Engineered TIL expressing membrane-bound IL-15 fusion protein and its application

Abstract

The present invention relates to a membrane-bound IL-15 fusion protein expressed on TIL cells. Specifically, the present invention uses a hypoxia response element HRE to induce the expression of a membrane-bound IL-15 (mIL-15) fusion protein, so that the TIL cell group expressing the fusion protein maintains the memory stem cell phenotype for a long time and enhances its anti-tumor activity; in the construction of the fusion protein, the CD86 transmembrane region and intracellular domain are used to further increase the proportion of stem cells in the TIL cells; in the molecular construction, truncated hEGFRt is used as a molecular switch to further improve its safety. The stemness and persistence of TIL cells expressing the fusion protein of the present invention are significantly improved, and long-lasting immune monitoring and treatment capabilities can be provided.

Description

Technical Field

[0001] The present invention relates to the technical field of gene engineering and cell therapy, and in particular to engineered TIL expressing membrane-bound IL-15 fusion protein and applications thereof. Background Art

[0002] The incidence of cancer is increasing year by year and is showing a trend of younger patients. Due to the complexity and heterogeneity of solid tumors, it is extremely difficult to cure them. In recent years, following surgery, radiotherapy, chemotherapy, and immunotherapy, cell therapy is gradually showing a strong therapeutic effect in the treatment of various cancers. With the rapid development of human tumor treatment technology, adoptive cell therapy (ACT) has gradually become one of the treatment methods for patients with advanced tumors. Chimeric antigen receptor T cell (CAR-T) therapy, engineered T cell receptor T cell (TCR-T) therapy, tumor infiltrating lymphocyte (TIL) therapy, etc. all belong to the category of ACT. Compared with CAR-T and TCR-T, TIL cell drugs have shown significant efficacy in the treatment of solid tumors and have become one of the most promising technical directions for conquering solid tumors.

[0003] Specific killer lymphocytes present in the tumor microenvironment are called tumor-infiltrating lymphocytes (TIL). TIL therapy is an adoptive cell therapy that uses the patient's own immune system to treat tumors, and has been successfully used in patients with metastatic melanoma and other solid tumors. TIL is composed of T cells with multiple TCR clones, which can not only directly recognize tumor-associated antigens, but also fight tumor-specific new antigens, making it more effective in dealing with tumor heterogeneity. TIL expresses chemokine receptors after being stimulated by tumor antigens in vivo, which makes it easier for TIL to locate in tumor tissue after reinfusion. In addition, TIL comes from the patient himself, which means that this method has low toxicity.

[0004] However, TIL therapy obviously has its limitations. Usually, the preparation method of TIL is to obtain a single cell suspension after mechanical treatment or enzymatic digestion of tumor tissue, select and culture T lymphocytes by adding high concentrations of IL2, and culture billions of TIL cells within 2-4 weeks before re-infusing them into the patient. Re-infusion therapy is accompanied by high-dose IL2 infusion to support the growth and activity of TIL injected into the body. However, high-dose IL2 can cause dose-limiting toxicity, requiring enhanced monitoring and care; low-dose IL2 may be beneficial to peripheral tolerance and regulatory T cell production, thereby inhibiting the anti-tumor response of TIL; in addition, the IL2 signaling pathway drives the proliferation of effector T cells and promotes terminal differentiation and exhaustion; the above disadvantages greatly limit the clinical application of TIL therapy.

[0005] To reduce the toxicity associated with high-dose IL2 and improve the in vivo survival and function of traditional TIL therapy, there is an urgent need to develop engineered TIL cells with improved stemness, persistence, and anti-tumor activity. Summary of the invention

[0006] The purpose of the present invention is to provide an engineered TIL cell with improved stemness, persistence and anti-tumor activity.

[0007] In a first aspect of the present invention, a fusion polypeptide is provided, wherein the fusion polypeptide comprises the following elements:

[0008] (i) interleukin 15 (IL-15) or a functionally active fragment thereof;

[0009] (ii) a transmembrane domain; and

[0010] (iii) intracellular domain;

[0011] Wherein, the intracellular domain is the CD86 intracellular domain.

[0012] In another preferred embodiment, the CD86 intracellular domain includes the full-length intracellular domain, or an active fragment thereof.

[0013] In another preferred embodiment, the IL-15 includes a full-length, mature form of IL-15, or an active fragment thereof.

[0014] In another preferred embodiment, the IL-15 includes wild type and mutant type.

[0015] In another preferred embodiment, the amino acid sequence of IL-15 is shown in SEQ ID NO:1.

[0016] In another preferred embodiment, the fusion polypeptide further comprises a hinge region.

[0017] In another preferred embodiment, the fusion polypeptide may further optionally include a signal peptide and / or a linker.

[0018] In another preferred embodiment, the linker is located between element (i) and element (ii), between element (ii) and element (iii), between element (i) and the hinge region, or between the hinge region and element (ii).

[0019] In another preferred embodiment, the linker includes a flexible linker or a rigid linker.

[0020] In another preferred embodiment, the amino acid sequence of the linker is as shown in SEQ ID NO:5.

[0021] In another preferred embodiment, the structural formula of the fusion polypeptide is as shown in formula (I):

[0022] X-IL15-LH-TM-Cyto(I)

[0023] in,

[0024] “-” each independently represents no peptide or a connecting peptide;

[0025] X is none or signal peptide;

[0026] IL15 is the interleukin 15 element;

[0027] L is none or a linker;

[0028] H is the hinge region;

[0029] TM is the transmembrane domain;

[0030] Cyto is an intracellular domain, wherein the intracellular domain is the CD86 intracellular domain.

[0031] In another preferred example, the amino acid sequence of the CD86 intracellular domain is shown in SEQ ID NO:2.

[0032] In another preferred example, the transmembrane domain is the CD86 transmembrane domain. Preferably, the amino acid sequence of the CD86 transmembrane domain is as shown in SEQ ID NO:3.

[0033] In another preferred example, the hinge region is the CD86 hinge region, and preferably, the amino acid sequence of the CD86 hinge region is as shown in SEQ ID NO:4.

[0034] In another preferred embodiment, the amino acid sequence of the signal peptide is shown in SEQ ID NO:6.

[0035] In another preferred example, the sequence of the fusion protein is shown in SEQ ID NO:7.

[0036] In the second aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide encodes the fusion polypeptide of the present invention.

[0037] In the third aspect of the present invention, a nucleic acid construct is provided, wherein the nucleic acid construct comprises a first expression cassette, wherein the first expression cassette comprises a hypoxia-responsive promoter and the polynucleotide according to the second aspect of the present invention.

[0038] In another preferred embodiment, the hypoxia-responsive promoter is a promoter comprising n hypoxia response elements HRE, that is, a promoter containing n×HRE elements, where n is an integer selected from 1-20.

[0039] In another preferred embodiment, n is an integer selected from 2 to 9; more preferably, n is an integer selected from 2 to 4; most preferably, n is 3.

[0040] In another preferred embodiment, the hypoxia-responsive promoter is selected from the following group: TK-mini promoter containing n×HRE elements, CMV-mini promoter containing n×HRE elements, IL2-mini promoter containing n×HRE elements.

[0041] In another preferred embodiment, the nucleic acid construct further comprises a molecular switch element sequence, and the molecular switch element is selected from the following group: hEGFRt, BCMA, and CD20.

[0042] In another preferred embodiment, the molecular switch element is hEGFRt.

[0043] In another preferred embodiment, the molecular switch element is connected to the first expression cassette via a cleavable connecting peptide sequence.

[0044] In another preferred embodiment, the cleavable connecting peptide is a self-cleaving 2A peptide, preferably a T2A peptide.

[0045] In another preferred example, the nucleic acid construct comprises a second expression cassette, and the second expression cassette comprises a second promoter and a molecular switch element coding sequence.

[0046] In another preferred example, the second promoter is SFFV promoter.

[0047] In the fourth aspect of the present invention, a vector is provided, characterized in that the vector comprises the polynucleotide as described in the second aspect of the present invention.

[0048] In another preferred embodiment, the vector includes a plasmid or a viral vector.

