Universal anti-tumor SynNotch synthetic receptor combination and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a universal anti-tumor SynNotch synthetic receptor combination, a Synn-MSC-BiTE / TriTE cell and an application of the SynNotch synthetic receptor combination and the Synn-MSC-BiTE / TriTE cell. According to the invention, BiTE / TriTE and MSC cells are integrated to obtain recombinant Syn-MSC-BiTE / TriTE cells, and a novel general tumor in-situ cell therapy, namely Syn-MSC-BiTE / TriTE, which takes MSC as a cell carrier and regulates BiTE / TriTE secretion mediating T cells through a SynNotch system to carry out tumor targeted killing is constructed. The receptor combination disclosed by the invention not only can be used for carrying out active permeation of solid tumors by utilizing the tumor chemotaxis of MSC, but also can be specifically and continuously expressed and accumulated in tumor tissues under the regulation and control of a SynNotch system, so that a powerful and long-acting tumor in-situ T cell anti-tumor reaction is mediated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a universal anti-tumor SynNotch synthetic receptor combination, Syn-MSC-BiTE / TriTE cells and applications. Background Art

[0002] Malignant tumors are one of the major diseases that seriously threaten the health of Chinese residents. According to data disclosed by the National Cancer Center of China: in 2016, the number of new cases of malignant tumors in China was 4.064 million, and 2.413 million people died of malignant tumors; in 2020, the number of new cases of malignant tumors increased to 4.568 million, and the number of tumor-related deaths increased to 3.002 million in the same year (GLOBOCAN2020). In recent years, with the continuous innovation of technology, the continuous deepening of clinical medical research, and the continuous standardization of clinical treatment, traditional chemotherapy / radiotherapy, surgical treatment combined with emerging targeted therapy, immunotherapy and other intervention methods have achieved remarkable results in the clinical treatment of malignant tumors. In particular, chimeric antigen receptor T-cell therapy (CAR-T) achieved 11 years of cancer-free survival in clinical trials of acute lymphoblastic leukemia (ALL), revealing the advent of the era of tumor immunotherapy. Unlike traditional therapies, immunotherapy is to regulate the patient's own immune system and reactivate the anti-tumor immune response to achieve targeted killing of tumor cells. Among them, immune checkpoint inhibitor (ICI) and adoptive cell transfer therapy (ACT) are the two most widely used tumor immunotherapy strategies in clinical practice.

[0003] Immune checkpoints are a class of membrane surface proteins that regulate the degree of immune cell activation. They can positively regulate the activation of immune cells and also serve as a negative feedback system to prevent overactivation of immune cells. During the activation of tumor antigen-specific T cells, the immunogenic death of tumor cells will release a large number of tumor-specific antigens (oncogenic virus antigens, new antigens produced by mutations) and be taken up, processed, and co-presented to the cell surface with HLA molecules by antigen presenting cells (APCs), and bind to specific T-cell receptors (TCRs), thereby activating the TCR signaling pathway. CD28 on the surface of T cells binds to B7 molecules (B7.1 or B7.2) on the surface of APCs, activating costimulatory signals to further activate T cells; at the same time, activated T cells will upregulate the expression of inhibitory ligand CTLA-4, competitively bind to B7 molecules on the surface of APCs, thereby inhibiting overactivation of T cells and playing a negative feedback regulatory role. Tumor cells and other immune cells can activate T cell immunosuppressive receptors (PD-1, TIM-3, LAG3, VISTA, etc.) by expressing a series of immunosuppressive ligands (PD-L1, B7H3, B7H4, etc.), inhibiting T cell activation and achieving immune escape. To date, 11 immune checkpoint inhibitors (5 PD-1 monoclonal antibodies, 3 PD-L1 monoclonal antibodies and 2 CTLA-4 monoclonal antibodies) have been approved by the FDA for the clinical treatment of various malignant tumors; 17 immune checkpoint inhibitors have also been approved for marketing in my country for the first-line, second-line and later-line treatment of various malignant tumors such as lung cancer, liver cancer, esophageal cancer, nasopharyngeal carcinoma, melanoma, and blood tumors.

[0004] However, the clinical application of immune checkpoint inhibitors still faces huge challenges: 1) Low clinical response rate: primary resistance mechanisms (low level of PD-L1 expression, low tumor mutation load and low level of tumor infiltrating lymphocytes) and secondary resistance mechanisms (loss of new antigens, defective antigen presentation mechanism, compensatory expression of other immune checkpoints and inhibitory tumor immune microenvironment) make the remission rate of ICI monotherapy only 10-25%; 2) Obvious immune-related side effects: The use of ICI relieves systemic T cell immunosuppression, enhances the activation and proliferation levels of T cells, and inhibits the immune regulatory function of Treg cells, disrupting the body's immune homeostasis and causing immune-related adverse events (irAEs). The results of a five-year follow-up of CheckMate 067 by the American Cancer Research Institute Dana-Farber showed that 59%, 22%, and 28% of patients with advanced melanoma who received nivolumab combined with ipilimumab or single-drug use, respectively, experienced grade 3-4 treatment-related adverse events; a meta-analysis of 36 ICI-related phase 2 / 3 clinical studies found that 54%-76% of patients experienced varying degrees of irAEs when receiving ICI treatment. Clinical intervention for irAEs is often through oral glucocorticoids or other immunosuppressants (such as infiximab, etc.), and transient immunosuppressive interventions can affect the efficacy of ICI. Therefore, finding new delivery pathways and implementing immune checkpoint intervention strategies that can target the tumor microenvironment are crucial to reducing the occurrence of irAEs adverse events and improving the effectiveness of ICI treatment.

