Engineered til expressing membrane-bound il-15 fusion protein and use thereof

Abstract

The present invention relates to a membrane-bound IL-15 fusion protein expressed on a TIL cell. Specifically, in the present invention, a hypoxia response element (HRE) is used to induce the expression of the membrane-bound IL-15 (mIL-15) fusion protein, so that a TIL cell population expressing the fusion protein persistently maintain a memory stem cell phenotype for a long time, thereby enhancing the anti-tumor activity thereof. During the construction of the fusion protein, a CD86 transmembrane region and an intracellular domain are used, thereby further improving the proportion of stem cells in the TIL cells. During molecule construction, a truncated form of hEGFRt is used as a molecular switch to further enhance the safety. The stemness and persistence of the TIL cells expressing the fusion protein of the present invention are significantly enhanced, and persistent immune surveillance and therapeutic efficacy can be provided.

Description

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

[0001] The present invention relates to the technical field of genetic 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. Among all types of cancer, solid tumors account for approximately 90%. Due to the complexity, variability, and strong heterogeneity of solid tumors, they are extremely difficult to cure. In recent years, following surgery, radiotherapy, chemotherapy, and immunotherapy, cell therapy has gradually demonstrated its powerful therapeutic effects 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 options 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 fall into the category of ACT. Compared with CAR-T and TCR-T, TIL cell drugs have demonstrated significant efficacy in the treatment of solid tumors and have become one of the most promising technical directions for conquering solid tumors.

[0003] The 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. It has been successfully used in patients with metastatic melanoma and other solid tumors. TIL is composed of T cells with multiple TCR clones. It can not only directly recognize tumor-associated antigens, but also fight against tumor-specific neoantigens, 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 be localized in tumor tissue after being reinfused. In addition, TIL comes from the patient himself, which means that this method has low toxicity.

[0004] However, TIL therapy obviously has its limitations. The usual method for preparing TIL is to obtain a single-cell suspension after mechanical treatment or enzymatic digestion of tumor tissue. T lymphocytes are selected and cultured by adding high concentrations of IL2. Billions of TIL cells are cultured within 2-4 weeks and then re-infused 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 lead to dose-limiting toxicity, requiring enhanced monitoring and care; low-dose IL2 may be beneficial to peripheral tolerance and the production of regulatory T cells, thereby inhibiting the anti-tumor response of TIL; in addition, the IL2 signaling pathway drives the proliferation of effector T cells, promoting 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, comprising 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 comprises 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): X-IL15-LH-TM-Cyto (I)

[0022] in,

[0023] “-” each independently represents no peptide or a connecting peptide;

[0024] X is none or signal peptide;

[0025] IL15 is the interleukin 15 element;

[0026] L is none or a linker;

[0027] H is the hinge region;

[0028] TM is the transmembrane domain;

[0029] Cyto is an intracellular domain, wherein the intracellular domain is the CD86 intracellular domain.

[0030] In another preferred example, the amino acid sequence of the CD86 intracellular domain is shown in SEQ ID NO: 2.

[0031] 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.

[0032] In another preferred embodiment, the hinge region is the CD86 hinge region. Preferably, the amino acid sequence of the CD86 hinge region is as shown in SEQ ID NO:4.

[0033] In another preferred embodiment, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 6.

[0034] In another preferred embodiment, the sequence of the fusion protein is shown in SEQ ID NO: 7.

[0035] In the second aspect of the present invention, a polynucleotide is provided, which encodes the fusion polypeptide of the present invention.

[0036] In the third aspect of the present invention, a nucleic acid construct is provided. 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.

[0037] In another preferred embodiment, the hypoxia-responsive promoter is a promoter comprising n hypoxia response elements (HREs), ie, a promoter comprising n×HRE elements, where n is an integer selected from 1 to 20.

[0038] 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.

[0039] 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, and IL2-mini promoter containing n×HRE elements.

[0040] 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.

[0041] In another preferred embodiment, the molecular switch element is hEGFRt.