[0049] In another preferred embodiment, the viral vector includes: a lentiviral vector, an adenoviral vector, and a yellow fever virus vector.

[0050] In another preferred embodiment, the vector is a plasmid.

[0051] In the fifth aspect of the present invention, an immune cell is provided, wherein the cell comprises the fusion polypeptide of the present invention, the polynucleotide described in the second aspect of the present invention, the nucleic acid construct described in the third aspect of the present invention, and / or the vector described in the fourth aspect of the present invention.

[0052] In another preferred embodiment, the immune cells are selected from the following group: tumor infiltrating cells (TIL), T cells, and NK cells.

[0053] In another preferred embodiment, the cells are tumor infiltrating cells (TIL).

[0054] In the sixth aspect of the present invention, a composition is provided, which comprises the fusion polypeptide described in the present invention, the polynucleotide described in the second aspect of the present invention, the nucleic acid construct described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, and / or the cell described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0055] In the seventh aspect of the present invention, a kit is provided, which comprises the fusion polypeptide of the present invention, the polynucleotide described in the second aspect of the present invention, the nucleic acid construct described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, the cell described in the fifth aspect of the present invention, and / or the composition described in the sixth aspect of the present invention.

[0056] In the eighth aspect of the present invention, provided is the use of the fusion polypeptide of the present invention, the polynucleotide of the second aspect of the present invention, the nucleic acid construct of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the cell of the fifth aspect of the present invention, the composition of the sixth aspect of the present invention, and / or the kit of the seventh aspect of the present invention in the preparation of a drug for preventing, alleviating and / or treating tumors.

[0057] In another preferred embodiment, the tumor is selected from the following group: lung cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, bladder cancer, liver tumor, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumor, and head and neck cancer.

[0058] In the ninth aspect of the present invention, a method for treating a disease is provided, the method comprising administering the immune cell described in the fifth aspect of the present invention or the composition described in the sixth aspect of the present invention to a subject.

[0059] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Lentiviral infection of TIL cells is shown. Figure 1 A shows a schematic diagram of the structures of plasmids 001, 002, 003, and 004; Figure 1 B shows the results of flow cytometry detection of the ratio of mIL-15 and hEGFRt after TILs were infected with lentiviruses with different plasmid structures.

[0061] Figure 2The ELISA test for the shedding of mIL-15 shows that there is no significant difference in the secretion of IL-15 after hypoxia treatment compared with the control group and the non-hypoxia group, indicating that there is no shedding of membrane-expressed IL-15.

[0062] Figure 3 Shows T SCM and flow cytometry results of T cell exhaustion. Figure 3 A shows a schematic diagram of the structure of trophoblast cells K562-mIL21-4-1BBL; Figure 3 B shows flow cytometry detection of mIL21 in K562 and K562-mIL21-4-1BBL cells; Figure 3 C shows that after TILs were infected with lentiviruses of different plasmid structures, they were co-cultured with trophoblast cells K562-mIL21-4-1BBL for 7 and 14 days, and cell counts and cell viability were recorded; Figure 3 D shows the flow cytometry analysis of TILs after lentivirus infection with different plasmid structures and co-culture with trophoblast cells K562-mIL21-4-1BBL for 18 days. SCM ; Figure 3 E shows that after TILs were infected with lentiviruses with different plasmid structures, they were co-cultured with trophoblast cells K562-mIL21-4-1BBL for 18 days, and IL7Ra was detected by flow cytometry; Figure 3 F shows that after TILs were infected with lentiviruses with different plasmid structures, they were co-cultured with trophoblast cells K562-mIL21-4-1BBL for 18 days, and the exhaustion of CD8+T cells was detected by flow cytometry.

[0063] Figure 4 The antitumor activity of TIL cells overexpressing mIL-15 was shown. Figure 4 A shows that TIL, 001-TIL and 002-TIL cells were co-cultured with tumor cells Calu6 for 24 h, and the activity of tumor cells was detected by CCK8 method; Figure 4 B shows that TIL, 001-TIL and 002-TIL cells were co-cultured with tumor cells Calu6 for 24 h, and the secretion of IFN-γ was detected by ELISA.

[0064] Figure 5 The flow cytometry results of mIL-15 are shown. Figure 5 A shows the flow cytometry detection of mIL-15+ cells after 002-TIL cells were treated with mAb, complement, and mAb for 4 h; Figure 5 B shows the flow cytometry detection of mIL-15+ cells after 002-TIL cells were treated with mAb, NK cells, and mAb for 24 h.

[0065] Figure 6 The results of the detection of TIL cells infected with 005 lentivirus are shown. Figure 6 A shows a schematic diagram of the 005 plasmid structure; Figure 6 B shows the ratio of mIL-15 and hEGFRt detected by flow cytometry after 005 lentivirus infected TIL; Figure 6 C shows the gMFI of mIL-15 and hEGFRt detected by flow cytometry after TILs were infected with 005 lentivirus; Figure 6 D shows the in vitro proliferation and survival of TIL and 005-TIL cells with and without the addition of IL2 (-IL2: without the addition of IL2; +IL2: with the addition of IL2).

[0066] Figure 7 The figure shows the results of 005 lentivirus infection of cervical cancer TIL cells. Figure 7 A shows the ratio of mIL-15+ and hEGFRt+ cells detected by flow cytometry after 005 lentivirus infected TIL; Figure 7 B shows the gMFI of mIL-15 and hEGFRt detected by flow cytometry after TILs were infected with 005 lentivirus; Figure 7 C shows the flow cytometry analysis of TILs after 055 lentivirus infection and co-culture with trophoblast cells K562-mIL21-4-1BBL for 18 days. SCM ; Figure 7 D shows that after TILs were infected with 055 lentivirus, they were co-cultured with trophoblast cells K562-mIL21-4-1BBL for 18 days, and IL7Ra was detected by flow cytometry; Figure 7 E shows that after TIL was infected with 055 lentivirus, it was co-cultured with trophoblast cells K562-mIL21-4-1BBL for 18 days, and the expression of exhaustion proteins in CD8+ T cells was detected by flow cytometry. DETAILED DESCRIPTION

[0067] After extensive and in-depth research, the inventors have provided for the first time a membrane-bound IL-15 fusion protein with a specific structure, wherein the fusion protein (fusion protein of the present invention) includes an IL-15 element and a CD86 transmembrane region and an intracellular domain. The fusion protein of the present invention unexpectedly and significantly increases the proportion of stem cells in TIL cells and significantly enhances their anti-tumor activity. In addition, the present invention also uses a hypoxia response element HRE to induce the expression of the fusion protein of the present invention, thereby allowing engineered TIL cells to express the fusion protein of the present invention in a hypoxic environment and maintain the long-term persistence of the memory stem cell phenotype. The present invention was completed on this basis.

[0068] Specifically, the inventors preferably use the hypoxia response element HRE to induce the expression of the fusion protein of the present invention in the modification of TIL cells, and use truncated hEGFRt as a molecular switch in the molecular construction to further improve its safety. The stemness and persistence of the engineered TIL cells of the present invention are significantly improved, and can provide long-lasting immune monitoring and treatment capabilities.

[0069] Fusion polypeptide of the present invention

[0070] As used herein, the terms "fusion polypeptide of the present invention", "fusion protein of the present invention", "membrane-bound IL-15 of the present invention" and "mIL-15 of the present invention" are used interchangeably and all refer to a fusion protein of the present invention comprising IL-15, a transmembrane domain and an intracellular domain.

[0071] Interleukin IL-15 is a pro-survival cytokine that maintains long-lived CD8+ memory T cell homeostasis, inhibits activation-induced cell death (AICD), enhances in vivo anti-tumor activity, and reverses T cell anergy. Monomeric IL-15 is a small, unstable protein with a short serum half-life that requires supraphysiological administration to obtain an in vivo response. In the present invention, IL-15 is linked to a transmembrane domain and an intracellular domain to obtain a membrane-bound IL-15 that has the function of maintaining the long-term persistence of the memory stem cell phenotype.