[0005] The efficacy of ICI is limited by many factors, such as the number of antigen-specific T cells in tumor tissue, the level of immune cell infiltration, and the expression level of immune checkpoints. Unlike the ICI immunotherapy strategy, ACT is achieved by isolating immune cells from tumor patients, amplifying them in vitro, functionally identifying them, and gene editing them, so that they have direct anti-tumor immune activity, and then returning them to the patient's body to achieve tumor treatment. Among them, T cell-dependent ACT therapies mainly include chimeric antigen receptor T cells (CAR-T) and T cell receptor-engineered T cells (TCR-T) therapy. In terms of mechanism of action, CAR-T therapy is similar to TCR-T therapy: CAR-T cells can recognize tumor-associated antigens on the surface of tumor cell membranes through the ScFv functional domain in the CAR molecule, and then transmit the signal to the ITAM domain in the CD3zeta chain in the CAR molecule, simulating TCR signal activation and mediating downstream signal transduction, thereby activating CAR-T cells. In clinical applications, CAR-T and TCR-T have their own advantages and disadvantages: 1) Antigen differences: CAR-T can only recognize tumor cell membrane surface antigens; TCR-T can recognize specific tumor antigen peptides / HLA complexes through TCR, so TCR-T can recognize different types of antigens such as tumor neoantigens generated by mutations, differentially expressed antigens, carcinoembryonic / cancer testicular antigens. 2) HLA restriction: CAR-T is not HLA-restricted and can be widely used in antigen-positive tumor patients; while TCR-T's antigen recognition is HLA-restricted and can only be applied to antigen-positive tumor patients with specific HLA typing. In addition, the individual differences in genomic mutations in tumor patients are extremely large, so the clinical application range of the same TCR-T is extremely limited. 3) Preparation difficulty: For each tumor patient, the preparation of TCR-T must go through multiple processes such as whole genome sequencing (looking for potential tumor neoantigen peptides), HLA binding ability prediction and verification, tumor antigen peptide / HLA complex specific binding TCR sequence screening and verification, and TCR-T preparation, which is time-consuming and difficult to prepare; while CAR has a simple molecular structure and a relatively simple preparation process. 4) Side effects: CAR-T can only recognize tumor-associated antigens, and the side effects of on-target off-tumor (OTOT) are obvious, and it is easy to cause cytokine storms; TCR-T can recognize tumor-specific neoantigens, and its specificity is significantly better than CAR-T, and it rarely produces cytokine storms.

[0006] So far, a number of CAR-T therapeutic products targeting BCMA and CD19 have been approved by the FDA for marketing; four products have also been approved in China, mainly for the clinical treatment of hematological tumors. In January 2022, the first TCR-T cell therapy product (Tebentafusp, trade name Kimmtrak) was approved by the FDA for the treatment of HLA-A*02:01-positive unresectable or metastatic uveal melanoma; so far, 63 TCR-T therapeutic drugs have entered the clinical trial stage, including 34 in Phase I, 23 in Phase II, and 5 clinical applications, mainly concentrated in solid tumors. Although T cell-dependent ACT therapy has shown significant efficacy in many preclinical studies and some clinical studies, the clinical application of both CAR-T and TCR-T still faces huge challenges: the lack of effective targets and high-specificity and high-affinity tumor targeting sequences greatly limits the application of CAR-T / TCR-T therapy in certain cancers; the endogenous resistance mechanism of tumor cells and the acquired immunotherapy tolerance mechanism seriously affect the efficacy of CAR-T / TCR-T; excessive activation of CAR-T cells can also trigger severe cytokine storms and cause nerve damage. It is reported that the cost of a single infusion of the first FDA-approved CAR-T drug (Tisagenlecleucel) is as high as $475,000. The high cost of treatment also makes it difficult for ACT therapy to truly benefit the people. Due to the existence of graft versus host disease (GvHD), the current CAR-T / TCR-T cell therapy must still follow the restrictions of autologous infusion of immune cells. Even for patients with the same type of tumor, each patient must go through the complete preparation process. However, due to different genetic backgrounds, different disease progression, and large differences in immune cell status among patients, there are large differences between batches of CAR-T / TCR-T cell preparation, product quality control is difficult, the preparation cycle is long, and the cost is high.

[0007] MSC was first discovered by Friedenstein's research team in 1968 and has been proven to be widely present in various tissues and organs throughout the body, showing excellent in vitro proliferation activity. The International Society for Cellular Therapy stipulates that MSC surface molecular markers must meet the expression (positive ≥ 95%) of CD105, CD73 and CD90, and not express (<2%) CD45, CD34, CD11b or CD14, CD19 or CD79a and HLA-DR. MSC grows adherently under normal culture conditions and has the ability to differentiate into osteoblasts, adipocytes or chondrocytes under specific culture conditions in vitro. A large number of studies have shown that under the action of chemokines / chemokine receptors, MSC can home to a variety of tumors, including breast cancer, liver cancer, lung cancer and other primary tumors, as well as primary metastatic tumors such as melanoma lung metastases, breast cancer lung metastases, pancreatic cancer lung metastases, etc., and are ideal materials for constructing tumor-targeted drug delivery systems. More importantly, MSCs express low levels of HLA-I molecules, and almost no HLA-II molecules, as well as immune regulation-related genes such as Fas ligand, T cell co-activators B7.1, B7.2, CD40 or CD40L, making MSCs very low in immunogenicity and safe for allogeneic transfusion. In Europe, two allogeneic MSC therapies have been approved by the EMA for the treatment of corneal damage and complex anal fistulas in patients with Crohn's disease; the US FDA has approved an allogeneic MSC cell product for the treatment of GvHD; in Japan, MSC therapy has also been approved for the treatment of spinal cord injury, arthritis, and GvHD. In addition, tumor-related clinical studies have also confirmed that allogeneic transfusion of MSCs in tumor patients has shown good safety (NCT02500047, NCT02008539, NCT01983709). Ease of access, good in vitro proliferation activity, low immunogenicity, and good biosafety all suggest the potential of MSCs as universal cell therapy cell carriers.