[0042] In another preferred embodiment, the molecular switch element is connected to the first expression cassette via a cleavable connecting peptide sequence.

[0043] In another preferred embodiment, the cleavable connecting peptide is a self-cleaving 2A peptide, preferably a T2A peptide.

[0044] In another preferred embodiment, 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.

[0045] In another preferred embodiment, the second promoter is the SFFV promoter.

[0046] 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.

[0047] In another preferred embodiment, the vector includes a plasmid or a viral vector.

[0048] In another preferred embodiment, the viral vector includes: a lentiviral vector, an adenoviral vector, and a yellow fever virus vector.

[0049] In another preferred embodiment, the vector is a plasmid.

[0050] In the fifth aspect of the present invention, an immune cell is provided, which comprises 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, and / or the vector of the fourth aspect of the present invention.

[0051] In another preferred embodiment, the immune cells are selected from the group consisting of tumor infiltrating cells (TIL), T cells, and NK cells.

[0052] In another preferred embodiment, the cells are tumor infiltrating cells (TIL).

[0053] 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.

[0054] 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.

[0055] 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 medicament for preventing, alleviating and / or treating tumors.

[0056] In another preferred embodiment, the tumor includes a solid tumor, a hematological tumor, or a combination thereof.

[0057] In another preferred embodiment, the tumor is selected from the group consisting of lung cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, bladder cancer, liver cancer, 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, comprising administering the immune cell according to the fifth aspect of the present invention or the composition according to the sixth aspect of the present invention to a subject.

[0059] In another preferred embodiment, the disease is tumor.

[0060] In another preferred embodiment, the tumor includes a solid tumor, a hematological tumor, or a combination thereof.

[0061] In another preferred embodiment, the tumor is selected from the group consisting of lung cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, bladder cancer, liver cancer, 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.

[0062] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 shows lentiviral infection of TIL cells. Figure 1A shows a schematic diagram of the plasmid structures of 001, 002, 003, and 004; Figure 1B shows the results of flow cytometry analysis of the ratio of mIL-15 and hEGFRt after TIL cells were infected with lentiviruses of different plasmid structures.

[0064] Figure 2 shows the shedding of mIL-15 detected by ELISA. 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 membrane-expressed IL-15 does not shed.

[0065] Figure 3 shows the T SCM Figure 3A shows a schematic diagram of the structure of trophoblast cells K562-mIL21-4-1BBL; Figure 3B shows the flow cytometry detection of mIL21 in K562 and K562-mIL21-4-1BBL cells; Figure 3C shows that after TILs were infected with lentiviruses with different plasmid structures, they were co-cultured with trophoblast cells K562-mIL21-4-1BBL for 7 and 14 days, and cell counting and cell viability were recorded; Figure 3D 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 T cell exhaustion was detected by flow cytometry. SCM ; Figure 3E shows that after TILs were infected with lentiviruses of 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 3F shows that after TILs were infected with lentiviruses of different plasmid structures, they were co-cultured with trophoblast cells K562-mIL21-4-1BBL for 18 days, and CD8+ T cell exhaustion was detected by flow cytometry.

[0066] Figure 4 shows the RNA-Seq and flow cytometry analysis of UN-TIL+IL2, TIL-mIL-15+IL2, and TIL-mIL-15-IL2 cells after 14 days of expansion on the K562-mIL21-4-1BBL cell culture platform. Figure 4A shows a volcano plot showing upregulated (red) or downregulated (blue) genes in TIL-mIL-15-IL2 cells compared with UN-TIL+IL2 and TIL-mIL-15+IL2 cells. Figure 4B shows KEGG pathway analysis showing the expression of differentially expressed genes in TIL-mIL-15-IL2 cells relative to UN-TIL+IL2 cells. Figure 4C shows GSEA analysis showing the gene signatures in TIL-mIL-15-IL2 cells relative to UN-TIL+IL2 cells. Figure 4D shows a heat map showing the expression of effector differentiation, effector function, memory stemness-related genes, inhibitory molecules, and survival genes. Figure 4E shows the expression of TCF1 in CD3+hEGFRt- and CD3+hEGFRt+ cells from UN-TIL+IL2, TIL-mIL-15+IL2, and TIL-mIL-15-IL2 cells (-IL2: no IL2 added; +IL2: IL2 added).