[0072] In the present invention, the preferred intracellular domain is the CD86 intracellular domain. The use of the CD86 intracellular domain can further increase the proportion of stem T cells and reduce the expression of T cell exhaustion molecules. The preferred transmembrane domain of the present invention is the CD86 transmembrane domain

[0073] The fusion protein of the present invention may further include a hinge region, a signal peptide and / or a linker. The preferred structural formula of the fusion protein of the present invention is shown in formula (I):

[0074] X-IL15-LH-TM-Cyto(I)

[0075] in,

[0076] “-” each independently represents no peptide or a connecting peptide;

[0077] X is none or signal peptide;

[0078] IL15 is interleukin 15;

[0079] L is none or a linker;

[0080] H is a hinge region; the preferred hinge region of the present invention is the CD86 hinge region;

[0081] TM is a transmembrane domain; the preferred transmembrane domain of the present invention is the CD86 transmembrane domain;

[0082] Cyto is an intracellular domain, and the preferred intracellular domain of the present invention is the CD86 intracellular domain.

[0083] Among them, the preferred amino acid sequence of IL-15 is as follows:

[0084] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISL ESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQ MFINTS (SEQ ID NO: 1).

[0085] Preferably, the amino acid sequence of the CD86 intracellular domain is as follows:

[0086] KWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKT SSCDKSDTCF (SEQ ID NO: 2).

[0087] Preferably, the amino acid sequence of the CD86 transmembrane domain is WITAVLPTVIICVMVFCLILW (SEQ ID NO: 3).

[0088] Preferably, the amino acid sequence of the CD86 hinge region is LEDPQPPPDHIP (SEQ ID NO: 4).

[0089] Preferably, the amino acid sequence of the linker is SGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 5).

[0090] Preferably, the amino acid sequence of the signal peptide is MDWTWILFLVAAATRVHS (SEQ ID NO: 6).

[0091] Preferably, the amino acid sequence of the fusion protein of the present invention is as follows:

[0092] MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQM FINTSSGGGSGGGGSGGGGSGGGGSGGGSLEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF(SEQ ID NO:7).

[0093] Nucleic acid constructs of the present invention

[0094] As used herein, the nucleic acid construct of the present invention can express the fusion protein of the present invention. The nucleic acid construct of the present invention comprises a first expression cassette encoding the fusion protein of the present invention, and the first expression cassette uses a hypoxia-responsive promoter.

[0095] The hypoxia-responsive promoter of the present invention is a promoter comprising n hypoxia-responsive elements HRE, i.e., n×HRE promoter. Hypoxia is a hallmark feature of solid tumors, and this unique environmental signal can be used for targeted cancer therapy. When constructing a nucleic acid molecule expressing the fusion protein of the present invention, the present invention uses a hypoxia-responsive promoter to express the fusion protein of the present invention in a hypoxic environment. There is no particular limitation on the type of promoter used to construct the hypoxia-responsive promoter of the present invention. Preferably, the present invention uses a TK mini promoter containing n×HRE.

[0096] Preferably, the present invention adopts 3×HRE-TK-mini promoter, whose sequence is shown in SEQ ID NO:8.

[0097] In one embodiment, the present invention uses 6×HRE-TK-mini promoter, whose sequence is shown in SEQ ID NO:9.

[0098] In one embodiment, the present invention uses 9×HRE-TK-mini promoter, whose sequence is shown in SEQ ID NO:10.

[0099] In one embodiment, the nucleic acid construct of the present invention further comprises a molecular switch element (also known as an immune brake element). When the cells expressing the nucleic acid construct present safety risks, the molecular switch element can be used as a target to eliminate the risky cells using corresponding drugs.

[0100] In one embodiment, the molecular switch element of the present invention can be connected to the first expression cassette via a cleavable connecting peptide; preferably, the cleavable connecting peptide is a self-cleaving peptide; preferably, the cleavable connecting peptide is T2A.

[0101] In one embodiment, the molecular switch element of the present invention is located in a second expression cassette, and the expression of the molecular switch element is controlled by a second promoter. In one embodiment, the second promoter is an SFFV promoter, which is a constitutive promoter that can stably and highly activate the expression of the molecular switch element. In one embodiment, the second expression cassette comprises a second signal peptide.

[0102] In one embodiment, the molecular switch or immune brake element of the present invention is hEGFRt. When the cells containing the nucleic acid construct of the present invention present safety risks, the hEGFRt therapeutic monoclonal antibody cetuximab can be injected to eliminate the target cells through ADCC and CDC, further improving safety; at the same time, the flow detection of hEGFRt molecules can also be used to indicate the positive rate of cells with target gene integration.

[0103] In a preferred embodiment, a hypoxia-responsive mIL-15 nucleic acid construct having a molecular switch element is constructed, which comprises the following sequences in sequence: n×HRE-TK-mini promoter sequence, a first signal peptide sequence, an IL-15 sequence, a linker sequence, a CD86 hinge region sequence, a CD86 transmembrane region-intracellular domain sequence, a SFFV promoter sequence, a second signal peptide sequence, and a hEGFRt sequence.

[0104] Preferably, in the n×HRE-TK-mini promoter, n is an integer selected from 2 to 9; more preferably, n is an integer selected from 2 to 4; most preferably, n is 3.

[0105] The vector of the present invention

[0106] In the present invention, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid connected to it. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to the viral genome. Some vectors can replicate autonomously in the host cells they are introduced into (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating together with the host genome, such as naked RNA polynucleotides that cannot replicate autonomously, naked DNA polynucleotides, polynucleotides composed of DNA and RNA in the same chain, poly-lysine-coupled DNA or RNA, peptide-coupled DNA or RNA, liposome-coupled DNA, etc. In addition, some vectors can guide the expression of genes effectively connected to them. This type of vector is referred to as a "recombinant expression vector" (or simply "expression vector") in the present invention. Generally speaking, the expression vector used in recombinant DNA technology is usually in the form of a plasmid. In this specification, "plasmid" and "vector" are used interchangeably because plasmid is the most commonly used form of vector.

[0107] As used herein, "the vector of the present invention" refers to a vector containing the nucleic acid construct of the present invention. Preferably, the vector of the present invention is a plasmid.

[0108] Engineered TIL cells of the present invention

[0109] As used herein, the terms "engineered TIL cells of the present invention" or "TIL cells of the present invention" are used interchangeably and refer to TIL cells that can express the fusion protein of the present invention and / or TIL cells containing the vector of the present invention.

[0110] The engineered TIL cells of the present invention express membrane-bound IL-15 (mIL-15), which can promote T SCM The formation of immature differentiation states can reduce the exhaustion of T cells. T cell subsets corresponding to the immature differentiation state can provide excellent clinical practical potential, T memory stem cells (T SCM ) is the lowest differentiated memory T cell subset identified so far, and this memory subset has the highest self-renewal ability and therapeutic potential. SCM The increase in proportion can maintain the long-term survival of the TIL cell population.

[0111] Pharmaceutical composition of the present invention

[0112] The pharmaceutical composition of the present invention may comprise the fusion protein of the present invention or the immune effector cell of the present invention (such as the engineered TIL cell of the present invention) and one or more pharmaceutically acceptable carriers, diluents, excipients and adjuvants. These compositions may be suitable for use in the treatment of therapeutic indications described herein.

[0113] The composition can be a liquid solution, suspension, emulsion, sustained release formulation or powder, and can be formulated with a pharmaceutically acceptable carrier. The composition can be formulated into a suppository using traditional adhesives and carriers such as triglycerides. "Pharmaceutically acceptable carrier" refers to a carrier matrix or vehicle that does not interfere with the effectiveness of the biological activity of the active ingredient and does not produce toxicity to the host or subject.

[0114] Fusion protein or immune effector cell can be delivered together with pharmaceutically acceptable vehicle.In one embodiment, vehicle can enhance stability and / or delivery properties.Vehicles such as artificial membrane vesicles (including liposomes, nonionic surfactant vesicles (noisomes), nano-microliposomes, etc.), microparticles or microcapsules or colloid preparations comprising pharmaceutically acceptable polymers.

[0115] The pharmaceutical composition comprising one or more fusion proteins or immune effector cells can be formulated into sterile injectable aqueous or oily suspensions according to methods known in the art and using appropriate one or more dispersing agents or wetting agents and / or suspending agents. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parent acceptable diluent or solvent.