[0008] Studies have found that the effect of unmodified MSCs on tumor cells is controversial. MSCs can regulate T cell subset differentiation by expressing inhibitory ligands such as PD-L1, HLA-G1, ICAM-1, and VCAM-1 through direct contact; they can also regulate the function and activation state of immune cells by secreting immunoregulatory cytokines or metabolites such as IDO, PGE2, IL-10, and TGF-b, and establish immune tolerance in the repair of inflammatory damage in the body, thereby maintaining immune homeostasis. On the contrary, RC Zhao's research team found that adipocyte-derived MSCs can negatively regulate the WNT signaling pathway in tumor cells by secreting dickkopf-1, thereby inhibiting tumor cell proliferation; and MSCs derived from healthy human bone marrow can specifically migrate to Kaposi's sarcoma, weaken the activation of Akt signals in tumor cells by direct contact, and inhibit the proliferation of Kaposi's sarcoma. Genetically modified MSCs can exert anti-tumor activity by releasing overexpressed IFN-b, while not affecting their classic phenotype and tumor chemotaxis characteristics. Based on previous studies, it was also found that the immune phenotypes and expression profiles of immune-regulatory cytokines and growth factors of MSCs from different tissue sources are completely different (research basis), suggesting that different tissue sources of MSCs will affect their immune regulation ability. Based on this, selecting MSCs with appropriate immune phenotypes and empowering them through genetic modification can enable them to have significant anti-tumor activity.

[0009] Bispecific T-cell engager (BiTE) is composed of two single-chain variable fragments (ScFv) connected by a small molecule peptide segment. It can spontaneously bind to CD3 molecules and tumor cell surface antigens, draw T cells closer to tumor cells, and then form immune synapses to exert targeted killing activity. BiTE has a small molecular weight (~55 kDa) and high flexibility. It can mediate effective anti-tumor response even at very low concentrations (10 pg / ml), and the effector cell to target cell ratio is extremely low (<1: 100). However, BiTE also faces many difficulties in clinical application: due to its small molecular weight, BiTE has a short half-life (~2 days), and multiple administrations to maintain the necessary blood drug concentration will cause a series of side effects such as CRS; in addition, the immunosuppressive microenvironment is also an important factor limiting the efficacy of BiTE.

[0010] This invention innovatively proposes a new type of universal tumor in situ cell therapy concept: combining BiTE / TriTE therapy with cell therapy, constructing a universal tumor in situ cell new system (Syn-MSC-BiTE / TriTE) with MSC as cell carrier, regulating BiTE / TriTE secretion through SynNotch system to mediate T cells for tumor targeted killing. Not only can the tumor chemotaxis of MSC be used for active penetration of solid tumors, but also the specific and continuous expression and accumulation in tumor tissues under the regulation of SynNotch system can mediate strong and long-lasting tumor in situ T cell anti-tumor response. Summary of the invention

[0011] The purpose of the present invention is to overcome the problems existing in the prior art and provide a universal anti-tumor SynNotch synthetic receptor combination, Syn-MSC-BiTE / TriTE cells and applications. The present invention combines BiTE / TriTE therapy with cell therapy to construct a universal tumor in situ cell therapy (Syn-MSC-BiTE / TriTE) with MSC as a cell carrier, and regulates BiTE / TriTE secretion through the SynNotch system to mediate T cells for tumor targeted killing, which can mediate a strong and long-lasting tumor in situ T cell anti-tumor response.

[0012] The purpose of the present invention and the solution to the technical problem are achieved by adopting the following technical solutions.

[0013] The first aspect of the present invention provides a universal anti-tumor SynNotch synthetic receptor combination, the synthetic receptor combination comprising: A primary synNotch synthetic receptor, wherein the primary synNotch synthetic receptor comprises a ScFv fragment targeting a tumor-specific antigen, a mouse Notch receptor core domain, a yeast transcription complex, and a suicide gene RQR8 domain connected in sequence; A secondary synNotch expression plasmid, wherein the secondary synNotch expression plasmid comprises a 5x UAG promoter and a functional protein connected in sequence, wherein the functional protein comprises the following (i) to (iii): (i) BiTE or TriTE; (ii) T cell chemokines; and (iii) immune checkpoint inhibitors; The functional proteins are connected via P2A peptides.

[0014] In a preferred embodiment of the present invention, the ScFv fragment and the mouse Notch receptor core domain are connected via the TM transmembrane domain, the yeast transcription complex and RQR8 are connected via the P2A peptide, and the ScFv fragment is also connected to a CMV promoter.

[0015] In a preferred embodiment of the present invention, the ScFv comprises a heavy chain variable region VH and a light chain variable region VL, and optionally comprises a connecting peptide for connecting the heavy chain variable region VH and the light chain variable region VL; the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.2, and the connecting peptide is SGGGGSGGGGSGGGGS; the amino acid sequence of the ScFv is shown in SEQ ID NO.3.

[0016] In a preferred embodiment of the present invention, the mouse Notch receptor core domain comprises Lin12-Notchrepeat, a heterodimer domain and a transmembrane region, and its amino acid sequence is shown in SEQ ID NO.10.

[0017] In a preferred embodiment of the present invention, the yeast transcription complex comprises any one of Gal4-VP64, ZFHD1-VP64, and Gal4-KRAB.

[0018] In a preferred embodiment of the present invention, the RQR8 domain comprises a CD8 TM transmembrane domain, a first CD20 mimotope domain, a CD34 epitope domain and a second CD20 mimotope domain of an anti-tumor monoclonal antibody connected sequentially.