[0067] Figure 5 shows the anti-tumor activity of TIL cells overexpressing mIL-15. Figure 5A shows that TIL, 001-TIL, and 002-TIL cells were co-cultured with Calu6 tumor cells for 24 hours, and tumor cell activity was detected by CCK8 assay. Figure 5B shows that TIL, 001-TIL, and 002-TIL cells were co-cultured with Calu6 tumor cells for 24 hours, and IFN-γ secretion was detected by ELISA.

[0068] Figure 6 shows the flow cytometry results of mIL-15. Figure 6A shows the flow cytometry detection of mIL-15+ cells in 002-TIL cells after treatment with mAb, complement, and mAb for 4 hours; Figure 6B shows the flow cytometry detection of mIL-15+ cells in 002-TIL cells after treatment with mAb, NK cells, and mAb for 24 hours.

[0069] Figure 7 shows the results of the detection of TIL cells infected with 005 lentivirus. Figure 7A shows a schematic diagram of the 005 plasmid structure; Figure 7B shows the ratio of mIL-15 and hEGFRt detected by flow cytometry after 005 lentivirus infection of TIL; Figure 7C shows the gMFI of mIL-15 and hEGFRt detected by flow cytometry after 005 lentivirus infection of TIL; Figure 7D shows the in vitro proliferation and survival of TIL and 005-TIL cells with and without the addition of IL2 (-IL2: no IL2 added; +IL2: IL2 added).

[0070] Figure 8 shows the results of cervical cancer TIL cells infected with 005 lentivirus. Figure 8A shows the ratio of mIL-15+ and hEGFRt+ cells detected by flow cytometry after 005 lentivirus infection of TIL; Figure 8B shows the gMFI of mIL-15 and hEGFRt detected by flow cytometry after 005 lentivirus infection of TIL; Figure 8C shows the gMFI of mIL-15 and hEGFRt detected by flow cytometry after 055 lentivirus infection of TIL and co-cultured with trophoblast cells K562-mIL21-4-1BBL for 18 days. SCM ; Figure 8D shows that after TIL was infected with 055 lentivirus, it was co-cultured with trophoblast cells K562-mIL21-4-1BBL for 18 days, and IL7Ra was detected by flow cytometry; Figure 8E 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

[0071] After extensive and in-depth research, the inventors have for the first time provided a membrane-bound IL-15 fusion protein with a specific structure. The fusion protein (the fusion protein of the present invention) includes an IL-15 element and a CD86 transmembrane region and intracellular domain. The fusion protein of the present invention unexpectedly significantly increases the proportion of stem cells in TIL cells and significantly enhances their anti-tumor activity. In addition, the present invention also uses the 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.

[0072] Specifically, the inventors engineered TIL cells, preferably using the hypoxia response element (HRE) to induce expression of the fusion protein of the present invention, and employed a truncated hEGFRt as a molecular switch in the molecular construction to further enhance safety. The engineered TIL cells of the present invention exhibit significantly enhanced stemness and persistence, providing long-lasting immune monitoring and therapeutic capabilities.

[0073] Fusion polypeptide of the present invention

[0074] 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 the fusion protein of the present invention comprising IL-15, a transmembrane domain and an intracellular domain.

[0075] Interleukin IL-15 is a pro-survival cytokine that maintains homeostasis of long-lived CD8+ memory T cells, inhibits activation-induced cell death (AICD), enhances anti-tumor activity in vivo, and reverses T cell anergy. Monomeric IL-15 is a small, unstable protein with a short serum half-life, requiring supraphysiological administration to achieve an in vivo response. In the present invention, IL-15 is linked to a transmembrane domain and an intracellular domain to obtain membrane-bound IL-15, which has the function of maintaining the long-term persistence of the memory stem cell phenotype.

