-TGF-BB was able to completely eliminate HepG2-E7 cells at effector-target ratios of 2:1, 1:2 and 1:8, and the killing ratio and time were positively correlated with the effector-target ratio; the SCG61-TGF-BB cytokine IFN-γ detection results at the above effector-target ratios were highly consistent with the killing results (Figure 36); Mock T had no effect on target cells.

[0402] In summary, SCG61-TGF-BB has significant killing and anti-tumor activity against Caski-E7 and HepG2-E7 in vitro, providing support and dosage basis for subsequent non-clinical experiments.

[0403] Example 31: In vivo killing, amplification, and persistence of SCG61-TGF-BB in the CaSki-E7 transplanted tumor model in immunodeficient mice

[0404] To understand the role of SCG61-TGF-BB in killing, amplifying, and maintaining CaSki-E7 tumor cells in vivo, 23 NPG-immunodeficient female mice were inoculated with CaSki-E7 cells on Day 7. The mice were randomly divided into five groups, three of which received low, medium, and high doses of SCG61-TGF-BB. Each group received a single injection of SCG61-TGF-BB via the tail vein on Day 0 at doses of 2×10 6 , 7×10 6 , 2×10 7 Positive T cells / mouse; negative control group received a single injection of 2×10 Mock T cells via tail vein 7 T cells / mouse; at the same time, mice in the model control group received a single injection of an equal amount of the test substance vehicle via tail vein injection and were observed for 4 weeks. The long and short diameters of tumors were measured twice weekly, and tumor volumes were calculated. Peripheral blood was collected weekly to assess the proliferation and survival of SCG61-TGF-BB TCR-T cells.

[0405] “NA” means not applicable.

[0406] The results showed that the tumor volumes of the model control group, negative control group, and SCG61-TGF-BB low-, medium-, and high-dose groups on Day 22 were 1266.94±104.49, 1229.65±225.45, 1117.53±165.11, 581.24±249.42, and 117.73±31.32 mm, respectively. 3 The tumor volume inhibition rates of the low, medium and high dose groups of SCG61-TGF-BB were 11.79%, 54.12% and 90.71%, respectively. It can be seen that the inhibition of tumor volume by SCG61-TGF-BB was dose-dependent.

[0407] After 1 week of administration, SCG61-TGF-BB high dose (2.0×10 7 TCR-T cells / animal) significantly inhibited tumor cell proliferation (P=0.00001, P<0.05); the medium dose (7×10 6 TCR-T cells / animal) also significantly inhibited the proliferation of tumor cells (P=0.055), but rebound occurred in 2 / 5 mice after 2 weeks; low dose (2×10 6 TCR-T cells / animal) can control tumor growth within 2 weeks, but 5 / 5 mice ultimately fail to suppress tumors (Figure 37). On Day 7 after reinfusion, SCG61-TGF-BB TCR-T expansion in the animal's peripheral blood reached its peak, and the number of cells was positively correlated with the dose and gradually decreased over time (Figure 38).

[0408] Under experimental conditions, SCG61-TGF-BB was intravenously administered (0.7-2.0×10 7 TCR-T cells / animal significantly inhibited the proliferation of CaSki-E7-LG tumor cells, and the lowest effective dose (0.7×10 7 TCR-T cells / animal).

[0409] Example 32: In vivo killing, amplification, and survival of SCG61-TGF-BB in the HepG2-E7-LG transplanted tumor model in immunodeficient mice

[0410] To understand the effect of SCG61-TGF-BB on the killing, proliferation, and survival of HepG2-E7-LG tumor cells in vivo, 25 NPG-immunodeficient female mice were inoculated with HepG2-E7-LG cells on Day 7. The mice were randomly divided into five groups, three of which were low-, medium-, and high-dose SCG61-TGF-BB groups. On Day 0, each group received a single injection of SCG61-TGF-BB via the tail vein at a dose of 2×10 6 , 7×10 6 , 2×10 7 Positive T cells / mouse; negative control group received a single injection of 2×10 Mock T cells via tail vein 7 T cells / mouse; at the same time, mice in the model control group received a single injection of an equal amount of the test substance vehicle via tail vein injection and were observed for 4 weeks. The long and short diameters of tumors were measured twice weekly, and tumor volumes were calculated. Peripheral blood was collected weekly to assess the proliferation and survival of SCG61-TGF-BB TCR-T cells.

[0411] “NA” means not applicable.