[0019] In a preferred embodiment of the present invention, the BiTE is an Anti-Trop2 ScFv obtained based on anti-Trop-2 mAb, and is connected to an Anti-CD3 ScFv obtained based on anti-CD3 mAb via a connecting peptide to obtain a bispecific T cell engager.

[0020] In a preferred embodiment of the present invention, the amino acid sequence of the Anti-Trop2 ScFv is shown as SEQ ID NO.4, the amino acid sequence of the Anti-CD3 ScFv is shown as SEQ ID NO.5, and the amino acid sequence of the BiTE is shown as SEQ ID NO.6.

[0021] In a preferred embodiment of the present invention, the TriTE is a trispecific T cell engager based on BiTE and connected to interleukin IL-15 via Sushi polypeptide.

[0022] In a preferred embodiment of the present invention, the amino acid sequence of the Sushi polypeptide is shown as SEQ ID NO.7, the amino acid sequence of the interleukin IL-15 is shown as SEQ ID NO.8, and the amino acid sequence of the TriTE is shown as SEQ ID NO.9.

[0023] In a preferred embodiment of the present invention, the T cell chemokines include CCL5 and CXCL19.

[0024] In a preferred embodiment of the present invention, the immune checkpoint inhibitors include Anti-PD-L1 and Anti-PD-1.

[0025] The second aspect of the present invention provides a coding gene, which encodes the universal anti-tumor SynNotch synthetic receptor combination as described in any of the above items.

[0026] In a preferred embodiment of the present invention, the nucleotide sequence of the gene encoding the SynNotch synthetic receptor combination is shown as SEQ ID NO.11.

[0027] The third aspect of the present invention provides an expression vector, which expresses the encoding gene described above.

[0028] The fourth aspect of the present invention provides a host cell, which comprises the SynNotch synthetic receptor combination, encoding gene, or expression vector described in any one of the above items.

[0029] In a preferred embodiment of the present invention, the host cell is an animal cell, a plant cell or a microbial cell.

[0030] The fifth aspect of the present invention provides a universal anti-tumor system, comprising recombinant target cells obtained by transfecting target cells with the SynNotch synthetic receptor combination, encoding gene, or expression vector described in any of the above items, wherein the target cells are immune cells or pluripotent stem cells.

[0031] In a preferred embodiment of the present invention, the target cells are mesenchymal stem cells MSC, and the recombinant target cells are Syn-MSC-BiTE / TriTE cells.

[0032] The sixth aspect of the present invention provides a universal anti-tumor method, comprising transfecting a target cell with the SynNotch synthetic receptor combination, encoding gene, or expression vector described in any of the preceding items to obtain a recombinant target cell, wherein the target cell is an immune cell or a pluripotent stem cell.

[0033] In a preferred embodiment of the present invention, the target cells are mesenchymal stem cells MSC, and the recombinant target cells are Syn-MSC-BiTE / TriTE cells.

[0034] In a preferred embodiment of the present invention, the method further comprises the step of treating the recombinant target cells by intravenous injection.

[0035] The seventh aspect of the present invention further provides a pharmaceutical composition comprising any of the above-mentioned SynNotch synthetic receptor combinations, encoding genes, expression vectors, host cells, or universal anti-tumor systems; and a pharmaceutically acceptable carrier; The medicament is used to treat a disease in a subject in need of the medicament, and the disease includes a tumor.

[0036] The eighth aspect of the present invention also provides a use of the SynNotch synthetic receptor combination, encoding gene, expression vector, host cell, or universal anti-tumor system described in any of the above items in the preparation of a drug for treating tumors.

[0037] By means of the above technical solution, the present invention has at least the following advantages: 1. The present invention obtains a universal cell therapy product, which can realize allogeneic transfusion and can be prepared once for multiple uses. It has small batch differences, short preparation cycle and low cost.

[0038] 2. The present invention increases the controllability and safety in vivo by adding suicide genes to the SynNotch receptor.

[0039] 3. Based on the tumor tropism of MSC and the gating characteristics of the SynNotch system, the present invention enables Syn-MSC-BiTE / TriTE to be specifically activated only in tumor tissues, exerting tumor in situ and antigen-specific anti-tumor responses, thus greatly avoiding the risk of systemic toxicity such as CRS.

[0040] 4. The present invention is based on the structural characteristics of BiTE / TriTE and can give full play to the anti-tumor immune response of T cells.

[0041] 5. The SynNotch synthetic receptor of the present invention can effectively recruit more T cells by releasing T cell chemokines in situ, and is also effective for patients with immune desert type.

[0042] 6. Under the action of immune checkpoint inhibitors, the anti-tumor response of T cells can be further enhanced in situ in the tumor.

[0043] 7. The Syn-MSC-BiTE / TriTE of the present invention exerts anti-tumor effects by recruiting and activating T cells rather than directly killing them. The dosage used is much lower than that of cell therapies such as CAR-T / TCR-T, which reduces the burden on patients and further reduces costs.

[0044] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The Syn-MSC-BiTE system of the present invention and a schematic diagram of the anti-tumor process of the system are shown; Figure 2 Flow cytometry analysis of the expression of classical CD molecules in MSCs is shown; Figure 3 Shown is a sequence analysis diagram of MSC immunophenotype from different sources; Figure 4 A schematic diagram of the structure of the SynNotch synthetic receptor combination of the present invention is shown; Figure 5 The activation of the Syn-MSC-BiTE / TriTE system was identified by fluorescence microscopy imaging and flow cytometry analysis; Figure 6 The cell viability was detected by Luciferase Assay; Figure 7 Tumor growth in mice is shown. DETAILED DESCRIPTION

[0046] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] [the term] As used in this disclosure, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one, two or more of the features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0048] As used in the present disclosure, the terms “comprising” or “including” are open expressions, namely including the contents specified in the present invention but not excluding other contents.

