A pharmaceutical composition for improving or treating ovarian cancer, and a preparation method and use thereof

Abstract

The application discloses a pharmaceutical composition for improving or treating ovarian cancer, and a preparation method and application thereof. The application provides an engineered cell and a new method which can significantly enhance the migration and infiltration ability of immune cells to the parenchyma part of ovarian cancer and relieve tumor immune escape, thereby solving the defects that immune cells are difficult to effectively infiltrate into the parenchyma of ovarian cancer in the prior art, and significantly improving the curative effect of immunotherapy for ovarian cancer.

Description

Technical Field

[0001] This invention relates to the field of biological immunotherapy technology, specifically to a pharmaceutical composition for improving or treating ovarian cancer, its preparation method, and its application. Background Technology

[0002] Ovarian cancer is the leading cause of cancer death among gynecological malignancies in women worldwide. Its main treatments include surgery and systemic therapy. Although traditional chemotherapy has shown some efficacy in initial treatment, the five-year survival rate remains unsatisfactory due to the often advanced stage of diagnosis and the prevalence of chemotherapy resistance and recurrence. Therefore, the development of new treatment strategies is particularly urgent. Drug treatment for ovarian cancer mainly relies on chemotherapy, targeted therapy, and small molecule drugs. Traditional chemotherapy drugs, such as platinum compounds (e.g., cisplatin and carboplatin) and taxanes (e.g., paclitaxel), are the cornerstone of first-line treatment for ovarian cancer and are often used in combination after cytoreductive surgery. These drugs directly kill tumor cells through cytotoxic effects. However, chemotherapy resistance in tumor cells is a significant challenge, with many patients experiencing disease recurrence and chemotherapy resistance after initial treatment. Studies have shown that fibroblasts can reduce the intranuclear accumulation of platinum drugs in ovarian cancer cells by releasing glutathione and cysteine, thereby leading to chemotherapy resistance. Targeted therapy inhibits tumor growth by acting on specific molecular targets or signaling pathways specific to tumor cells. For example, PARP inhibitors (such as olaparib) and anti-angiogenic drugs (such as bevacizumab) have shown potential in the treatment of ovarian cancer. Bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor (VEGF), can block the nutrient supply to tumors by inhibiting tumor angiogenesis. Furthermore, since the sustained activation of the STAT3 signaling pathway promotes tumor development, progression, and metastasis, small molecule inhibitors such as STAT3 inhibitors (such as Napabucasin and JMS-053) are also considered potential targets for ovarian cancer treatment. The field of drug discovery also focuses on finding anticancer compounds with cytotoxic effects from natural products; for example, cleerodane diterpenoids extracted from edible plants have been found to be cytotoxic to human ovarian cancer cells and can induce apoptosis.

[0003] Cell therapy, particularly chimeric antigen receptor CAR-T cell therapy, has shown significant efficacy in the treatment of hematologic malignancies as a novel immunotherapy and holds promise for the treatment of ovarian cancer. CAR-T cells are created by genetically engineering a patient's own T cells to specifically recognize and attack tumor cells. Studies on CAR-T cell therapy for ovarian cancer have already been conducted. For example, by targeting ovarian cancer cell-specific antigens (such as MSLN), CAR-T cells can enhance their efficiency in killing tumor cells. Furthermore, primary NK cells can effectively lyse poorly differentiated stem cell-like tumor cells (PDSLCs), but their cytotoxic potential is lower when treating well-differentiated tumors. Currently, researchers are exploring ways to enhance the anti-tumor activity of NK cells through engineering. For example, hyperactivated NK (sNK) cells have been proposed for targeting high-grade serous ovarian cancer (HGSOC) to overcome the resistance of PDSLCs to conventional therapies.

[0004] Despite the immense potential of cell therapy in ovarian cancer, numerous challenges remain, with immunosuppression in the tumor microenvironment and poor CAR-T cell infiltration into solid tumors being major limiting factors. The tumor microenvironment (TME) of ovarian cancer is a complex ecosystem containing various immunosuppressive cells and molecules, such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and immune checkpoint molecules (e.g., PD-1 / PD-L1), which collectively suppress the anti-tumor activity of immune cells. T-cell exhaustion (TEX), the gradual loss of T cell function within the TME, severely impacts the efficacy of immunotherapy for ovarian cancer. Unlike hematologic malignancies, one of the greatest challenges in cell therapy for solid tumors is the difficulty in effectively infiltrating tumor tissue with CAR-T cells. The dense structure of the tumor stroma and the physical barriers formed by suppressor cells and molecules hinder CAR-T cells from reaching and killing tumor cells.