[0412] The results showed that the tumor volumes of the Day 22 model control group, negative control group, and SCG61-TGF-BB low-, medium-, and high-dose groups were 2400.34±726.22, 1986.19±720.41, 1252.83±202.74, 439.02±365.64, and 132.34±175.87 mm, respectively. 3 The tumor volume inhibition rates of the low-, medium-, and high-dose SCG61-TGF-BB groups were 47.81%, 81.71%, and 94.49%, respectively (Figure 39). This shows that the medium- and high-dose SCG61-TGF-BB groups can significantly inhibit the proliferation of tumor cells (P<0.05), and the inhibition of tumor volume is dose-dependent.

[0413] On Day 7 after transfusion, SCG61-TGF-BB TCR-T cell proliferation in the peripheral blood of animals reached its peak, and the number of cells was positively correlated with the dose and gradually decreased over time ( FIG40 ).

[0414] Under experimental conditions, SCG61-TGF-BB was intravenously administered (0.7-2.0×10 7 TCR-T cells / animal significantly inhibited the proliferation of HepG2-E7-LG tumor cells, and the lowest effective dose (0.7×10 7 TCR-T cells / animal).

[0415] The new generation of HPV-targeted TCR-T therapy transduces a T cell receptor targeting HPV-16 E7 and a PD-1 or TGFβRII co-stimulatory fusion receptor into T cells, specifically recognizing and killing HPV-16-infected cells. It is suitable for a variety of malignancies caused by human papillomavirus (HPV), including cervical cancer, head and neck cancer, anal cancer, vaginal cancer, vulvar cancer, and penile cancer. By targeting the antigen expression unique to HPV infection, it kills tumor cells and simultaneously eliminates the viral infection.

[0416] In vitro studies have shown that HPV-targeted TCR-T cells specifically express the TCR and have a low mismatch rate. HPV-targeted TCR-T cells can recognize key binding sites for HPV-16 and HPV-52 epitope peptides and exhibit strong binding activity. HPV-targeted TCR-T cells have a specific killing effect on tumor cells expressing HLA-A*02:01 and HPV-16 / 52E7. A higher effector-target ratio indicates a stronger tumor-killing ability and more cytokines released. HPV-targeted TCR-T cells do not recognize and kill HLA-A*02:01-negative or HPV-16 E7-negative tumor cells, demonstrating their targeted specificity and safety. HPV-targeted TCR-T cells can maintain their anti-tumor effects and proliferation capacity after multiple rounds of tumor stimulation.

[0417] In vivo studies have shown that different doses of HPV-targeted enhanced TCR-T cells showed significant inhibitory effects on tumor growth in different HPV-16 E7-positive tumor NCG mouse CDX models. 7 Mice treated with HPV-enhanced TCR-T cells (100 cells) showed significant tumor suppression, while mice in the medium and low dose groups also showed some tumor suppression. This indicates that HPV-enhanced TCR-T cells can maintain their anti-cancer effects in mice.

[0418] In summary, the results of preclinical trials have preliminarily confirmed the good safety of HPV-targeted enhanced TCR-T cells and their killing effect on HPV-16 E7+ target cells. The preclinical animal trial dose of this drug reached 2×10 7 No obvious toxic side effects were observed with HPV-targeted enhanced TCR-T cells, supporting further exploration of their safety and tolerable dose in humans.

[0419] The above is a detailed introduction to the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method and core idea of ​​the present application. At the same time, for those skilled in the art, based on the idea of ​​the present application, there may be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A modified immune cell, characterized in that The modified immune cells contain a T cell receptor (TCR) targeting the HPV E7 antigen and a switch-stimulating molecule.

2. The modified immune cell according to claim 1, wherein The TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain; wherein, The amino acid sequence of αCDR3 of the TCR α chain variable domain is as shown in SEQ ID NO: 3, or a variant thereof, wherein one or two amino acids are replaced by other amino acids; and; The amino acid sequence of the βCDR3 of the TCR β chain variable domain is shown in SEQ ID NO: 6, or a variant thereof, in which one or two amino acids are replaced by other amino acids.

3. The modified immune cell according to claim 2, characterized in that The TCR α chain variable domain comprises complementarity determining regions αCDR1, αCDR2 and αCDR3, and the TCR β chain variable domain comprises complementarity determining regions βCDR1, βCDR2 and βCDR3, wherein: αCDR1, αCDR2 and αCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and βCDR1, βCDR2 and βCDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; or CDR variants in which one or two amino acids in one or more CDRs are replaced by other amino acids.