[0049] As used in this disclosure, the term "coding gene" refers to a nucleic acid fragment that provides a template that can be used to produce a gene product. In certain embodiments, the gene fragment includes regulatory sequences preceding and following the coding sequence.

[0050] As used in the present disclosure, the terms "nucleic acid", "nucleotide" or "nucleic acid sequence", "nucleic acid molecule", "nucleic acid fragment" or "polynucleotide" are used interchangeably. A polynucleotide molecule is a biopolymer composed of nucleotide monomers covalently bonded in a chain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides with different biological functions. DNA consists of two polynucleotide chains, each of which is helical. In nature, RNA usually occurs in a single-stranded form that folds on itself. Exemplary types of RNA include double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), messenger RNA (mRNA), antisense RNA, transfer RNA (tRNA), small nuclear RNA (snRNA) and ribosomal RNA (rRNA).

[0051] As used in this disclosure, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0052] As used in the present disclosure, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0053] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

[0054] As used in this disclosure, the term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other untoward reactions when administered to animals or humans, as appropriate. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, etc. that can be used as media for pharmaceutically acceptable substances.

[0055] As used in the present disclosure, the terms "cancer", "malignancy", "neoplasm", "tumor" and "cancer" are used interchangeably and refer to a disease, disorder or condition in which cells exhibit or exhibit relatively abnormal, uncontrolled and / or autonomous growth, so that they exhibit or exhibit an abnormally elevated proliferation rate and / or an abnormal growth phenotype. In some embodiments, for example, as proposed herein, a tumor may include one or more cancers. In some embodiments, for example, as proposed herein, a tumor may be or include precancerous (e.g., benign), malignant, pre-metastatic, metastatic and / or non-metastatic cells. In some embodiments, for example, as proposed herein, a tumor may be or include a solid tumor. In some embodiments, for example, as proposed herein, a tumor may be or include a melanoma. In general, examples of different types of cancer known in the art include, for example, colorectal cancer, hematopoietic cancers including leukemias, lymphomas (Hodgkin and non-Hodgkin), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, solid tissue cancers, squamous cell carcinomas of the oral cavity, pharynx, larynx and lung, liver cancer, genitourinary cancers such as prostate cancer, cervical cancer, bladder cancer, uterine cancer and endometrial cancer, as well as renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer and cancers of the nervous system, benign lesions such as papilloma, etc.

[0056] As used in this disclosure, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The recombinant cell or pharmaceutical composition of the present invention can be administered by any common route, as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous injection, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0057] As used in this disclosure, the term "treatment" refers to the use of drugs to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a chimeric polypeptide, recombinant cell, pharmaceutical composition or drug to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a chimeric polypeptide, recombinant cell or pharmaceutical composition described herein to an individual in need.

[0058] The present invention achieves the purpose of activating T cells to exert antigen-specific anti-tumor response by constructing a universal anti-tumor SynNotch synthetic receptor combination and recombining it with MSC cells to obtain a Syn-MSC-BiTE / TriTE system. The Syn-MSC-BiTE / TriTE receptor combination of the present invention enters the tumor tissue under the action of MSC tumor chemotaxis. After recognizing Trop-2 on the surface of the tumor cell membrane, the SynNotch receptor will be activated, the yeast transcription complex in the membrane will be cut, and then enter the cell nucleus. After recognizing the UAG sequence in another plasmid, the transcription and translation of the downstream functional gene are initiated. After the expression of the downstream functional gene, it plays a role in recruiting T cell infiltration, mediating T cells to exert antigen-specific tumor killing function, and relieving PD-1-mediated T cell inhibition in the tumor tissue.

[0059] The technical solution of the present disclosure is described in detail below.

[0060] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0061] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0062] Figure 1 The Syn-MSC-BiTE / TriTE system of the present invention and a schematic diagram of the anti-tumor process of the system are shown. Figure 1 As shown, the anti-tumor process of the Syn-MSC-BiTE / TriTE system of the present invention includes: MSC phenotype determination and function screening; SynNotch system construction; Syn-MSC-BiTE cell construction and Syn-MSC-BiTE cell treatment strategy. The following is a detailed description through specific examples.

[0063] Example 1: MSC phenotype determination and screening 1. Expand and purify MSCs from umbilical cord using in vitro culture The umbilical cord (from the Department of Obstetrics and Gynecology of Nanjing Drum Tower Hospital, donated voluntarily by pregnant women) was cut into small pieces of about 2 cm, and washed with DPBS CTS containing 2% SP until there was no blood. After washing, the three blood vessels in the umbilical cord were removed: From the cross-section of the umbilical cord, it can be seen that there are three blood vessels in the umbilical cord, namely two arteries and one vein. The artery has a thick wall, a small lumen, and good elasticity. The artery can be pulled hard until it is completely pulled out. Cut the umbilical cord after removing the blood vessels into pieces of about 1 mm 3 After the tissue block is cut into pieces, stick it directly into a T75 culture flask, invert it for 4 hours, then place the culture flask upright, and add 6 mL of human mesenchymal stem cell culture medium (do not cover the tissue block) (MSC expansion medium: DMEM-LG, Gibco, catalog number 10567014; 10% FBS, Gibco, 2664025; 1x double antibody, Gibco, 15140122; bFGF (5 μg / mL), Gibco, CTP0263). The distance between each tissue block is 0.1 cm, and the tissue blocks need to be evenly spread; add the culture medium drop by drop to avoid washing the tissue block. After 5 days, add 6 mL of fresh human mesenchymal stem cell culture medium to the culture flask. After about 10-14 days, cells will crawl out and form colonies CFU-F. Gently tap the culture bottle to make the tissue pieces fall off and discard them. Gently wash the cell surface with PBS 4 mL / T75 culture bottle, replace with 10 mL of fresh complete culture medium, and continue to culture in the incubator. When the cells reach 80%-90% confluence, subculture them to obtain umbilical cord-derived MSCs.