[0005] Adoptive cellular immunotherapy, particularly chimeric antigen receptor T-cell (CAR-T) therapy, has achieved some success in the treatment of hematologic malignancies. However, its application in the treatment of solid tumors still faces significant challenges, with efficacy falling far short of expectations. The fundamental bottleneck lies in the inability of immune cells to effectively migrate and infiltrate tumor tissue. Existing technologies attempt to address this issue, but all have significant limitations and shortcomings: most current CAR-T technologies focus on enhancing T-cell activation intensity and sustained killing ability by optimizing co-stimulatory signaling domains (such as 4-1BB and CD28) and introducing novel signaling elements. However, even with strong in vitro killing capabilities, if T cells cannot effectively migrate and reach the tumor site, their therapeutic effect is ineffective. This "heavy killing, light migration" design approach is a major reason for the poor treatment outcomes of solid tumors. Some studies have attempted to enhance the migration ability of T cells by overexpressing other chemokine receptors (such as CXCR3, CCR2, and CCR4). However, this strategy has significant drawbacks: these chemokine receptors are typically involved in multiple physiological and inflammatory processes, and their ligands are not specifically highly expressed in the tumor microenvironment. Therefore, overexpression of these receptors may lead to T cell nonspecific migration to other tissues, even causing off-target toxicity, instead of specifically accumulating at the tumor site; the chemokine secretion profile exhibits significant heterogeneity among different individuals, different tumor types, and even different stages of the same tumor. A strategy based on a specific chemokine (such as CXCL9 / 10) may be effective for some patients, but completely ineffective for other tumors that do not secrete this chemokine, limiting its widespread application.

[0006] The immunosuppressive microenvironment of solid tumors is another key factor hindering therapeutic efficacy. Existing technologies often attempt to "counter" or "break down" this suppressive environment (e.g., by expressing PD-1 antibodies), but few solutions utilize the inherent characteristics of the tumor microenvironment for therapeutic purposes. For example, many solid tumors (such as ovarian cancer, colorectal cancer, and liver cancer) have metabolic abnormalities, producing and accumulating specific metabolites that are mechanisms for tumor immune escape. Existing technologies have failed to transform this "disadvantage" into a usable "targeted navigation signal." To bypass the migration barrier, some protocols use intratumoral injection to directly deliver CAR-T cells. While this method ensures local concentration, it is invasive, complex, and unsuitable for deep or metastatic lesions, thus failing to become a universal solution.

[0007] In summary, existing technologies share a common core deficiency: they fail to endow immune cells with a powerful active homing capability that can be efficiently guided by signals that are ubiquitous and specific in the tumor microenvironment.

[0008] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0009] In view of the technical problems existing in the current technology, the present invention, after in-depth research, provides a new method that can significantly enhance the migration and infiltration ability of immune cells into the parenchyma of ovarian cancer and eliminate tumor immune escape, thereby improving the efficacy of ovarian cancer immunotherapy. The solution of the present invention can at least solve the technical defects of the prior art in that immune cells are unable to effectively infiltrate into the parenchyma of ovarian cancer. Specifically, the present invention includes the following:

[0010] In a first aspect, the present invention provides engineered cells for improving and / or treating ovarian cancer, wherein the engineered cells comprise a nucleic acid molecule containing a chimeric antigen receptor coding sequence and an immune escape inhibitor protein coding sequence, or a single carrier containing the nucleic acid molecule, wherein the chimeric antigen receptor comprises a polypeptide that recognizes ovarian cancer antigens.

[0011] In some embodiments, the engineered cells for improving and / or treating ovarian cancer according to the present invention include T cells, NK cells, macrophages and / or dendritic cells.

[0012] In some embodiments, the engineered cells for improving and / or treating ovarian cancer according to the present invention, wherein the T cells include cytotoxic T cells or chimeric antigen receptor T cells.

[0013] In some embodiments, the engineered cells for improving and / or treating ovarian cancer according to the present invention, wherein the chimeric antigen receptor further includes a transmembrane domain, an intracellular signal transduction domain, a hinge region, and an intracellular co-stimulatory domain.

[0014] In some embodiments, the engineered cells for improving and / or treating ovarian cancer according to the present invention further include a coding sequence for a linker peptide between the coding sequence of the chimeric antigen receptor and the coding sequence of the immune escape inhibitory protein.