[0076] 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.

[0077] 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): X-IL15-LH-TM-Cyto (I)

[0078] in,

[0079] “-” each independently represents no peptide or a connecting peptide;

[0080] X is none or signal peptide;

[0081] IL15 is interleukin 15;

[0082] L is none or a linker;

[0083] H is a hinge region; the preferred hinge region of the present invention is the CD86 hinge region;

[0084] TM is a transmembrane domain; the preferred transmembrane domain of the present invention is the CD86 transmembrane domain;

[0085] Cyto is an intracellular domain, and the preferred intracellular domain of the present invention is the CD86 intracellular domain.

[0086] Among them, the preferred amino acid sequence of IL-15 is as follows:

[0087] Preferably, the amino acid sequence of the CD86 intracellular domain is as follows:

[0088] Preferably, the amino acid sequence of the CD86 transmembrane domain is WITAVLPTVIICVMVFCLILW (SEQ ID NO: 3).

[0089] Preferably, the amino acid sequence of the CD86 hinge region is LEDPQPPPDHIP (SEQ ID NO: 4).

[0090] Preferably, the amino acid sequence of the linker is SGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 5).

[0091] Preferably, the amino acid sequence of the signal peptide is MDWTWILFLVAAATRVHS (SEQ ID NO: 6).

[0092] Preferably, the amino acid sequence of the fusion protein of the present invention is as follows:

[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, wherein 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., an n×HRE promoter. Hypoxia is a hallmark 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 an oxygen-deficient 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 uses the 3×HRE-TK-mini promoter, whose sequence is shown in SEQ ID NO:8.

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

[0098] In one embodiment, the present invention uses the 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 a cell expressing the nucleic acid construct presents a safety risk, the molecular switch element can be used as a target to eliminate the risky cell using a corresponding drug.

[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 expression of the molecular switch element is controlled by a second promoter. In one embodiment, the second promoter is the SFFV promoter, which is a constitutive promoter that can stably and efficiently drive 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. If cells containing the nucleic acid construct of the present invention present a safety risk, the hEGFRt therapeutic monoclonal antibody cetuximab can be injected to eliminate the target cells through ADCC and CDC, further improving safety. Flow cytometry 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, an SFFV promoter sequence, a second signal peptide sequence, and a hEGFRt sequence.

[0104] Preferably, n in the n×HRE-TK-mini promoter 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 a viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (e.g., non-additive 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 autonomously replicate, 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, certain vectors can direct 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 typically in the form of a plasmid. In this specification, "plasmid" and "vector" are used interchangeably, as 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 T cells reduces 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, which has the highest self-renewal capacity 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 can 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 can be suitable for use in the treatment of therapeutic indications as 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 conventional 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 is non-toxic to the host or subject.

[0114] The fusion protein or immune effector cell can be delivered together with a pharmaceutically acceptable vehicle. In one embodiment, the 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 colloidal preparations comprising pharmaceutically acceptable polymers.

[0115] The pharmaceutical composition comprising one or more fusion proteins or immune effector cells can be prepared 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 effects of drugs, including but not limited to immunological adjuvants, which enhance or diversify the immune response to antigens.

[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, a "subject" can be a clinical patient, a clinical trial volunteer, an experimental animal, or the like. The subject may be suspected of having a disease characterized by cell proliferation or have a risk of developing a disease characterized by cell proliferation, be diagnosed with a disease characterized by cell proliferation, or be 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 compositions 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 pancreatic 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 this 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 the 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 only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A 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] Nucleotide sequences of the elements in Table A