[0116] In the present invention, the term "adjuvant" generally refers to any substance that assists or regulates the action of a drug, including but not limited to immunological adjuvants, which enhance or diversify the immune response to an antigen.

[0117] In the present invention, the term "subject" can be a mammal in need of treatment, such as a human or veterinary patient (e.g., a rodent, such as a mouse or rat, cat, dog, cow, horse, sheep, goat, or other livestock). In some embodiments, the "subject" can be a clinical patient, a clinical trial volunteer, an experimental animal, and the like. The subject may be suspected of having a disease characterized by cell proliferation or having a disease characterized by cell proliferation, diagnosed as having a disease characterized by cell proliferation, or a control subject confirmed not to have a disease characterized by cell proliferation. As described herein, diagnostic methods for diseases characterized by cell proliferation and the clinical division of such diagnosis are known to those skilled in the art.

[0118] The pharmaceutical composition of the present invention can be used to treat tumors. In the present invention, the term "tumor" or "tumor cell" generally refers to or describes a physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of tumors include, but are not limited to, carcinomas, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas and islet cell carcinomas), mesotheliomas, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas and melanomas. "Tumor cells" can further include "solid tumors", which refer to tumors selected from the group consisting of gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancer.

[0119] The main advantages of the present invention include:

[0120] (1) The present invention constructs for the first time a membrane-bound interleukin-15 fusion protein containing the CD86 intracellular domain, and the stemness, persistence and anti-tumor activity of TIL cells expressing the fusion protein are significantly improved.

[0121] (2) When constructing a nucleic acid construct for expressing a fusion protein, the present invention uses a hypoxia-responsive promoter, i.e., a promoter containing an HRE element, which can induce the expression of the fusion protein of the present invention under a hypoxic environment that is characteristic of solid tumors.

[0122] (3) The present invention adds an immune molecule switch, such as hEGFRt, when constructing a nucleic acid construct expressing a fusion protein, thereby avoiding the potential toxicity of sustained expression of IL-15 and improving the safety of clinical treatment.

[0123] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not indicated in the following examples are usually based on conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions. Unless otherwise stated, reagents and cell lines are commercially available products.

[0124] The sequence information involved in the examples is shown in Table A.

[0125] Table A Nucleotide sequences of the elements

[0126]

[0127]

[0128]

[0129] Example 1: Infection of TIL cells with lentivirus containing target gene

[0130] 1. Plasmid construction

[0131] According to the characteristics of the hypoxic microenvironment of solid tumors, 3×HRE, 6×HRE, and 9×HRE elements were used to induce expression, and four structures were designed and constructed, named constructs 001, 002, 003, and 004, respectively, and full gene synthesis was performed. Figure 1 As shown in A, the nucleotide sequence of IL-15 in each construct is as described in SEQ ID NO: 12; construct 001 is used as a control, the structure uses CD80 transmembrane domain and CD80 intracellular domain, and the promoter uses 3×HRE-TK-mini promoter; constructs 002, 003 and 004 use CD86 transmembrane domain and CD86 intracellular domain, and the promoters use 3×HRE-TK-mini promoter, 6×HRE-TK-mini promoter, and 9×HRE-TK-mini promoter respectively. The four constructs all contain the second promoter SFFV, the second signal peptide SP2 and the molecular switch element hEGFRt. The plasmid construction methods containing the four constructs are as follows:

[0132] (1) Download the CDS sequence of the relevant gene from NCBI, and then perform codon optimization on some gene sequences to enhance their expression. These optimization methods include but are not limited to: human codon usage preference, moderate GC content, stable mRNA secondary structure, etc., eliminate repetitive sequences and cryptic splicing sites and unnecessary restriction enzyme sites, and prevent the depletion of tRNA pool in cells;

[0133] (2) The optimized sequence was directly cloned into the lentiviral shuttle vector pLV-EF1a-c-MYC-IRES-EGFP by GenScript through whole gene synthesis, and the sequence from EFI1a promoter to EGFP in the vector was replaced with the target sequence. The newly constructed vector was named: pLV-HRE-mIL-15.

[0134] 2. Lentivirus Preparation

[0135] HEK-293T cells were transfected with the pLV-HRE-mIL-15, psPAX2 and pMD2.G vector plasmids containing the mIL-15 gene obtained in step 1. The transfection system was configured as follows: (1) Centrifuge tube A: Opti-MEM 500μl + main plasmid 10μg + pMD2.G 5μg + psPAX2 5μg + p3000 40μl; (2) Centrifuge tube B: OPti-MEM 500μl + Lipo3000 40μl. Slowly drip the mixture in centrifuge tube B into centrifuge tube A, gently mix with a pipette, and let stand at room temperature for 15-20 minutes;

[0136] The transfection system was added to HEK-293T cells. After 4 hours of transfection, the supernatant was discarded and 10 ml of DMEM complete medium was added to each dish; the dish was placed in a 37°C, 5% CO2 incubator for continued culture; after 48 hours, the culture supernatant of the transfected 293T cells was collected; the cells were centrifuged at 400 g for 5 minutes; the cells were filtered with a 0.45 μm filter, and the filtrate obtained was the original solution of the recombinant lentivirus; the lentivirus was concentrated using an Utra-15 centrifugal filter device, and the cells were centrifuged at 5000 rpm for 50 minutes; the concentrated lentivirus was packaged and stored in a -80°C refrigerator for later use.

[0137] 3. Lentivirus titer determination

[0138] Count the HEK-293T cells after washing and digestion. Dilute the concentrated lentiviral stock solution in 10-fold gradient dilutions (50×, 500×, 5000×, 50000×). Take 2.5×10 5 HEK-293T cells were added to the corresponding centrifuge tubes, each containing 500 μl of culture medium-virus-cell mixture, and then transferred to a 24-well plate after mixing; after culturing in a 37°C, 5% CO2 incubator for 24 hours, the culture medium was replaced; after 48 hours, the infected HEK-293T cells were collected for hypoxia treatment, and the proportion of cells with positive transgene expression was detected by flow cytometry to calculate the virus titer. Lentivirus titer calculation method: virus titer = (m×2.5×10 5 × dilution factor) / transfected volume, where m is the proportion of cells positive for transgene expression.

[0139] 4. Preparation of lung cancer TIL cells

[0140] Place the lung cancer tumor block in a 100 mm culture dish and wash with PBS. Use sterile ophthalmic scissors or a scalpel to remove the necrotic area and connective tissue on the tumor block, and then cut it into 1-3 mm 3Small pieces; culture the lung cancer tumor pieces in RPMI1640 medium containing 10% AB serum, 1% glutamine, 1% double antibody, and 6000IU / mL IL2. Observe the cells under a microscope. If there are no obvious adherent cells, change the medium every other day; if the lymphocytes do not gradually increase or the cancer cells do not decrease, continue to change the medium; if the lymphocyte density increases significantly, such as the appearance of cell clusters, expand the wells and continue to culture in a 37°C, 5% CO2 incubator; continue to culture for no more than 10 days, and collect TIL cells. The collected cell suspension is filtered with a 40μm filter to remove the tumor pieces; centrifuged at 1000rpm for 5min, and resuspended in an appropriate amount of REP complete medium for use.

[0141] 5. Lentiviral infection of TIL cells

[0142] According to the titer of lentivirus, TIL cells were infected with MOI=10; lentivirus (MOI=10), Lentiboost (100×), TIL cells (3.5×10 5 ) was added to REP complete medium, with 200 μl of medium-virus-cell mixture in each centrifuge tube. After mixing, the mixture was transferred to a 48-well plate and cultured in a 37°C, 5% CO2 incubator. After 24 hours of infection, the cells were collected and centrifuged at 1000 rpm, and the virus solution was discarded. An appropriate amount of REP complete medium was added to resuspend the mixture, and the mixture was transferred to a 24-well plate and cultured in a 37°C, 5% CO2 incubator.