[0015] In some embodiments, the engineered cells for improving and / or treating ovarian cancer according to the present invention, wherein the ovarian cancer antigen includes at least one of MSLN, HER2, EGFR, VEGF / VEGFR, EPCAM, MUC1, BRCA1 / BRCA2, ATM, ATR, CHEK1 / 2, RAD51, PALB2, CLDN18.2, GPC3, TROP2, AXL, c-MET, FOLR2 / 3, α-FRα, and PD-L1.

[0016] In some embodiments, the engineered cells for improving and / or treating ovarian cancer according to the present invention, wherein the nucleic acid molecule has the sequence shown in SEQ ID NO.3.

[0017] A second aspect of the invention provides a pharmaceutical composition for improving and / or treating ovarian cancer, comprising the engineered cells described in the first aspect.

[0018] A third aspect of the present invention provides a method for preparing engineered cells for improving and / or treating ovarian cancer, comprising the step of introducing nucleic acid molecules or carriers into cells, wherein the nucleic acid molecules or carriers are those mentioned in the first aspect.

[0019] A fourth aspect of the invention provides the use of engineered cells in the preparation of medicaments for improving and / or treating ovarian cancer, wherein the engineered cells are the engineered cells described in the first aspect.

[0020] The technical effects of this invention include, but are not limited to: In some embodiments, the present invention uses a single vector to construct lentiviruses, which can significantly reduce toxicity to cells, thereby ensuring high survival rates of engineered cells.

[0021] In some embodiments, the immune escape inhibitor protein of the present invention can utilize the oxidized cholesterol ligands that are highly expressed in the tumor microenvironment as a "navigation signal," and by overexpressing the immune escape inhibitor protein in immune cells, immune cells can be recruited to the tumor site more efficiently and precisely.

[0022] In vitro and in vivo experiments of this invention demonstrate that overexpression of immune escape inhibitory protein can significantly enhance the ability of CAR-T cells to migrate into the tumor parenchyma; in the SKOV3 ovarian cancer mouse model, CAR-T cells overexpressing immune escape inhibitory protein exhibit stronger and more durable tumor clearance ability than conventional CAR-T cells; in addition, this invention is not only applicable to CAR-T cells, but also to other types of immune cells, and not only to ovarian cancer, but may also be applicable to other solid tumors that produce oxidized cholesterol. Attached Figure Description

[0023] Figure 1 To detect the expression efficiency of CAR and immune escape inhibitory proteins in cells after different lentiviral transductions by flow cytometry.

[0024] Figure 2 To detect the viability of cells 72 h after transduction with different lentiviruses using trypan blue.

[0025] Figure 3To detect the migration ability of CAR-T cells overexpressing immune escape inhibitory proteins under 7α,25-OHC chemotaxis.

[0026] Figure 4 To detect the migration ability of CAR-T cells overexpressing immune escape inhibitory proteins under chemotactic conditions in SKOV3 cell culture supernatant.

[0027] Figure 5 To detect the killing ability of CAR-T cells overexpressing immune escape inhibitory proteins in vitro.

[0028] Figure 6 The results show the anti-tumor ability of CAR-T cells overexpressing immune escape inhibitory proteins.

[0029] Figure 7 Flow cytometry was used to detect intratumoral CD3 expression of immune escape inhibitory proteins in CAR-T therapy groups. + T cell infiltration status. Detailed Implementation

[0030] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementations of the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, the numerical ranges in this invention should be understood to specifically disclose the upper and lower limits of the range and every intermediate value between them. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0033] In this invention, the terms "binding," "recognition," "targeting," "specific binding," and "specific binding" are used interchangeably and refer to the reaction between an antibody or its antigen-binding fragment and the antigen it targets. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody can bind at a rate of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (KD) binds to the antigen.

[0034] In this invention, the term "treatment" refers to therapeutic and preventative or therapeutic measures aimed at preventing or slowing (reducing) undesirable physiological changes or disorders, such as the progression of ovarian cancer. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and relief (whether partial or complete). "Treatment" also means extended survival compared to the expected survival without treatment. Those in need of treatment include those who already have the condition or disorder, those who are susceptible to the condition or disorder, or those who need to prevent the condition or disorder.

[0035] As used herein, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as desired by an investigator or clinician. Furthermore, the term "therapeutic effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of the disease or condition, compared to a corresponding subject who did not receive that amount. The term also includes, within its scope, amounts that effectively enhance normal physiological function. Generally, the effective amount as used herein varies depending on various factors, such as the given drug or compound, pharmaceutical formulation, route of administration, type of disease or symptom, subject being treated, etc., but can still be conventionally determined by those skilled in the art. The effective amount of the drug of the present invention can be readily determined by those skilled in the art using conventional methods known in the art.