[0126] Example 1: Infection of TIL cells with lentivirus containing target gene

[0127] 1. Plasmid construction

[0128] Based on the characteristics of the hypoxic microenvironment of solid tumors, four constructs, designated constructs 001, 002, 003, and 004, were designed and constructed using 3×HRE, 6×HRE, and 9×HRE elements for inducible expression. These constructs were then synthesized. As shown in Figure 1A, the nucleotide sequence of IL-15 in each construct is as described in SEQ ID NO:12. Construct 001 served as a control and employed the CD80 transmembrane and intracellular domains, driven by the 3×HRE-TK-mini promoter. Constructs 002, 003, and 004 employed the CD86 transmembrane and intracellular domains, driven by the 3×HRE-TK-mini promoter, 6×HRE-TK-mini promoter, and 9×HRE-TK-mini promoter, respectively. All four constructs contained a secondary promoter, SFFV, a secondary signal peptide, SP2, and the molecular switch element, hEGFRt. The plasmids containing each of the four constructs were constructed as follows:

[0129] (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., elimination of repetitive sequences and cryptic splicing sites and unnecessary restriction enzyme sites, while preventing the depletion of tRNA pool in cells;

[0130] (2) The optimized sequence was directly cloned into the lentiviral shuttle vector pLV-EF1a-c-MYC-IRES-EGFP (Wuhan Miaoling) by whole gene synthesis by GenScript, and the sequence from the EFI1a promoter to EGFP in the vector was replaced with the target sequence. The newly constructed vector was named: pLV-HRE-mIL-15.

[0131] 2. Lentivirus Preparation

[0132] 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 500 μl + main plasmid 10 μg + pMD2.G 5 μg + psPAX2 5 μg + p3000 40 μl; (2) Centrifuge tube B: OPti-MEM 500 μl + Lipo3000 40 μl. Slowly drip the mixture in centrifuge tube B into centrifuge tube A, gently mix with a pipette, and let it stand at room temperature for 15-20 minutes.

[0133] 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 cells were placed in a 37°C, 5% CO2 incubator for further culture. After 48 hours, the transfected 293T cell culture supernatant was collected and centrifuged at 400g for 5 minutes. The filtrate obtained was filtered through a 0.45μm filter to obtain the original solution of the recombinant lentivirus. The lentivirus was concentrated using an Utra-15 centrifugal filter device and centrifuged at 5000rpm for 50 minutes. The concentrated lentivirus was aliquoted and stored in a -80°C refrigerator for later use.

[0134] 3. Lentiviral titer determination

[0135] Count the HEK-293T cells after washing and digestion. Dilute the concentrated lentiviral stock solution in 10-fold gradient dilution (50×, 500×, 5000×, 50000×). Take 2.5×10 5 Add 500 μl of culture medium-virus-cell mixture to the corresponding centrifuge tubes, mix well and transfer to 24-well plates; culture in a 37°C, 5% CO2 incubator for 24 hours, then replace the culture medium; after 48 hours, collect the infected HEK-293T cells and treat them with hypoxia. Flow cytometry is used to detect the proportion of cells with positive transgene expression and 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.

[0136] 4. Preparation of lung cancer TIL cells

[0137] Place the lung cancer tumor 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, and then cut it into 1-3 mm pieces. 3Small pieces of lung cancer tumors were cultured in RPMI1640 medium supplemented with 10% AB serum, 1% glutamine, 1% double-antibody, and 6000 IU / mL IL-2. Observe the cells under a microscope. If no adherent cells were evident, change the medium every other day. If there was no gradual increase in lymphocytes or decrease in cancer cells, continue changing the medium. If the lymphocyte density increased significantly, such as the appearance of cell clusters, expand the wells and continue culturing in a 37°C, 5% CO2 incubator. Continue culturing for no more than 10 days before harvesting TIL cells. Filter the harvested cell suspension through a 40μm filter to remove the tumor mass. Centrifuge at 1000 rpm for 5 minutes, resuspend in an appropriate amount of REP complete medium, and set aside.

[0138] 5. Lentiviral infection of TIL cells

[0139] According to the titer of lentivirus, TIL cells were infected with MOI=10; lentivirus (MOI=10), Lentiboost (100×), TIL cells (3.5×10 5 ) were added to REP complete medium, with 200 μl of medium-virus-cell mixture in each centrifuge tube. After mixing, the cells were transferred to a 48-well plate and cultured in a 37°C, 5% CO2 incubator. After 24 h of infection, the cells were collected and centrifuged at 1000 rpm, and the virus solution was discarded. The cells were resuspended in an appropriate amount of REP complete medium, transferred to a 24-well plate, and cultured in a 37°C, 5% CO2 incubator.