[0143] 6. Hypoxia treatment of lentiviral-infected TIL cells

[0144] After culturing the TIL cells infected with lentivirus in step 5 for 72 h, the cells were collected for cell counting; 1×10 6 The remaining TIL cells (as the non-hypoxic control group) were transferred to a new 24-well plate and placed in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours of hypoxia, the cells were counted and 5×10 cells were taken from the experimental group and the control group, respectively. 5 The cells were detected by flow cytometry.

[0145] 7. Flow cytometry

[0146] Take the cell suspension from step 6 and count the cells. Take 5×10 5 The cells were centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended in 100 μl PBS containing 2% FBS, followed by adding 2.5 μl Fc to block Fc receptors and incubated at room temperature for 10 min; the cells were divided into two groups, the all-negative group and the group to be tested, with a cell volume of 50 μl in each group, i.e. 2.5×10 5cells; added IL-15 (Beijing Baixinyi Biotechnology Co., Ltd., A09D21-9E) and cetuximab injection (Merck) antibody mixture to the test group and incubated at room temperature for 15 minutes; after the incubation, added 1 ml of PBS containing 2% FBS, centrifuged at 400g for 5 minutes; discarded the supernatant, added 50 μl of FITC goat anti-human IgG Fcγ antibody (BioLegend, 398006) and Alexa Fluor 647 donkey anti-rabbit IgG antibody (BioLegend, 4064) antibody mixture prepared with PBS containing 2% FBS to the cells of the test group, incubated at room temperature for 10 minutes; added 50 μl of 7-AAD mixture prepared with PBS containing 2% FBS to the cells of the test group, incubated at room temperature for 7 minutes; added 1 ml of PBS containing 2% FBS to the all-negative group and the test group, centrifuged at 400g for 5 minutes; discarded the supernatant, added 200 μl of PBS containing 2% FBS to resuspend the cells, and tested on the machine.

[0147] 8. Results

[0148] After flow cytometry staining of TIL cell suspension infected with lentivirus containing the target gene, analysis was performed using a Beckman flow cytometer. Figure 1 B shows that plasmid construction and lentiviral infection were successful, and 001-TIL, 002-TIL, 003-TIL and 004-TIL were obtained respectively. Compared with uninfected TIL, the proportion of mIL-15+ cells in the TIL group infected with lentivirus was lower without hypoxia treatment; after hypoxia treatment, the proportion of mIL-15+ cells in TIL increased. At the same time, compared with the non-hypoxia group, the proportion of hEGFRt+ cells in the TIL cells infected with lentivirus after hypoxia treatment did not change significantly due to the regulation of the constitutive promoter SFFV.

[0149] The results showed that 3×HRE, 6×HRE, and 9×HRE can all induce the expression of mIL-15. Unexpectedly, among the three promoters, the 3×HRE promoter produced the highest proportion of mIL-15+ cells under hypoxia-induced conditions.

[0150] Example 2: Detection of IL-15 in the supernatant of TIL cells infected with lentivirus

[0151] TIL cells were infected with lentivirus for 72 hours and then treated with hypoxia. After 24 hours of hypoxia, the culture supernatants of non-hypoxic and hypoxic cells were collected. 100 μl of diluted standard and sample were added, and 100 μl of 1× dilution buffer R was added as the control well. The well was covered with a sealing film and incubated at room temperature (18-25°C) for 2 hours. Washing: Remove the liquid in the well, add 300 μl of 1× washing buffer working solution, stay for 1 minute and then discard the liquid in the well. Repeat 3 times, and pat dry on filter paper each time. Add 100 μl of detection antibody biotinylated antibody human IL-15 (Dayou, 1111502) working solution; cover the sealing film, incubate at room temperature (18-25°C) for 1 hour; wash the plate 3 times; add 100 μl of streptavidin-HRP working solution; cover the sealing film, incubate at room temperature (18-25°C) for 30 minutes; wash the plate 3 times. Add 100 μl TMB and incubate at room temperature (18-25°C) in the dark for 5-30 min; quickly add 100 μl stop solution to terminate the reaction.

[0152] After lung cancer TIL cells were infected with lentivirus containing the target gene for 72 hours and treated with hypoxia, the culture supernatants of non-hypoxic and hypoxic group cells were collected, and the shedding of mIL-15 was detected by ELISA.

[0153] The results are as follows Figure 2 As shown, compared with the control group and the non-hypoxia group, there was no significant difference in the secretion of IL-15 after hypoxia treatment, indicating that the IL-15 expressed by the membrane of the present invention did not fall off.

[0154] Example 3: mIL-15 promotes T SCM Cell formation, reducing T cell exhaustion

[0155] The lentiviruses with different plasmid structures infected TILs to obtain 001-TIL, 002-TIL, 003-TIL, and 004-TIL, respectively. After co-culture with trophoblast cells K562-mIL21-4-1BBL for 7, 14, and 18 days, the cells were counted and T SCM , detection of T cell exhaustion.

[0156] 1. Construction of trophoblast cells K562-mIL21-4-1BBL

[0157] Construction of plasmids containing target genes IL21 and 4-1BBL: Full gene synthesis (GenScript) containing IL21, 4-1BBL, IRES, mCherry and Puro sequences was performed, and then cloned into the pLV-EF1a-c-MYC-IRES-EGFP vector to replace the original c-MYC-IRES-EGFP sequence; after obtaining the plasmid, lentivirus packaging was performed and its titer was calculated; according to the titer of the lentivirus, K562 cells were infected at MOI=10; the lentivirus-infected K562 cells were treated with puromycin (2μg / ml) to obtain trophoblast cells K562-mIL21-4-1BBL; the expression of IL21 on K562-mIL21-4-1BBL cells was detected by flow cytometry.

[0158] The amino acid sequence of mIL21-4-1BBL is shown below:

[0159] MEYASDASLDPEAPWPPARARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTK ELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPPSPRSEGACRAEGRG SLLTCGDVEENPGPHMMRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRR QKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT(SEQID NO:20).

[0160] 2. Co-culture of lentivirus-infected TIL cells with K562-mIL21-4-1BBL

[0161] K562-mIL21-4-1BBL cells treated with puromycin were collected and centrifuged at 1000 rpm for 10 min. After centrifugation, the supernatant was discarded and washed three times with PBS. The cells were counted and RPMI1640 complete medium was added to adjust the cell density to 1×10 6 / ml; add 6μg / ml mitomycin C, mix well and transfer to a T75 culture flask; place in a 37°C, 5% CO2 incubator for 18-24h.

[0162] Day 1: After 72 hours of lentivirus infection, TIL cells were treated with hypoxia. After 24 hours of hypoxia, cells in the non-hypoxic and hypoxic groups were collected, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The culture medium used for TIL cells not infected with lentivirus was REP complete medium (containing IL2 and anti-CD3), and the culture medium used for TIL cells infected with lentivirus was REP complete medium (without IL2 but containing anti-CD3). The cells were resuspended, mixed, and the cells were counted. K562-mIL21-4-1BBL cells treated with mitomycin C were taken and centrifuged at 1000 rpm for 10 minutes. After centrifugation, the supernatant was discarded and washed three times with PBS. K562-mIL21-4-1BBL cells were resuspended with REP complete medium and the cells were counted. TIL cells and K562-mIL21-4-1BBL cells were co-cultured in a 24-well plate at a ratio of 1:25. The cells were placed at 37°C and 5% Culture in a CO2 incubator; change the medium every 3 days (no anti-CD3 is added in subsequent changes); and perform subculture when the cell confluence reaches 80%.

[0163] Day 7 and 14: Collect the co-cultured cells and centrifuge at 1000 rpm for 5 min; discard the supernatant, add the corresponding REP complete medium to resuspend the cells, and count the cells.

[0164] Day 18: TIL cells were collected, counted, and treated with hypoxia for 24 h; centrifuged at 1000 rpm for 5 min; the supernatant was discarded, and the corresponding REP complete medium was added to resuspend the cells and counted; 1×10 6 The cells were detected by flow cytometry.