[0036] engineered cells In a first aspect, the present invention provides an engineered cell that expresses a chimeric antigen receptor and an immune escape inhibitor protein, comprising a nucleic acid molecule containing a chimeric antigen receptor coding sequence and an immune escape inhibitor protein coding sequence, or a single vector containing the nucleic acid molecule.

[0037] The engineered cells of this invention can function as host cells, containing the nucleic acid molecules or vectors described in this invention. Those skilled in the art will understand that host cells include both T cells ultimately used for disease treatment and various cells used in the production of CAR-T cells, such as E. coli cells.

[0038] In this invention, immune cells include, but are not limited to, T cells, NK cells, macrophages, or dendritic cells. Among them, T cells can be various T cells well known in the art, especially various T cells commonly used in cell immunotherapy, including but not limited to peripheral blood T lymphocytes, cytotoxic cytotoxic T cells, helper T cells, inhibitory / regulatory T cells, γδ T cells, cytokine-induced killer cells, and tumor-infiltrating lymphocytes, as well as any one or more combinations of the above cells.

[0039] The nucleic acid molecules of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. In this invention, the coding sequence refers to the portion of the nucleic acid sequence that directly identifies its protein product (e.g., CAR, single-stranded antibody, hinge region, transmembrane region, intracellular signaling region, immune escape inhibitory protein, etc.). The boundary of the coding sequence is typically defined by the ribosome binding site (for prokaryotic cells) immediately upstream of the 5' open reading frame of the mRNA and the transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA. The coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0040] In this invention, the chimeric antigen receptor has the meaning known in the art; it is an artificially modified receptor that can anchor specific molecules (such as antibodies) that recognize tumor cell surface antigens onto immune cells (such as T cells), enabling the immune cells to recognize tumor antigens and kill tumor cells.

[0041] The chimeric antigen receptors suitable for use in this invention are preferably various CARs targeting ovarian cancer antigens. Typically, a CAR sequentially comprises a polypeptide that binds to the ovarian cancer antigen, a hinge region, a transmembrane region (sometimes referred to herein as a transmembrane domain), an intracellular signal transduction domain, and an intracellular co-stimulatory domain. The polypeptide that binds to the ovarian cancer antigen can be a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is an antibody or its antigen-binding fragment.

[0042] In this invention, ovarian cancer antigens include, but are not limited to, MSLN, HER2, EGFR, VEGF / VEGFR, EPCAM, MUC1, BRCA1 / BRCA2, ATM, ATR, CHEK1 / 2, RAD51, PALB2, CLDN18.2, GPC3, TROP2, AXL, c-MET, FOLR2 / 3, α-FRα, PD-L1, etc.

[0043] In a preferred embodiment, the polypeptide binding to ovarian cancer antigens of the present invention is an antibody or its antigen-binding fragment that specifically binds to any of the aforementioned ovarian cancer antigens. The antibody is preferably a monoclonal antibody, and the antigen-binding fragment is preferably a single-chain antibody. The single-chain antibody may contain a heavy chain variable region and a light chain variable region, or may consist of a heavy chain variable region, a light chain variable region, and an optional linker. In the present invention, the antibody may be a human antibody, including human-mouse chimeric antibodies or humanized antibodies. The antibody may be a secretory antibody or a membrane-anchored antibody, preferably a membrane-anchored antibody.

[0044] In one specific implementation, the ovarian cancer antigen is HER2, and the antibody targeting it is a monoclonal antibody or single-chain antibody that specifically binds to HER2.

[0045] In this invention, other components of the CAR, such as the transmembrane domain, intracellular signal transduction domain, hinge region, and intracellular costimulatory domain, can use the transmembrane domain, intracellular signal transduction domain, hinge region, and intracellular costimulatory domain conventionally used to construct various types of CARs, and are not particularly limited thereto.

[0046] In this invention, the hinge region refers to the area between the CH1 and CH2 functional regions of the immunoglobulin heavy chain. This region is rich in proline, does not form α-helices, and is prone to stretching and to some extent twisting, which is beneficial for the complementary binding between the antibody's antigen-binding site and the antigen epitope. The hinge region suitable for this invention can be selected from the CD8 extracellular hinge region, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the CD28 extracellular hinge region, the IgG4 Fc CH2CH3 hinge region, and the CD4 extracellular hinge region. In some embodiments, this invention uses the CD8 α-hinge region.