[0140] 6. Hypoxia treatment of lentiviral-infected TIL cells

[0141] After culturing the TIL cells infected with the lentivirus in step 5 for 72 h, the cells were collected and counted; 1×10 6 The remaining TIL cells (as the control group) were transferred to a new 24-well plate and cultured in a 37°C, 5% CO2 incubator. 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.

[0142] 7. Flow cytometry

[0143] 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 of PBS containing 2% FBS. Subsequently, 2.5 μl of Fc was added to block Fc receptors and incubated at room temperature for 10 min. The cells were divided into two groups, a negative group and a test group, with a cell volume of 50 μl in each group, i.e., 2.5 × 10 5cells; IL-15 (Beijing Baixinyi Biotechnology Co., Ltd., A09D21-9E) and cetuximab injection (Merck) antibody mixture were added to the test group and incubated at room temperature for 15 minutes; after the incubation, 1 ml of PBS containing 2% FBS was added and centrifuged at 400g for 5 minutes; the supernatant was discarded, and 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 in PBS containing 2% FBS was added to the cells of the test group and incubated at room temperature for 10 minutes; 50 μl of 7-AAD mixture prepared in PBS containing 2% FBS was added to the cells of the test group and incubated at room temperature for 7 minutes; 1 ml of PBS containing 2% FBS was added to the all-negative group and the test group and centrifuged at 400g for 5 minutes; the supernatant was discarded, and 200 μl of PBS containing 2% FBS was added to resuspend the cells and detect them on the instrument.

[0144] 8. Results

[0145] After flow cytometry staining of TIL cell suspensions infected with lentivirus containing the target gene, analysis was performed using a Beckman flow cytometer. The results, as shown in Figure 1B, indicate that plasmid construction and lentiviral infection were successful, resulting in 001-TIL, 002-TIL, 003-TIL, and 004-TIL, respectively. Compared with uninfected TIL, the proportion of mIL-15+ cells in the TIL group infected with lentivirus without hypoxia treatment was lower; after hypoxia treatment, the proportion of mIL-15+ cells in TIL increased. Furthermore, compared with the non-hypoxia group, the proportion of hEGFRt+ cells in the TIL group infected with lentivirus after hypoxia treatment did not change significantly due to the regulation of the constitutive promoter SFFV.

[0146] The results showed that 3×HRE, 6×HRE, and 9×HRE were all able to induce the expression of mIL-15. Surprisingly, among the three promoters, the 3×HRE promoter produced the highest proportion of mIL-15+ cells under hypoxia-induced conditions.

[0147] Example 2: Detection of IL-15 in the supernatant of TIL cells infected with lentivirus

[0148] 72 hours after lentiviral infection, TIL cells were subjected to hypoxia. After 24 hours of hypoxia, culture supernatants from 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 a control well. The wells were sealed with film and incubated at room temperature (18-25°C) for 2 hours. Wash the wells: Remove all liquid from the wells and add 300 μl of 1× Wash Buffer. Allow to stand for 1 minute, then discard the liquid. Repeat three times, patting dry on filter paper each time. Add 100 μl of biotinylated detection antibody (human IL-15) (Dayou, 1111502) working solution. Cover the wells with film and incubate at room temperature (18-25°C) for 1 hour. Wash the wells three times. Add 100 μl of Streptavidin-HRP working solution. Cover the wells with film and incubate at room temperature (18-25°C) for 30 minutes. Wash the wells three 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.

[0149] Lung cancer TIL cells were infected with lentivirus containing the target gene for 72 hours and then treated with hypoxia. The culture supernatants of cells in the non-hypoxia and hypoxia groups were collected, and the shedding of mIL-15 was detected by ELISA.

[0150] The results are shown in FIG2 . 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 experience shedding.