[0165] 3. Flow cytometry

[0166] (1) Flow cytometry detection of trophoblast cells K562-mIL21-4-1BBL:

[0167] K562 cells infected with lentivirus treated with puromycin were collected and the cells were counted. 5×10 5The cells were centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended in 100 μl PBS containing 2% FBS, 2.5 μl Fc was added to block Fc receptors, and incubated at room temperature for 10 min; the cells were divided into two groups, the all-negative group and the group to be tested, with a cell volume of 50 μl in each group, i.e. 2.5×10 5 cells; add Alexa Fluor 647 mouse anti-human IL21 (BioNTech, 562043) antibody mixture to the test group (add 1.25 μl antibody to each sample) and incubate at room temperature for 8 min; after the incubation, add 50 μl of 7-AAD mixture prepared with PBS containing 2% FBS to the cells of the test group and incubate at room temperature for 7 min; add 1 ml of PBS containing 2% FBS to the all-negative group and the test group, centrifuge at 400 g for 5 min; discard the supernatant, add 200 μl PBS containing 2% FBS to resuspend the cells, and detect on the machine.

[0168] (2) Flow cytometry detection of TIL cells:

[0169] After TIL cells were co-cultured with trophoblast cells for 18 days, cells were collected and counted. 7.5×10 5 The cells were centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were resuspended in 150 μl PBS containing 2% FBS, followed by the addition of 4 μl Fc to block Fc receptors, and incubated at room temperature for 10 min; the cells were divided into three groups: a negative group, a group to be tested, and a group to be tested. The cell volume of each group was 50 μl, i.e., 2.5×10 5 cells; add Alexa to the group to be tested 700 anti-human CD45RA antibody (BioLegend, 304120), APC anti-human CD62L antibody (BioLegend, 304810), Brilliant Violet 510 TM Anti-human CD3 antibody (BioLegend, 317332) and APC / Cyanine7 anti-human CD8 antibody (BioLegend, 344714) antibody mixture (1.25 μl antibody for each sample) were incubated at room temperature for 8 min; FITC anti-human CD127 (IL-7Rα) antibody (BioLegend, 351312), PE anti-human CD279 (PD-1) antibody (BioLegend, 621608), APC anti-human CD366 (Tim-3) antibody (BioLegend, 345012), Brilliant Violet 510 TMAnti-human CD3 antibody (BioLegend, 317332) and APC / Cyanine7 anti-human CD8 antibody (BioLegend, 344714) antibody mixture (1.25 μl antibody was added to each sample) and incubated at room temperature for 8 min. After incubation, 50 μl of 7-AAD mixture prepared with PBS containing 2% FBS was added to the cells of the first group and the second group to be tested, and incubated at room temperature for 7 min. 1 ml of PBS containing 2% FBS was added to the negative group and the group to be tested, and centrifuged at 400 g for 5 min. The supernatant was discarded, and 200 μl of PBS containing 2% FBS was added to resuspend the cells, and the cells were tested on the machine.

[0170] 4. Results

[0171] like Figure 3 A shows the schematic diagram of the structure of trophoblast cells K562-mIL21-4-1BBL; Figure 3 B shows that compared with K562 cells, the proportion of mIL21+ cells in trophoblast cells K562-mIL21-4-1BBL is 84.3%, indicating that the trophoblast cells K562-mIL21-4-1BBL were successfully constructed. Figure 3 C shows that compared with the control group TIL cells cultured with IL2, there was no significant difference in the proliferation and viability of the mIL-15-overexpressing TIL cells cultured without IL2, indicating that TIL overexpressing mIL-15 can promote its proliferation; among them, after hypoxia treatment of 002-TIL, cell proliferation was better than that of the non-hypoxia group.

[0172] like Figure 3 D shows that compared with the control group TIL cells cultured with IL2, 001-TIL and 002-TIL cells overexpressed mIL-15 under the condition of no IL2 culture. SCM Compared with the non-hypoxic group, the proportion of T cells in 001-TIL and 002-TIL cells after hypoxia treatment increased significantly. SCM Compared with 001-TIL (CD80-TM and CD80-Cyto), the T of 002-TIL structured with CD86-TM and CD86-Cyto in the present invention is increased. SCM The proportion increased significantly.

[0173] like Figure 3 E shows that compared with the control group TIL cells cultured with IL2, the proportion of IL7Ra+ cells in 002-TIL cells overexpressing mIL-15 increased. SCM The proportion of IL7Ra+ cells increased, indicating that overexpression of mIL-15 can enhance their stemness. Figure 3F shows that compared with the control group TIL cells cultured with IL2, under the condition of no IL2 culture, 001-TIL and 002-TIL cells overexpressed mIL-15, and the expression levels of PD-1 and Tim3 in CD8+T cells were significantly reduced; compared with 001-TIL (CD80-TM and CD80-Cyto), the expression levels of PD-1 and Tim3 in CD8+T cells of 002-TIL with the structure of CD86-TM and CD86-Cyto in the present invention were significantly reduced.

[0174] The results showed that TIL cells overexpressed mIL-15, promoting their T SCM The generation of cells and the expression of their exhaustion proteins were reduced; and the effect of using CD86-TM-CD86-Cyto was significantly better than that of CD80-TM-CD80-Cyto.

[0175] Example 4: Anti-tumor activity of TIL cells overexpressing mIL-15

[0176] In order to verify whether overexpression of mIL-15 in TIL cells promotes its anti-tumor activity and the difference between CD80-TM and CD80-Cyto and CD86-TM and CD86-Cyto in the function of mIL-15, 001-TIL and 002-TIL cells were co-cultured with tumor cells Calu6, and CCK8 and ELISA were used to detect tumor activity and IFN-γ secretion. The experimental method is as follows:

[0177] 1. CCK8 method to detect tumor cell activity

[0178] Take 1×10 5 Calu6 cells were inoculated in 24-well plates. TIL cells were infected with lentivirus for 72 hours and then subjected to hypoxia treatment. After 24 hours of hypoxia, TIL cells in the non-hypoxia and hypoxia groups were collected and centrifuged at 1000 rpm for 5 minutes. REP complete medium was added to adjust the cell density to 1.5×10 6 / ml; discard the culture supernatant of Calu6 cells in the 24-well plate; add 1ml of non-hypoxic and hypoxic TIL cell suspension (i.e. 1.5×10 6 TIL cells); after culturing in a 37°C, 5% CO2 incubator for 24 hours, the cell culture supernatant was collected; the cells were washed once with PBS; and the activity of tumor cells was detected using the CCK8 method.

[0179] 2. ELISA to detect the secretion of IFN-γ

[0180] Centrifuge the cell culture supernatant collected above at 400g for 5 minutes, and transfer the supernatant to a new centrifuge tube; determine the number of strips required according to the number of experimental wells (blank and standard); add 100μl of diluted standard and sample, and add 100μl 1× dilution buffer R as the control well; add 100μl biotinylated antibody human IFN-γ (Dayou, 1110002) working solution; cover with a sealing film and incubate at room temperature (18-25℃) for 1h. Wash the plate: deduct the liquid in the well, add 300μl 1× washing buffer working solution, stay for 1min and then discard the liquid in the well; repeat 3 times, and pat dry on filter paper each time. Add 100μl streptavidin-HRP working solution; cover with a sealing film and incubate at room temperature (18-25℃) for 20min; repeat washing 3 times. Color development: add 100 μl TMB, incubate at room temperature (18-25°C) in the dark for 5-30 min; Termination reaction: quickly add 100 μl stop solution to terminate the reaction.

[0181] 3. Results

[0182] The results are as follows Figure 4 , as shown in Table 1 and Table 2.

[0183] like Figure 4 A shows that compared with tumor cell Calu6 cultured alone, the activity of tumor cell Calu6 was significantly reduced when 001-TIL and 002-TIL cells were co-cultured with tumor cell Calu6; and compared with 001-TIL, 002-TIL cells had stronger anti-tumor activity.

[0184] like Figure 4 B shows that compared with TIL cells overexpressing mIL-15 cultured alone, the secretion of IFN-γ in the culture supernatant of 001-TIL and 002-TIL cells co-cultured with tumor cells Calu6 significantly increased; and compared with 001-TIL, the secretion of IFN-γ in 002-TIL cells was higher.