[0047] In this invention, the transmembrane region can be of natural or synthetic origin. The transmembrane region may originate from the α, β, or ζ chains of CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or T-cell receptors. Preferably, the transmembrane region is derived from CD8α.

[0048] In this invention, the intracellular signal transduction region is responsible for activating at least one normal effector function of immune effector cells expressing chimeric antigen receptors. For example, the effector function of T cells can be cytolytic activity or helper activity, including cytokine secretion. While the entire intracellular signal transduction region can generally be used, in many cases, using the whole chain is unnecessary. Regarding the use of a truncated portion of the intracellular signal transduction region, as long as it transduces effector function signals, such a truncated portion can be used instead of the whole chain. Therefore, the intracellular signal transduction region includes any truncated form of the intracellular signal transduction region sufficient to transduce effector function signals. In some embodiments, the signal transduction region is derived from at least one of CD3ζ, FcRγ (FCER1G), FcRβ (Fcε Rib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD137, CD79a, CD79b, and CD66d. Preferably, the intracellular region is derived from the human CD3ζ intracellular region.

[0049] In addition to antigen-specific signal stimulation, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. The "co-stimulatory signal transduction region" can be the cytoplasmic portion of the co-stimulatory molecule. The term "co-stimulatory molecule" refers to an associated binding chaperone on immune cells (such as T cells) that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response mediated by the immune cell, such as, but not limited to, proliferation and survival. Co-stimulatory signal transduction regions can originate from CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, and PD. L2, PD Intracellular signaling regions of at least one of L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, and 4-1BB. In some embodiments, the co-stimulatory signaling region is derived from 4-1BB. 1BB.

[0050] In this invention, the number of signal transduction regions and / or co-stimulation signal transduction regions can be one or more, and there is no particular limitation on this.

[0051] In some embodiments, the amino acid sequence of the chimeric antigen receptor of the present invention typically includes, from the N-terminus to the C-terminus, a single-chain antibody targeting ovarian cancer antigens, a hinge region, a transmembrane region, and an intracellular signaling region. An exemplary chimeric antigen receptor amino acid sequence may be shown in SEQ ID NO.1, and its coding sequence is shown in SEQ ID NO.2.

[0052] Through in-depth research, this invention has discovered that immune escape inhibitory proteins can synergistically promote the therapeutic effect of ovarian cancer, including but not limited to enhancing the infiltration of immune cells into tumor tissue, increasing the sensitivity of tumors to immunotherapy and / or relieving and reversing tumor immune escape.

[0053] In this invention, immune escape inhibitory proteins can be linked to the CAR of this invention via adapters commonly used in the art. For example, in some embodiments, the adapter is a conventional P2A sequence, but other types of self-cleaving peptide adapters can also be used to link to the CAR.

[0054] In this invention, the coding sequences of the chimeric antigen receptor and the immune escape inhibitor protein can be located in the same expression cassette or different expression cassettes. An expression cassette refers to the complete element required to express the gene of interest, including the promoter, gene coding sequence, and PolyA tailing signal sequence. The nucleic acid molecule described in this invention can be two independent nucleic acid molecules, each containing a CAR coding sequence and the immune escape inhibitor protein coding sequence, which can be the CAR expression cassette and the immune escape inhibitor protein expression cassette, respectively; alternatively, the CAR coding sequence and the immune escape inhibitor protein coding sequence can be linked into a single nucleic acid molecule by a linker or linker peptide sequence, i.e., two expression cassettes are linked into the same nucleic acid molecule by a suitable linker sequence.

[0055] The present invention also provides a vector containing the coding sequences of the CAR and immune escape inhibitory proteins described herein, and one or more regulatory sequences operatively linked to these sequences. The regulatory sequence may be a suitable promoter sequence. The promoter sequence is typically operatively linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence exhibiting transcriptional activity in a selected host cell, including mutant, truncated, and heterozygous promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to that host cell. The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator functional in a selected host cell may be used in this invention.

[0056] In some embodiments, the vector can be a cloning vector, an expression vector, or a homologous recombination vector. Specifically, the coding sequences of the CAR and immune escape inhibitory protein of the present invention can be cloned into various types of vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and granules. Cloning vectors can be used to provide the coding sequences of the CAR and immune escape inhibitory protein of the present invention, such as a nucleic acid molecule containing the coding sequence of the CAR and the coding sequence of the immune escape inhibitory protein. Expression vectors can be provided to cells in the form of viral vectors. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0057] In this invention, a suitable vector typically contains a replication origin, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers that function in at least one organism. For example, when using a retroviral vector, the retroviral vector typically contains a replication origin, a 3'LTR, a 5'LTR, the coding sequences for the CAR and immune escape inhibitory proteins described in this invention, and optional markers.