[0064] Using the same method as above, we expanded and purified gingival-derived MSCs (G-MSCs), adipose-derived MSCs (AD-MSCs), decidua-derived MSCs (D-MSCs), chorionic-derived MSCs (PC-MSCs), amniotic-derived MSCs (A-MSCs), dental pulp-derived MSCs (DP-MSCs), and bone marrow-derived MSCs (BM-MSCs).

[0065] 2. MSC phenotype analysis The results of phenotype analysis of MSCs from different tissues are shown in Figure 2 .like Figure 2As shown, the expression of classical CD molecules in MSCs by flow cytometry analysis was as follows: CD73, eBioscience™, 12-0739-42; CD90, eBioscience™, 12-0739-42; CD105, eBioscience™, 12-1057-42; HLA-DR, eBioscience™, 12-9956-42; CD14, eBioscience™, 12-0149-42; CD19, eBioscience™, 12-0199-42; CD34, eBioscience™, 12-0349-42; CD45, eBioscience™, 12-0459-42.

[0066] 3. RNA-seq analysis of the immune phenotype of MSCs from different tissues Each group collects 10 7 Cells were washed three times with cold PBS and centrifuged to remove the supernatant. 1 mL Trizol (ThermoFisher, 15596026CN) was added to lyse the cells, and 200 μL tetrachloromethane was added, and the tube was inverted to mix and extract RNA. Centrifuge at 12000 g for 15 min, the supernatant was transferred to a new enzyme-free EP tube, 500 μL isopropanol was added, and the tube was inverted to mix. Centrifuge at 12000 g for 10 min. The supernatant was discarded and 75% ethanol was added for washing. Centrifuge at 12000 g for 5 min. The supernatant was discarded and the precipitate was RNA. The RNA purity, integrity and concentration were measured; fragmentation and reverse transcription were performed into cDNA to construct a sequencing library; Qubit2.0 and Agilent 2100 were used to detect the library concentration and insert size. Sequencing was then performed using a high-throughput sequencing platform (NovaSeq 6000), and the results are shown in Figure 3 . Figure 3 As shown, analysis of the sequencing results showed that adipose-derived MSC (AD-MSC) has an immune-promoting phenotype and is a good cell carrier for universal cell therapy.

[0067] Example 2: Design of SynNotch synthetic receptor combinations like Figure 4 As shown, the SynNotch synthetic receptor combination designed in this embodiment includes: The primary synNotch synthetic receptor comprises a ScFv fragment targeting a tumor-specific antigen, a mouse Notch receptor core domain, a yeast transcription complex, and a suicide gene RQR8 domain connected in sequence; wherein the ScFv fragment and the mouse Notch receptor core domain are connected via a TM transmembrane domain, the yeast transcription complex and RQR8 are connected via a P2A peptide, and a CMV promoter is also connected to the ScFv fragment.

[0068] The secondary synNotch expression plasmid comprises a 5x UAG promoter and a functional protein connected in sequence, wherein the functional protein includes the following (i) to (iii): (i) BiTE; (ii) T cell chemokines; and (iii) immune checkpoint inhibitors; The above functional proteins are connected by P2A peptide.

[0069] Among them, the sequence information corresponding to each domain in the SynNotch synthetic receptor combination is shown in Table 1: Table 1 SynNotch synthetic receptor combination sequence information Example 3: Construction of dual plasmids CMV-ScFv-TM-Notch Core+ Gal4-(GGS)3-VP64-P2A-RQR8, 5xUAG-CCL5-P2A-CXCL19-P2A-BiTE-P2A-anti-PD-1 1. CMV-ScFv-TM-Notch Core+ Gal4-(GGS) 3 - Construction of VP64-P2A-RQR8 plasmid 1) The heavy and light chain variable regions V H 、V L The ScFv sequence was obtained by connecting the connecting peptide SGGGGSGGGGSGGGGS (the specific sequence information is shown in Table 1).

[0070] 2) The obtained ScFv was integrated with the Notch Core domain in mouse Notch, and then fused with the yeast transcription complex (Gal4 / VP64) to construct the SynNotch receptor Notch Core+ Gal4-(GGS) 3 -VP64.

[0071] 3) The obtained SynNotch receptor was fused with P2A peptide to express the RQR8 structure to obtain Notch Core + Gal4-(GGS)3-VP64-P2A-RQR8. Among them, RQR8 is a suicide gene, which can be eliminated in vivo by Rituximab and play the role of a suicide gene.

[0072] 4) Integrate promoter CMV, ScFv, TM, Notch Core + Gal4-(GGS)3-VP64-P2A-RQR8 to obtain CMV-ScFv-TM-Notch Core + Gal4-(GGS) 3 -VP64-P2A-RQR8 plasmid.

[0073] 2. Construction of 5x UAG-CCL5-P2A-CXCL19-P2A-BiTE-P2A-anti-PD-1 plasmid The CMV promoter in the vector was replaced with a 5x UAG sequence that can be recognized by the yeast transcription complex Gal4-VP64, and the functional gene was integrated into the open reading frame after 5x UAG. The functional genes include: 1) ScFv obtained based on anti-Trop-2 mAb (IMMU-132), and BiTE connected to ScFv obtained based on anti-CD3 mAb (blinatumomab) through a linker; 2) T cell chemokines: CCL5 and CXCL19; 3) anti-PD-1 single domain antibody. The functional genes were connected by P2A peptide to construct a 5x UAG-CCL5-P2A-CXCL19-P2A-BiTE-P2A-anti-PD-1 plasmid.