[0151] Example 3: mIL-15 promotes T SCM Cell formation, reducing T cell exhaustion

[0152] TILs were infected with lentiviruses with different plasmid structures 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.

[0153] 1. Construction of trophoblast cells K562-mIL21-4-1BBL

[0154] Plasmids containing the target genes IL21 and 4-1BBL were constructed: the sequences containing IL21, 4-1BBL, IRES, mCherry, and Puro were fully synthesized (GenScript) and then cloned into the pLV-EF1a-c-MYC-IRES-EGFP vector, replacing the original c-MYC-IRES-EGFP sequence. After obtaining the plasmid, lentiviral packaging was performed, and its titer was calculated. Based on the lentiviral titer, K562 cells were infected at an MOI of 10. Lentivirus-infected K562 cells were treated with puromycin (2 μg / ml) to obtain trophoblast cells K562-mIL21-4-1BBL. IL21 expression in K562-mIL21-4-1BBL cells was detected by flow cytometry.

[0155] The amino acid sequence of mIL21-4-1BBL is shown below:

[0156] 2. Co-culture of lentivirus-infected TIL cells with K562-mIL21-4-1BBL

[0157] Puromycin-treated K562-mIL21-4-1BBL cells were collected and centrifuged at 1000 rpm for 10 min. After centrifugation, the supernatant was discarded and the cells were washed three times with PBS. The cells were counted and the cell density was adjusted to 1×10 cells by adding RPMI1640 complete medium. 6 / ml; add 6 μg / ml mitomycin C, mix well and transfer to a T75 culture flask; place in a 37°C, 5% CO2 incubator and culture for 18-24 hours.

[0158] Day 1: After 72 hours of lentivirus infection, TIL cells were subjected to hypoxia treatment; after 24 hours of hypoxia, cells from the non-hypoxia and hypoxia 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 the centrifugation, the supernatant was discarded and the cells were 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 (anti-CD3 is not added in subsequent changes); subculture when the cell confluence reaches 80%.

[0159] 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.

[0160] 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 cells were resuspended in the corresponding REP complete medium and counted; 1×10 6 The cells were detected by flow cytometry.

[0161] 3. Flow cytometry

[0162] (1) Flow cytometry detection of trophoblast cells K562-mIL21-4-1BBL:

[0163] K562 cells infected with lentivirus treated with puromycin were collected and counted, and 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 of PBS containing 2% FBS. 2.5 μl of Fc was added to block Fc receptors and incubated at room temperature for 10 min. The cells were divided into two groups, a negative group and a test group, 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 (1.25 μl antibody for 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 of PBS containing 2% FBS to resuspend the cells, and detect on the machine.

[0164] (2) Flow cytometry detection of TIL cells:

[0165] After co-culture of TIL cells and trophoblast cells for 18 days, cells were collected and counted, and 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 of PBS containing 2% FBS. Then 4 μl of Fc was added to block Fc receptors and incubated at room temperature for 10 min. The cells were divided into three groups: a completely 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 was added to each sample) and 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 TM Anti-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 minutes. After the incubation, 50 μl of 7-AAD mixture prepared with PBS containing 2% FBS was added to the cells of the first and second groups to be tested and incubated at room temperature for 7 minutes. 1 ml of PBS containing 2% FBS was added to the negative group and the group to be tested, and the cells were centrifuged at 400 g for 5 minutes. The supernatant was discarded, and 200 μl of PBS containing 2% FBS was added to resuspend the cells and tested.

[0166] 4. Results

[0167] Figure 3A shows a schematic diagram of the structure of trophoblast K562-mIL21-4-1BBL. Figure 3B shows that compared with K562 cells, the proportion of mIL21+ cells in the trophoblast K562-mIL21-4-1BBL was 84.3%, indicating that the trophoblast K562-mIL21-4-1BBL was successfully constructed. Figure 3C shows that compared with the control TIL cells cultured with IL2, there was no significant difference in the proliferation and viability of TIL cells overexpressing mIL-15 when cultured without IL2, indicating that overexpression of mIL-15 in TILs can promote their proliferation. Specifically, after hypoxia treatment, 002-TIL cells proliferated better than the non-hypoxia group.