[0058] In this invention, suitable promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter sequences, early promoters of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), long terminal repeat (LTR) promoters of human immunodeficiency virus (HIV), MoMuLV promoters, avian leukemia virus promoters, immediate early promoters of Epstein-Barr virus (EBV), promoters of Russ's sarcoma virus, and human gene promoters, such as, but not limited to, actin promoters, myosin promoters, heme promoters, and creatine kinase promoters.

[0059] In this invention, selectable markers include any one or both of selectable marker genes or reporter genes to facilitate the identification and selection of expressing cells from a cell population infected with a viral vector. Useful selectable marker genes include, for example, antibiotic resistance genes. Suitable reporter genes include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein.

[0060] The preparation of the nucleic acid molecules of the present invention is not particularly limited. They can be obtained by PCR amplification or by directly synthesizing the nucleic acid molecules described in the present invention.

[0061] In a preferred embodiment, the nucleotide sequence of the nucleic acid molecule containing the coding sequence of CAR and immune escape inhibitory protein of the present invention is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0062] Pharmaceutical Composition One aspect of the present invention provides a pharmaceutical composition comprising CAR-T cells as described herein and optionally pharmaceutically acceptable excipients. In this invention, pharmaceutically acceptable excipients refer to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, including but not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. More specifically, suitable pharmaceutically acceptable excipients may be those commonly used in the art for CAR-T cell administration.

[0063] In this invention, the pharmaceutical composition contains a therapeutically effective amount of CAR-T cells. A therapeutically effective amount refers to a dose that can achieve therapeutic, preventative, alleviating, and / or remission of a disease or symptom in a subject. Those skilled in the art can determine the therapeutically effective amount based on factors such as the patient's age, sex, the nature and severity of the disease, and other physical conditions. In this invention, the subject or patient generally refers to a mammal, particularly a human.

[0064] In some embodiments, the pharmaceutical composition may contain at least one additive selected from: a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, a growth inhibitor, and an active pharmaceutical agent required for the specific indication to be treated. The specific amount of the additive may be adjusted as needed.

[0065] Methods for preparing engineered cells One aspect of the present invention provides a method for preparing engineered cells, comprising the step of introducing nucleic acids or vectors into mammalian cells. It should be noted that methods for introducing nucleic acids or vectors into mammalian cells are known in the art, and the vectors can be transfected into immune cells by physical, chemical, or biological methods. Physical methods for introducing vectors into immune cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, electroporation, etc. Chemical means for introducing nucleic acids or vectors into immune cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as an in vitro delivery medium is a liposome (e.g., an artificial membrane vesicle). Biological methods for introducing nucleic acids or vectors into immune cells include the use of DNA and RNA vectors. In some embodiments, transduced or transfected immune effector cells proliferate in vitro after the introduction of nucleic acids or vectors.

[0066] In some embodiments, the preparation method of the present invention further includes further evaluation or screening of transduced or transfected immune cells to select modified immune cells (engineered immune cells).

[0067] application The present invention also provides the use of engineered cells in the preparation of medicaments for the treatment and / or improvement of ovarian cancer.

[0068] The present invention further provides the use of engineered cells according to the invention in combination with other drugs in the preparation of a combination drug for ovarian cancer. Preferably, the other drugs include diagnostic agents, preventive agents, and / or therapeutic agents.

[0069] In this invention, the administration method of engineered cells or drugs containing them is particularly limited, and is preferably administered systemically.

[0070] Example 1. Isolation and activation of primary human T cells Peripheral blood was collected from healthy donors and anticoagulated with sodium heparin. Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation: blood was diluted with an equal volume of PBS and carefully added to the surface of human lymphocyte separation medium (Ficoll-Paque PLUS), and centrifuged at 800×g for 30 minutes at room temperature. The middle white membrane layer (monuclear cell layer) was aspirated, washed twice with PBS containing 10% FBS, and resuspended in complete T cell culture medium (X-VIVO15™ medium containing 50 U / mL IL-2). Using CD3 / CD28 T cell activation and expansion magnetic beads (Gibco™ Dynabeads™ CD3 / CD28), at a cell-to-bead ratio of 1:1, PBMCs and magnetic beads were co-incubated at 37°C, 5% CO2 for 48-72 hours to activate T cells.