[0074] Example 4: Preparation of Syn-MSC-BiTE cells The dual-plasmid SynNotch system constructed in Example 3 was transfected into the adipose-derived MSCs screened in Example 1 by electrofection. The specific transfection process was as follows: the transfection was carried out using the Lonza 4D transfection system and the matching transfection kit (P1 Primary Cell NucleofactorTM Kit, V4XP-1012, Lonza.). The transfection operation is described in the kit manual. 6 Cells were added with 1 μg of plasmid. Syn-MSC-BiTE cells were then constructed through resistance screening and in vitro amplification.

[0075] The specific resistance screening method is: 1. Change the culture medium 24 hours after transfection and add puromycin (final concentration is 1 μg / mL) to the MSC complete culture medium.

[0076] 2. After 48 hours of culture, reduce the concentration of puromycin to 0.5 μg / mL. Add appropriate amount of MSC culture medium (DMEM-LG configuration, containing 10% Qualitied MSC FBS, 1×Penicillin-Streptomycin and 12.5 ng / mL bFGF) for culture and expansion to obtain Syn-MSC-BiTE cells.

[0077] Example 5: Activity verification by co-culture with CD44v5-positive gastric cancer cell line MKN-45 1. CD44v5 positive gastric cancer cell line MKN-45 (from China Center for Type Culture Collection, GDC0220) was cultured for 10 6 cell / mL in 12-well plates; 2. Add the Syn-MSC-BiTE cells obtained in Example 4 (10 5 After 24 hours of contact culture, the expression of EGFP and mCherry was observed under a fluorescence microscope.

[0078] 3. Absorb 10 5 The cells were washed three times with PBS, reselected with 200 μL PBS, and the expression of EGFP (FL1) and mCherry (FL2) was detected by flow cytometry.

[0079] 4. Results Figure 5 .

[0080] like Figure 5 As shown, Figure 5 A is the expression of EGFP (green fluorescent protein) and mCherry (red fluorescent protein) observed by fluorescence microscope. Figure 5 B is the flow cytometry analysis of EGFP and mCherry expression. Figure 5 As shown in A and 5B, EGFP is stably expressed but mCherry is not expressed when it is not in contact with Trop-2 positive target cells, and mCherry is expressed only after contact with target cells. This shows that the SynNotch system is activated only after adding Trop-2 positive target cells.

[0081] Example 6: Therapeutic effect of Syn-MSC-BiTE cells 1. CD44v5 positive gastric cancer cell line MKN45 was cultured at 5×10 4 The cell / well concentration was plated in a 96-well plate.

[0082] 2. Divide the above 96-well plates into two groups: SynNotch group and Vector group; The SynNotch group: 3 Syn-MSC-BiTE cells were added at cell / well concentration; Vector group: according to 10 3 MSCs transfected with blank plasmid Vector were added at a concentration of 1 cell / well.

[0083] 3. Then, different concentrations of primary T cells (isolated from peripheral blood voluntarily donated by healthy individuals) were added to the SynNotch group and Vector group well plates according to different effector-target ratios for culture.

[0084] 4. Luciferase Assay was used to detect cell viability. The specific operation was as follows: after direct contact culture for 48 hours, Luciferin (final concentration of 1 mM, Promega, E6552) was added, reacted at room temperature for 5 min, and the luciferase signal strength was detected by an ELISA reader. The untreated ctrl group (MSC group transfected with blank plasmid Vector) was used as the control, and cell viability = ([luciferase signal strength of SynNotch group] / [luciferase signal strength of Vector group])*100%. The targeted killing function of cells was analyzed.

[0085] 5. See the results Figure 6 .

[0086] like Figure 6 As shown in the figure, compared with the Vector group, when the effector-target ratio was 0.32:1, it was observed that the morphology of tumor cells MKN45 in the SynNotch group was significantly shrunk and cell fragments appeared ( Figure 6A); Luciferase Assay results showed that Syn-MSC-BiTE could mediate significant anti-tumor activity ( Figure 6 B), CBA detection found that IFNg release also increased significantly ( Figure 6 C), which suggests that T cells are significantly activated. This shows that Syn-MSC-BiTE can activate T cells to exert antigen-specific anti-tumor response at a low target ratio.

[0087] Example 7: Verification of the therapeutic effect of Syn-MSC-BiTE cells Animal model construction: First, 1-5×10E6 MKN-45 cells were intraperitoneally injected into immunodeficient mice (6-8 weeks old, female, BALB / c nude) to construct a gastric cancer abdominal dissemination tumor model mouse. Two days later, the luciferase substrate D-luciferin (100 ug / mouse) was intraperitoneally injected, and the distribution of tumor cells and bioluminescence intensity were analyzed by in vivo imaging. An increase in the distribution area of ​​tumor tissue or an increase in signal intensity indicates that the model was successfully constructed.

[0088] The constructed model mice were randomly divided into four groups, namely, Mock group, MSC+PBMC group, PBMC group and Syn-MSC-BiTE+PBMC group, with 6 mice in each group. The treatment conditions of mice in each group were as follows: Mock group: Day 2, solvent control (100 ul saline) was injected into the tail vein; Day 3, 100 ul saline (containing 5×10E6 PBMCs from healthy people / mouse) was injected into the tail vein; MSC+PBMC group: Day 2, 100 ul of normal saline (containing 5×10E6 untreated MSC / mouse) was injected into the tail vein; Day 3, 100 ul of normal saline (containing 5×10E6 PBMC from healthy individuals / mouse) was injected into the tail vein; PBMC group: Day 2, 100 ul of normal saline (containing 5×10E6 untreated MSCs / mouse) was injected into the tail vein; Day 3, 100 ul of normal saline was injected into the tail vein; Syn-MSC-BiTE+PBMC group: On Day 2, the Syn-MSC-BiTE cells obtained in Example 4 were intravenously infused into the above model mice at a rate of 5×10E6 cells / mouse. On Day 3, healthy human PBMCs (5×10E6 cells / mouse) were injected through the tail vein.