4. The modified immune cell according to claim 2, characterized in that The TCR alpha chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; and / or The TCR beta chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

8.

5. The modified immune cell according to claim 1, wherein The modified immune cells express the following structures: AT cell receptor (TCR), wherein the TCR specifically targets the HPV E7 antigen polypeptide; Preferably, the TCR is capable of binding to an HPV E7 antigen polypeptide presented by HLA-A*02, more preferably, the TCR is capable of binding to an HPV E7 antigen polypeptide presented by HLA-A*02:01; more preferably, the polypeptide comprises the amino acid sequence YMLDLQPET or YILDLQPET, and B. A conversion stimulatory molecule, comprising: an extracellular domain (ECD), a transmembrane region (TM) and an intracellular domain (ICD), wherein the ECD is selected from the ECD of an immunosuppressive molecule, and the ICD is selected from the ICD of a co-stimulatory molecule.

6. The modified immune cell according to claim 5, characterized in that The conversion stimulating molecule comprises: The extracellular domain (ECD) of an immunosuppressive protein, wherein the ECD is fused to the intracellular domain (ICD) of a co-stimulatory molecule that mediates an immune cell activation signal; wherein the extracellular domain of the immunosuppressive protein binds to its immunosuppressive factor or ligand to generate an immune cell activation signal rather than an immune cell inactivation signal in the modified immune cell.

7. The modified immune cell according to claim 6, characterized in that The immunosuppressive protein is any one or a combination of PD-1, CTLA4, BTLA, TIM3, TIGIT, TGFβ receptor and any other protein having immunosuppressive function or associated with immunosuppressive signaling pathway, and the ECD sequence of the immunosuppressive protein may have at least one amino acid mutation; and / or The costimulatory molecule comprises: any one of CD28, 4-1BB, ICOS, CD27, IL-12R, CD3, and OX40 proteins or a combination thereof, and the costimulatory molecule ICD sequence may have at least one amino acid mutation.

8. The modified immune cell according to claim 6, characterized in that The ECD is TGFβ receptor II ECD; the sequence of the TGFβ receptor IIECD is shown in SEQ ID NO:

12.

9. The modified immune cell according to claim 6, characterized in that The ECD is PD-1ECD; more preferably, the PD-1ECD sequence has an amino acid mutation, where the alanine at position 132 is mutated to leucine; most preferably, the ECD amino acid sequence is as shown in SEQ ID NO:

9.

10. The modified immune cell according to claim 6, characterized in that The ICD is 4-1BB ICD; more preferably, the amino acid sequence of the 4-1BB ICD is as shown in SEQ ID NO:

11.

11. The modified immune cell according to claim 5, characterized in that The TCR comprises a TCR α chain and a TCR β chain, wherein the TCR α chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; and / or The TCR beta chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

8.

12. The modified immune cell according to claim 6, characterized in that The ECD and the ICD are connected by a transmembrane region sequence; preferably, the transmembrane region comprises a transmembrane domain of a protein selected from the group consisting of: α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154 and combinations thereof, and the transmembrane region sequence may have at least one amino acid mutation; more preferably, the transmembrane region sequence is a CD8 transmembrane region sequence; more preferably, the CD8 transmembrane region sequence is as shown in SEQ ID NO:

10.

13. The modified immune cell according to claim 12, characterized in that The immune cell includes the HPV E7 antigen-targeting TCR and a conversion stimulatory molecule with a structure of PD1 (ECD)-CD8 (TM)-4-1BB (ICD). More preferably, the immune cell comprises the amino acid sequence shown in SEQ ID NO:

15.

14. The modified immune cell according to claim 12, characterized in that The immune cell includes the HPV E7 antigen-targeting TCR and a conversion stimulatory molecule with a structure of TGFβRII (ECD)-CD8 (TM)-4-1BB (ICD). More preferably, the immune cell comprises the amino acid sequence shown in SEQ ID NO:

17.

15. The modified immune cell according to claim 3 or claim 7, characterized in that The immune cells are selected from lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, and mast cells; preferably, the immune cells are T cells.

16. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid sequence encoding the TCR according to any one of claims 1 to 4.

17. The nucleic acid molecule according to claim 16, wherein The nucleic acid molecule further encodes the nucleic acid sequence of the conversion stimulating molecule according to any one of claims 1-14.