[0071] 2. Construction and Packaging of Lentiviral Vectors (1) CAR vector construction: scFv targeting human HER2 (derived from commercially available trastuzumab) was designed and sequentially linked with the FLAG tag, CD8α hinge region, CD8 transmembrane domain, 4-1BB co-stimulatory domain and CD3ζ signaling domain through molecular cloning technology to construct a CAR expression cassette, which was then cloned into a lentiviral transfer plasmid (pCDH vector) located under the EF1α strong promoter.

[0072] (2) Construction of the immune escape inhibitory protein vector: The complete coding sequence of the human immune escape inhibitory protein was cloned into the lentiviral transfer plasmid (pCDH vector), located under the EF1α strong promoter.

[0073] (3) Construction of CAR-immune escape inhibitory protein vector: Based on the CAR vector, the complete coding sequence of human immune escape inhibitory protein is linked using P2A peptide.

[0074] (4) Lentiviral Packaging: Using a third-generation lentiviral packaging system, the above-mentioned transfer plasmids and packaging plasmids (pLP1, pLP2, pLP-VSVG) were co-transfected into HEK293FT cells at a ratio of 2:2:1:3. At 48 and 72 hours after transfection, cell supernatants containing lentiviral particles were collected, the virus was concentrated by ultracentrifugation, and aliquoted and stored at -80°C.

[0075] 3. T cell gene modification and in vitro expansion Viral transduction was performed 48-72 hours after T cell activation. Different concentrations of viral solution were added to the experimental and control wells at an MOI of 20, along with polybrene to promote infection. Adequate amounts of fresh culture medium and IL-2 (50-100 U / mL) were added, and the cells were cultured at 37°C and 5% CO2. After 8-12 hours of transduction, the culture medium was replaced with freshly prepared complete culture medium containing IL-2 (100-200 U / mL). Cells were passaged or replenished with fluid every 2-3 days according to cell density, maintaining a cell density of 0.5-2 × 10⁶ cells / day. 6 cells / mL, continuously amplified and cultured for 3-14 days.

[0076] 4. Transduction efficiency and phenotypic identification Around day 3 of amplification culture, a subset of cells was harvested for flow cytometry analysis. The expression efficiency of CAR was detected using an anti-FLAG antibody. The overexpression efficiency of the immune escape inhibitor protein (EBI2) was detected using a commercially available anti-human EBI2 antibody (Biolegend).

[0077] The results showed that double-positive cell populations could be successfully detected in the experimental groups (CAR-T+ immune escape inhibitor protein and immune escape inhibitor protein-CAR-T), and the expression efficiency was higher than 50%, indicating that the cell preparation was successful.

[0078] 5. In vitro migration experiment Migration experiments were performed using Transwell chambers (24-well plates, 5.0 μm polycarbonate membranes). The lower chamber was filled with culture medium containing different concentrations (0, 50, 100, 150 nM) of the immune escape inhibitor ligand 7α,25-dihydroxycholesterol (7α,25-OHC), or serum-free culture supernatant of SKOV3 cells (as a source of tumor-derived chemokines). T cells from both experimental and control groups were resuspended in serum-free medium and added to the upper chamber. After incubation at 37°C for 4 hours, cells in the lower chamber were collected and counted.

[0079] 6. In vitro tumor killing experiment SKOV3 cells were used as target cells and co-incubated with various effector T cells (E:T) at a ratio of 10:1. After 24 hours, the in vitro specific killing ability of CAR-T cells was evaluated by the lactate dehydrogenase (LDH) release assay.

[0080] 7. In vivo anti-tumor efficacy studies Animal model establishment: Six-week-old female NSG-immunodeficient mice were selected. Each mouse was subcutaneously injected with 5 × 10⁵ NSG molecules on its right side. 6 Mice were randomly divided into 3 groups (n=5) when the tumor volume reached approximately 100-150 mm³.

[0081] Cell reinfusion: 1 × 10⁻⁶ cells were reinfused into each mouse via the tail vein in a single dose. 7 Mice in the PBS group were injected with an equal volume of PBS as a negative control. The corresponding genetically engineered T cells were suspended in 200 μL PBS.

[0082] Treatment monitoring: The long and short diameters of the tumor were measured every 3 days using calipers, and the volume was calculated according to the formula (volume = long diameter × short diameter). 2 Calculate the tumor volume using ×0.5) and plot the growth curve.