[0185] Table 1. Antitumor activity of different TIL cells

[0186] Tumor cell activity (%) Calu6 TIL cells+Calu6 Control TIL 100 67.79 001-TIL 100 40.79 002-TIL 100 12.875

[0187] Table 2. IFN-γ secretion of different TIL cells

[0188]

[0189] The experimental results show that overexpression of mIL-15 in TIL cells can promote their anti-tumor activity, and the anti-tumor activity and IFN-γ secretion of 002-TIL are significantly better than those of 001-TIL. It can be seen that the effect of using CD86-TM and CD86-Cyto is significantly better than that of CD80-TM and CD80-Cyto.

[0190] Example 5: Cytotoxicity

[0191] In clinical practice, in order to further improve the safety of TIL cell therapy overexpressing mIL-15, the present invention provides a membrane surface domain (hEGFRt) as an immune brake element and / or recognition element, and the hEGFRt-specific monoclonal antibody cetuximab can mediate complement-dependent cytotoxicity (CDC) and NK cell-mediated cytotoxicity (ADCC) to effectively lyse TIL cells expressing mIL-15.

[0192] CDC is the binding of the Fc end of the antibody to the complement molecule and the activation of the complement molecule, the binding of the Fab end of the antibody to the target cell, and then the formation of a membrane attack complex, which ultimately leads to the lysis of the target cell. In the present invention, the hEGFRt expressed by the TIL cells binds to cetuximab and the added complement molecules, resulting in the lysis of the TIL cells.

[0193] ADCC refers to the binding of the Fab segment of an antibody to the antigenic epitopes of cells infected with lentivirus or tumor cells, and the binding of its Fc segment to the FcR on the surface of killer cells (NK cells, macrophages, neutrophils, etc.), mediating the direct killing of target cells by killer cells. It is an important mechanism for the action of anti-tumor therapeutic antibody drugs.

[0194] The experimental method is as follows:

[0195] (1) Complement-dependent cytotoxicity (CDC) assay

[0196] The lentivirus-infected TIL cells were co-cultured with K562-mIL21-4-1BBL cells for 18 days and subjected to hypoxia treatment. The cells were collected and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the corresponding REP complete medium was added to resuspend the cells and count the cells. 5×10 5 The cells were used for CDC experiment; a 48-well plate was used, the culture system was 200 μl, and they were divided into 3 experimental groups, namely: TIL cells, TIL cells + 100 μg / ml cetuximab, TIL cells + 25% complement + 100 μg / ml cetuximab; after being cultured in a 37°C, 5% CO2 incubator for 4 hours, the cells were collected for flow cytometry detection.

[0197] (2) Antibody-dependent cellular cytotoxicity (ADCC) assay

[0198] TIL cells infected with lentivirus were co-cultured with K562-mIL21-4-1BBL cells for 18 days and subjected to hypoxia treatment. The cells were collected and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the corresponding REP complete medium was added to resuspend the cells and count the cells. 1×10 6 The cells were used for ADCC experiment; a 24-well plate was used, the culture system was 1 ml, and they were divided into 4 experimental groups, namely: TIL cells, TIL cells + 100 μg / ml cetuximab, TIL cells + NK cells (efficacy-target ratio 1:10) + 100 μg / ml cetuximab, TIL cells + NK cells (efficacy-target ratio 1:20) + 100 μg / ml cetuximab; after culturing in a 37 ° C, 5% CO2 incubator for 24 hours, the cells were collected for flow cytometry detection.

[0199] The experimental results are as follows Figure 5 Compared with 002-TIL cells and 002-TIL cells and cetuximab, the proportion of mIL-15+ TIL cells was significantly reduced in 002-TIL cells treated with complement and cetuximab for 4 hours ( Figure 5 A). Compared with 002-TIL cells and 002-TIL cells and cetuximab, the proportion of mIL-15+ TIL cells was significantly decreased in 002-TIL cells treated with NK cells (1:10 and 1:20) and cetuximab for 24 h ( Figure 5 B).

[0200] This shows that the molecular brake element based on the action mechanism of the hEGFRt extracellular region has higher safety and effectiveness.

[0201] Example 6: Different connection methods of immune brake element hEGFRt

[0202] The immune brake element hEGFRt in the present invention is connected with the constitutive promoter SFFV and also provides a polypeptide linker sequence T2A that can be cut in vivo for connection. The DNA expression frame of the fusion protein formed by hEGFRt through T2A connection is translated into a fusion polypeptide in vivo, which is also cut in vivo while being translated in vivo. hEGFRt is transported to the extracellular space through the signal peptide and is fixed to the cell membrane surface through the transmembrane region of its C-terminal region. Figure 6 As shown in A, construct 005 was constructed, in which the immune brake elements were connected by the polypeptide linker sequence T2A, and the SFFV promoter sequence in construct 002 was replaced with the T2A sequence through molecular cloning technology.

[0203] like Figure 6As shown in B, lentivirus packaging was performed, and TIL cells were infected for 5 days and then treated with hypoxia for 24 hours, and then mIL-15 and hEGFRt were detected by flow cytometry. The results showed that the plasmid construction and lentivirus infection were successful. Compared with uninfected TIL, the proportion of mIL-15+ cells in TIL cells infected with 005 lentivirus was lower without hypoxia treatment; after hypoxia treatment, the proportion of mIL-15+ cells in TIL increased.

[0204] like Figure 6 As shown in C, compared with uninfected TIL, the gMFI of mIL-15 in TIL infected with 005 lentivirus was lower without hypoxia treatment; after hypoxia treatment, the gMFI of mIL-15 in TIL increased significantly; at the same time, compared with the non-hypoxia group, the gMFI of hEGFRt in TIL infected with lentivirus was significantly increased after hypoxia treatment. Figure 6 As shown in D, under the condition of adding IL2, both uninfected TIL and TIL infected with 005 lentivirus can continue to proliferate until the 13th day, and the cell viability is ≥90%; under the condition of not adding IL2, compared with uninfected TIL, TIL infected with 005 lentivirus can continue to proliferate until the 13th day, and the cell viability is ≥90%.

[0205] The results showed that the immune brake element hEGFRt can also be induced to express when connected by T2A; in the absence of IL2, TIL infected with 005 lentivirus can continue to proliferate, and the cell viability is ≥90%. The results showed that the immune brake element hEGFRt can also be induced to express when connected by T2A; in vitro, overexpression of mIL-15 can promote the proliferation of TIL cells and avoid clinical side effects caused by the use of IL2.

[0206] Example 7: Lentivirus infection of cervical cancer TIL cells

[0207] In this example, it is further studied whether the expression of mIL-15 of the present invention can promote the stemness of cervical cancer TIL cells or reduce the expression of their exhaustion proteins. The method is as follows:

[0208] Preparation of cervical cancer TIL cells: Place the cervical cancer tumor block in a 100 mm culture dish and wash it with PBS. Use sterile ophthalmic scissors or a scalpel to remove the necrotic area and connective tissue on the tumor block, and then cut it into 1-3 mm 3Small pieces; Cervical cancer tumor pieces are cultured in RPMI1640 medium containing 10% AB serum, 1% glutamine, 1% double antibody, and 6000IU / mL IL2. If there are no obvious adherent cells when observing the cells under a microscope, change the medium every other day; if the lymphocytes do not gradually increase or the cancer cells do not decrease, continue to change the medium; if the density of lymphocytes increases significantly, such as the appearance of cell clusters, expand the wells and continue to culture in a 37°C, 5% CO2 incubator; continue to culture for no more than 10 days, and collect the cells. The collected cell suspension is filtered with a 40μm filter to remove the tumor pieces; centrifuged at 1000rpm for 5min, and an appropriate amount of REP complete medium is added to resuspend and set aside.

[0209] The method described in Example 1 was used to introduce the 005 construct into the cervical cancer TIL cells, and the stemness of the TIL cells and the expression of the depletion protein were observed.

[0210] like Figure 7 As shown in A, cervical cancer TIL cells were infected with 005 lentivirus for 5 days and then treated with hypoxia for 24 hours, and then mIL-15 and hEGFRt were detected by flow cytometry. The results showed that compared with uninfected TIL, the proportion of mIL-15+ and hEGFRt+ cells in TIL cells infected with 005 lentivirus was lower without hypoxia treatment; after hypoxia treatment, the proportion of mIL-15+ and hEGFRt+ cells in TIL increased.