18. A carrier, characterized in that The vector comprises the nucleic acid molecule according to claim 16 or 17.

19. A pharmaceutical composition, characterized in that The composition contains a pharmaceutically acceptable carrier and the modified immune cell according to any one of claims 1 to 15, the nucleic acid molecule according to claim 16 or 17, and the vector according to claim 18.

20. Use of the modified immune cell according to any one of claims 1 to 15, the nucleic acid molecule according to claim 16 or 17, the vector according to claim 18 or the pharmaceutical composition according to claim 19 in the preparation of a medicament for preventing or treating diseases related to HPV infection.

21. The use according to claim 20, characterized in that The HPV infection-related diseases include various malignant tumors caused by human papillomavirus (HPV), including one or more of cervical cancer, oropharyngeal cancer, head and neck cancer, vaginal cancer, vulvar cancer, penile cancer and anal cancer.

22. A T cell receptor targeting HPV E7 antigen or a fragment thereof, characterized in that: The T cell receptor or fragment thereof comprises a TCR α chain variable domain and a TCR β chain variable domain selected from the group consisting of: (1) the TCR α chain variable domain comprises the following three CDRs: αCDR1 shown in SEQ ID NO: 1, αCDR2 shown in SEQ ID NO: 2, and αCDR3 shown in SEQ ID NO: 3, or CDR variants thereof in which one or two amino acids are replaced; and (2) The TCR β chain variable domain comprises the following three CDRs: βCDR1 shown in SEQ ID NO: 4, βCDR2 shown in SEQ ID NO: 5, and βCDR3 shown in SEQ ID NO: 6, or the above CDR variants in which one or two amino acids are replaced.

23. The T cell receptor or fragment thereof according to claim 22, characterized in that The TCR α chain variable domain comprises the following three CDRs: αCDR1 shown in SEQ ID NO: 1, αCDR2 shown in SEQ ID NO: 2, and αCDR3 shown in SEQ ID NO: 3; and The TCR β chain variable domain comprises the following three CDRs: βCDR1 shown in SEQ ID NO: 4, βCDR2 shown in SEQ ID NO: 5, and βCDR3 shown in SEQ ID NO:

6.

24. The T cell receptor or fragment thereof according to claim 22, characterized in that The TCR alpha chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; and / or The TCR beta chain variable domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

8.

25. The T cell receptor or fragment thereof according to claim 22, wherein It can bind to the HPV E7 antigen polypeptide presented by HLA-A*02, more preferably, it can bind to the HPV E7 antigen polypeptide presented by HLA-A*02:

01.

26. The T cell receptor or fragment thereof according to claim 25, characterized in that The polypeptide comprises the amino acid sequence YMLDLQPET or YILDLQPET.

27. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid sequence encoding the T cell receptor or a fragment thereof according to any one of claims 22 to 26 or a complementary sequence thereof.

28. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 27, wherein the vector is selected from the group consisting of a plasmid, a binary vector, a DNA vector, an mRNA vector, a retroviral vector, a lentiviral vector, a transposon-based vector, and an artificial chromosome.

29. An isolated polypeptide encoded by the nucleic acid molecule of claim 27 or the vector of claim 28.

30. An isolated cell, characterized in that The cell comprises the T cell receptor or fragment thereof according to any one of claims 22 to 26, the nucleic acid molecule according to claim 27, the vector according to claim 28 or the polypeptide according to claim 29. Preferably, the cell comprises the amino acid sequence shown by SEQ ID NO:

13.

31. A pharmaceutical composition, characterized in that The composition contains a pharmaceutically acceptable carrier and the T cell receptor or fragment thereof according to any one of claims 22 to 26, the nucleic acid molecule according to claim 27, the vector according to claim 28, the polypeptide according to claim 29, or the cell according to claim 30.

32. Use of the T cell receptor or fragment thereof according to any one of claims 22 to 26, the nucleic acid molecule according to claim 27, the vector according to claim 28, the polypeptide according to claim 29, the cell according to claim 30 or the pharmaceutical composition according to claim 31 in the preparation of a medicament for preventing or treating related diseases caused by HPV infection.

33. The use according to claim 32, wherein The HPV infection-related diseases include various malignant tumors caused by human papillomavirus (HPV), including one or more of cervical cancer, oropharyngeal cancer, head and neck cancer, vaginal cancer, vulvar cancer, penile cancer and anal cancer.

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