[0083] 8. Analysis of Immune Infiltration in Tumor Tissue Mice were sacrificed at the experimental endpoint, and tumor tissue was removed. After being cut into small pieces, the tissue was digested and centrifuged using a Miltenyi tissue dissociation kit and separated using Ficoll-Paque PLUS human lymphocyte separation medium to obtain tumor-infiltrating immune cells. Flow cytometry was used to detect CD3+ in the tumor tissue. + The number of infiltrating T cells.

[0084] II. Experimental Results Figure 1 The results showed that all three different expression vectors were successfully constructed: a single overexpression plasmid of immune escape inhibitor protein, a single overexpression plasmid of CAR, and a CAR-immune escape inhibitor protein fusion co-expression plasmid. Further experiments confirmed that CAR-T cells with high expression of immune escape inhibitor protein could be obtained under both the conditions of separate overexpression of CAR and immune escape inhibitor protein and co-expression of both.

[0085] Figure 2 The results showed that when using lentiviruses that overexpressed CAR and immune escape inhibitory protein respectively, the survival rate of T cells decreased significantly, indicating significant cytotoxicity under this condition; while the CAR-immune escape inhibitory protein fusion co-expression scheme significantly improved cell survival, indicating that it is a more feasible construction strategy suitable for subsequent functional experiments.

[0086] Figure 3 The results showed that under 7α,25-OHC stimulation, the migration ability of immune escape inhibitory protein-CAR-T cells was significantly enhanced, and the effect showed a concentration-dependent trend. Figure 4 The results showed that SKOV3 cell supernatant significantly promoted the migration of immune escape inhibitory protein-CAR-T cells, suggesting that these tumor cells have the ability to secrete 7α,25-OHC or ligand molecules with similar activity.

[0087] Figure 5 The results showed that after co-culturing with target cells SKOV3, the LDH release in the immune escape inhibitor protein-CAR-T cell group was significantly higher than that in the control group, indicating that the introduction of immune escape inhibitor protein enhanced the killing effect of CAR-T cells on target cells.

[0088] Figure 6 The results showed that in the SKOV3 subcutaneous xenograft tumor model (CDX), compared with the PBS-treated group and the control CAR-T cell group, intravenous infusion of immune escape inhibitory protein-CAR-T cells significantly inhibited the growth of mouse tumors, indicating that immune escape inhibitory protein-CAR-T cells have good anti-tumor activity in vivo.

[0089] Figure 7 The results showed that, compared with the PBS-treated group and the control CAR-T cell group, the CD3+ level in the tumor tissue of mice in the immune escape inhibitory protein-CAR-T cell group was significantly higher. + The significantly increased number of infiltrating T cells demonstrates that the high expression of immune escape inhibitory proteins effectively enhances the infiltration ability of CAR-T cells at the tumor site.

[0090] The sequence information involved in this invention is as follows: Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A pharmaceutical composition for improving and / or treating ovarian cancer, characterized in that, It includes engineered cells, said engineered cells being chimeric antigen receptor T cells, said engineered cells containing nucleic acid molecules composed of a coding sequence for a chimeric antigen receptor and a coding sequence for an immune escape inhibitory protein; or The engineered cells contain a single carrier of the nucleic acid molecules; The chimeric antigen receptor includes a single-chain antibody targeting HER2, a CD8α hinge region, a CD28 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 intracellular signal transduction domain. The immune escape inhibitory protein is GPR183. The coding sequence of the chimeric antigen receptor and the coding sequence of GPR183 are linked by the coding sequence of a linker peptide. The sequence of the nucleic acid molecule is shown in SEQ ID NO.

3.

2. The pharmaceutical composition for improving and / or treating ovarian cancer according to claim 1, characterized in that, The pharmaceutical composition further includes a pharmaceutically acceptable carrier.

3. The pharmaceutical composition for improving and / or treating ovarian cancer according to claim 2, characterized in that, T cells include cytotoxic T cells or chimeric antigen receptor T cells.

4. A method for preparing a pharmaceutical composition for improving and / or treating ovarian cancer, characterized in that, include: (1) Preparing a nucleic acid molecule or vector, wherein the nucleic acid molecule or vector is the nucleic acid molecule or vector of any one of claims 1-3, wherein the vector contains the nucleic acid molecule, and the sequence of the nucleic acid molecule is shown in SEQ ID NO.3; (2) Introduce the nucleic acid molecule or the vector into the cell.

5. The use of the reagent in the preparation of medicaments for improving and / or treating ovarian cancer, characterized in that, The reagent comprises engineered cells as described in any one of claims 1-3, wherein the engineered cells contain nucleic acid molecules as shown in SEQ ID NO. 3.

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