[0211] like Figure 7 As shown in B, compared with uninfected TIL, the gMFI of mIL-15 and hEGFRt in TIL cells infected with 005 lentivirus increased after hypoxia treatment.

[0212] like Figure 7 Figure C shows that under non-hypoxic and hypoxic conditions, compared with the control group TIL cells cultured with IL2, 005-TIL cells cultured without IL2 overexpressed mIL-15. SCM The proportion of cells increased significantly.

[0213] like Figure 7 Figure D shows that under non-hypoxic and hypoxic conditions, the proportion of IL7Ra+ cells in 005-TIL cells overexpressing mIL-15 without IL2 was significantly increased compared with the control group TIL cells cultured with IL2. SCM The proportion of IL7Ra+ cells increased, indicating that mIL-15 can enhance their stemness.

[0214] At the same time, if Figure 7E shows that under non-hypoxic and hypoxic conditions, compared with the control group TIL cells cultured with IL2, 005-TIL cultured without IL2 overexpressed mIL-15, and the expression levels of TIGIT and Tim3 in CD8+T cells were significantly reduced.

[0215] The results show that the expression of mIL-15 in the present invention in cervical cancer TIL cells can significantly promote their T SCM The generation of cells and significantly reduced the expression of their depleted proteins.

[0216] discuss

[0217] Long-term immune surveillance by persistent TILs may be key to achieving durable responses in adoptive cell therapy (ACT). In mouse and nonhuman primate models, TILs from central memory (T CM ) or naive (T N ) T cell subsets have shown higher therapeutic potential in adoptively transferred effector CD8+ T cells. Therefore, T cell subsets corresponding to the immature differentiated state have attracted much attention due to their potential to provide excellent clinical utility. T memory stem cells (T SCM ) is the least differentiated memory T cell subset identified so far, so it is important to prepare T cells suitable for cell therapy. SCM is crucial.

[0218] IL-15 is a pro-survival cytokine that is required for the maintenance of long-lived CD8+ memory T cell homeostasis, inhibits activation-induced cell death (AICD), enhances antitumor activity in vivo, and reverses T cell anergy. In contrast to IL2, IL-15 does not lead to the expansion of Tregs. In addition, IL-15 is required for the generation of innate T cells that participate in immune surveillance and hinder tumor growth. IL-15 is considered an immunotherapeutic agent for cancer treatment, acting on natural killer (NK) cells or T cells. Monomeric IL-15 is a small, unstable protein with a short serum half-life that requires supraphysiological administration to obtain an in vivo response. Physiological transport of IL-15 requires the use of the IL-15 receptor alpha chain (IL-15Rα), and recombinant soluble IL-15 binds to recombinant soluble IL-15Rα to enhance the antitumor activity of T cells.

[0219] Based on the above research foundation, in order to improve the persistence and memory potential of infused T cells, the patent application 202210316484.5 adopts the Tet-on system, and the inducer tetracycline is added to induce the expression of IL-7 or / and IL-15. Although this method can increase the proliferation of TIL cells, the Tet-on system contains the C-terminus of the herpes simplex virus (HSV) VP16 protein, which is immunogenic; and the system requires the inducer tetracycline or tetracycline derivatives to induce the secretion and expression of IL-7 and IL-15, and there are side effects caused by the inducer. Patent application US2022 / 0133801A1 uses Acetazolamide to induce TIL cells to express mIL15. This method avoids the toxicity of high-dose IL2 and promotes the anti-tumor activity of TIL cells. However, this method introduces drugs and has potential clinical side effects. At the same time, after adding small molecule drugs, IL-15 can be expressed in multiple organs and tissues, lacking the selectivity of tumor microenvironment enrichment expression, and there are certain safety risks.

[0220] Hypoxia is a hallmark of solid tumors because of insufficient blood supply, and oxygen concentrations may vary between 0.02% and 2% O2, compared to 2% to 9% O2 in normal tissues (Sharma A, Arambula JF, Koo S, Kumar R, Singh H, Sessler JL, et al. Hypoxia-targeted drug delivery. Chem Soc Rev. 2019; 48(3): 771-813.). Targeted therapy using this unique environmental signal is a strategy in cancer treatment. Therefore, using the hypoxic microenvironment of solid tumors, in TIL cell transformation, the present invention uses HRE to induce expression of membrane-bound IL-15 (mIL-15) to maintain the long-term persistence of the memory stem cell phenotype, and the use of CD86 transmembrane region and intracellular domain further increases the proportion of stem cells in TIL cells, enhancing their anti-tumor activity; truncated hEGFRt is used in molecular construction to further improve its safety. mIL-15 provides an ideal signaling pathway that allows TIL cells infused into patients to prolong their persistence, thereby providing long-lasting immune surveillance and therapeutic potential.

[0221] All documents mentioned in the present invention are cited as references in the present invention, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present invention.

Claims

1. A fusion polypeptide, characterized in that: The structural formula of the fusion polypeptide is shown in formula (I): X-IL15-LH-TM-Cyto(I) in, "-" is none independently; X is none or signal peptide; IL15 is an interleukin 15 element, and the amino acid sequence of interleukin 15 is shown in SEQ ID NO: 1; L is a linker; H is a hinge region, the amino acid sequence of which is shown in SEQ ID NO: 4; TM is a transmembrane domain, and the amino acid sequence of the transmembrane domain is shown in SEQ ID NO: 3; Cyto is an intracellular domain, wherein the intracellular domain is a CD86 intracellular domain, and the amino acid sequence of the CD86 intracellular domain is shown in SEQ ID NO:

2.

2. The fusion polypeptide according to claim 1, characterized in that The X is a signal peptide.

3. The fusion polypeptide according to claim 2, characterized in that The amino acid sequence of the signal peptide is shown in SEQ ID NO:

6.

4. The fusion polypeptide according to claim 1, characterized in that The amino acid sequence of the linker is shown in SEQ ID NO:

5.

5. The fusion polypeptide according to claim 1, characterized in that The sequence of the fusion polypeptide is shown in SEQ ID NO:

7.

6. A polynucleotide, characterized in that The polynucleotide encodes the fusion polypeptide of claim 1.

7. A nucleic acid construct, characterized in that The nucleic acid construct comprises a first expression cassette comprising a hypoxia-responsive promoter and the polynucleotide of claim 6.

8. The nucleic acid construct according to claim 7, wherein The hypoxia-responsive promoter is a promoter comprising n hypoxia response elements HRE, that is, a promoter containing n×HRE elements, where n is an integer selected from 1-20.

9. The nucleic acid construct according to claim 7, wherein The nucleic acid construct further comprises a molecular switch element sequence, and the molecular switch element is selected from the following group: hEGFRt, BCMA, and CD20.

10. A carrier, characterized in that The vector comprises the polynucleotide according to claim 6.

11. An immune cell, characterized in that: The cell comprises the fusion polypeptide of claim 1, the polynucleotide of claim 6, the nucleic acid construct of claim 7, and / or the vector of claim 10.

12. The cell according to claim 11, wherein The cells are tumor infiltrating cells (TIL).

13. A composition, characterized in that The composition comprises the fusion polypeptide of claim 1, the polynucleotide of claim 6, the nucleic acid construct of claim 7, the vector of claim 10, and / or the cell of claim 11, and a pharmaceutically acceptable carrier.

14. A kit, characterized in that: The kit comprises the fusion polypeptide of claim 1, the polynucleotide of claim 6, the nucleic acid construct of claim 7, the vector of claim 10, the cell of claim 11, and / or the composition of claim 13.

15. Use of the fusion polypeptide of claim 1, the polynucleotide of claim 6, the nucleic acid construct of claim 7, the vector of claim 10, the cell of claim 11, the composition of claim 13, and / or the kit of claim 14 in preparing a medicament for alleviating and / or treating a tumor selected from the group consisting of lung cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, bladder cancer, breast cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumor, and head and neck cancer.

16. The use according to claim 15, characterized in that The tumor is colorectal cancer.

17. The use according to claim 15, characterized in that The tumor is colon cancer or rectal cancer.

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