[0089] The tumor growth of each group of mice was detected and analyzed every day using a live imaging system.

[0090] The results are as follows Figure 7Shown: The constructed Syn-MSC-BiTE cells showed effective anti-tumor activity in a tumor-bearing mouse model.

[0091] Since MSC lack HLA-DR and hardly express MHC-I class molecules, allogeneic transfusion can be achieved. Syn-MSC-BiTE enters the tumor tissue under the action of MSC tumor chemotaxis. After recognizing Trop-2 on the surface of the tumor cell membrane, the SynNotch receptor will be activated, the yeast transcription complex in the membrane will be cut, and then enter the cell nucleus. After recognizing the UAG sequence in another plasmid, the transcription and translation of downstream functional genes are initiated. After the expression of downstream functional genes, they play a role in recruiting T cell infiltration in tumor tissues, mediating T cells to play antigen-specific tumor killing functions, and relieving PD-1-mediated T cell inhibition.

[0092] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A universal anti-tumor SynNotch synthetic receptor combination, characterized in that: The synthetic receptor combination includes: A primary synNotch synthetic receptor, wherein the primary synNotch synthetic receptor comprises a ScFv fragment targeting a tumor-specific antigen, a mouse Notch receptor core domain, a yeast transcription complex, and a suicide gene RQR8 domain connected in sequence; A secondary synNotch expression plasmid, wherein the secondary synNotch expression plasmid comprises a 5x UAG promoter and a functional protein connected in sequence, wherein the functional protein comprises the following (i) to (iii): (i) BiTE or TriTE; (ii) T cell chemokines; and (iii) immune checkpoint inhibitors; The functional proteins are connected via P2A peptides.

2. The synthetic receptor combination according to claim 1, characterized in that The ScFv fragment and the mouse Notch receptor core domain are connected via the TM transmembrane domain, the yeast transcription complex and RQR8 are connected via the P2A peptide, and the ScFv fragment is also connected to a CMV promoter.

3. The synthetic receptor combination according to claim 1, characterized in that The ScFv comprises a heavy chain variable region V H and light chain variable region V L , and optionally comprising a heavy chain variable region V H and light chain variable region V L The connecting peptide of the heavy chain variable region V H The amino acid sequence of the light chain variable region V L The amino acid sequence of is shown in SEQ ID NO.2, and the connecting peptide is SGGGGSGGGGSGGGGS; the amino acid sequence of the ScFv is shown in SEQ ID NO.

3.

4. The SynNotch synthetic receptor combination according to claim 1, characterized in that The mouse Notch receptor core domain comprises Lin12-Notch repeat, heterodimer domain and transmembrane region, and its amino acid sequence is shown in SEQ ID NO.10; The yeast transcription complex includes any one of Gal4-VP64, ZFHD1-VP64, and Gal4-KRAB.

5. The SynNotch synthetic receptor combination according to claim 1, characterized in that The RQR8 domain comprises a CD8 TM transmembrane domain, a first CD20 mimotope domain, a CD34 epitope domain and a second CD20 mimotope domain targeting an anti-tumor monoclonal antibody, which are sequentially connected.

6. The SynNotch synthetic receptor combination according to claim 1, characterized in that The BiTE is an Anti-Trop2 ScFv obtained based on anti-Trop-2 mAb, which is connected to an Anti-CD3ScFv obtained based on anti-CD3 mAb through a connecting peptide to obtain a bispecific T cell engager; the amino acid sequence of the Anti-Trop2 ScFv is shown in SEQ ID NO.4, the amino acid sequence of the Anti-CD3 ScFv is shown in SEQ ID NO.5, and the amino acid sequence of the BiTE is shown in SEQ ID NO.

6.

7. The SynNotch synthetic receptor combination according to claim 6, characterized in that The TriTE is a trispecific T cell adapter connected to interleukin IL-15 via Sushi polypeptide based on BiTE; The amino acid sequence of the Sushi polypeptide is shown in SEQ ID NO.7, the amino acid sequence of the interleukin IL-15 is shown in SEQ ID NO.8, and the amino acid sequence of the TriTE is shown in SEQ ID NO.

9.

8. The SynNotch synthetic receptor combination according to claim 1, characterized in that The T cell chemokines include CCL5 and CXCL19; The immune checkpoint inhibitors include Anti-PD-L1 and Anti-PD-1.

9. A coding gene encoding the universal anti-tumor SynNotch synthetic receptor combination according to any one of claims 1 to 8; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

11.

10. An expression vector, characterized in that: The expression vector expresses the coding gene according to claim 9.

11. A host cell, characterized in that The host cell comprises the SynNotch synthetic receptor combination according to any one of claims 1 to 8, the encoding gene according to claim 9, or the expression vector according to claim 10.

12. A universal anti-tumor system, characterized in that: The invention comprises a recombinant target cell obtained by transfecting a target cell with the SynNotch synthetic receptor combination according to any one of claims 1 to 8, the encoding gene according to claim 9, or the expression vector according to claim 10, wherein the target cell is an immune cell or a pluripotent stem cell.

13. A pharmaceutical composition, characterized in that A method comprising the SynNotch synthetic receptor combination according to any one of claims 1 to 8, the encoding gene according to claim 9, the expression vector according to claim 10, the host cell according to claim 11, or the universal anti-tumor system according to claim 12; and a pharmaceutically acceptable carrier; The medicament is used to treat a disease in a subject in need of the medicament, and the disease includes a tumor.

14. Use of the SynNotch synthetic receptor combination according to any one of claims 1 to 8, the encoding gene according to claim 9, the expression vector according to claim 10, the host cell according to claim 11, or the universal anti-tumor system according to claim 12 in the preparation of a drug for treating tumors.

Images