[0168] As shown in Figure 3D, 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, T SCMCompared with the non-hypoxia group, the proportion of T cells in 001-TIL and 002-TIL cells increased significantly after hypoxia treatment. SCM Compared with 001-TIL (CD80-TM and CD80-Cyto), the T of 002-TIL structure using CD86-TM and CD86-Cyto in the present invention is increased. SCM The proportion increased significantly.

[0169] As shown in Figure 3E, compared with the control 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 its stemness. At the same time, as shown in Figure 3F, 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 of PD-1 and Tim3 in CD8+ T cells was significantly reduced; compared with 001-TIL (CD80-TM and CD80-Cyto), the expression of PD-1 and Tim3 in CD8+ T cells of the structure 002-TIL using CD86-TM and CD86-Cyto in the present invention was significantly reduced.

[0170] The results showed that TIL cells overexpressed mIL-15, which promoted their T SCM The generation of cells was inhibited and the expression of their exhaustion proteins was reduced; and the effect of using CD86-TM-CD86-Cyto was significantly better than that of CD80-TM-CD80-Cyto.

[0171] Example 4: TIL-mIL15 cells exhibit a poorly differentiated phenotype

[0172] To further confirm the effect of mIL15 on T cell differentiation, exhaustion, and apoptosis, transcriptome sequencing and flow cytometry analysis were performed on UN-TIL+IL2, TIL-mIL15+IL2, and TIL-mIL15-IL2 cells expanded for 14 days based on the K562-mIL21-4-1BBL cell culture platform.

[0173] 1. RNA-Seq analysis

[0174] Based on the K562-mIL21-4-1BBL cell platform, 5×10 6The cells were used for transcriptome sequencing to analyze the differences in gene expression. Total RNA was extracted using the EASYspinPlus Tissue / Cell RNA Rapid Extraction Kit (Beijing Adlai Biotechnology, #RN2802). cDNA libraries were constructed using the Ultra™ RNA Library Prep Kit. Insert sizes of the cDNA libraries were measured using an Agilent 2100 bioanalyzer. If the insert sizes met expectations, the effective concentrations of the cDNA libraries were accurately quantified by qRT-PCR, followed by Illumina sequencing. Data analysis was performed using the Reads Per Kilobase per Million mapped reads (RPKM). Differentially expressed genes were identified as having a log2 (fold change) >1.5 or a log2 (fold change) <-1.5, with edgeR indicating statistical significance (p < 0.05).

[0175] 2. Flow cytometry detection of TCF1

[0176] (1) Surface staining: After TIL cells were co-cultured with K562-mIL21-4-1BBL cells for 14 days, the cells were collected and counted. 2×10 6 cells, and Brilliant Violet 510 TM staining with anti-human CD3 antibody (BioLegend, 317332) and eFluor 600 EGFR antibody (eBioscience, 50-9509-42);

[0177] (2) TCF1 staining: After surface staining, cells were subjected to Zombie staining and washed with 1× PBS. After fixing the cells with True-Nuclear Transcription Factor Buffer Set (BioLegend, #424401), they were stained with PE-anti TCF1 (BioLegend, 655208) antibody.

[0178] 3. Results

[0179] As shown in Figure 4A, RNA-seq analysis identified 1,344 upregulated genes and 51 downregulated genes in TIL-mIL15+IL2 cells compared with UN-TIL+IL2 cells, while 1,858 upregulated genes and 160 downregulated genes were identified in TIL-mIL15-IL2 cells. Compared with TIL-mIL15+IL2 cells, 164 upregulated genes and 239 downregulated genes were identified in TIL-mIL15-IL2 cells. As shown in Figure 4B, enrichment of multiple signaling pathways was also observed in TIL-mIL15-IL2 cells, among which the JAK-STAT, mTOR, and PI3K-Akt signaling pathways were closely related to the regulation of mIL15. As shown in Figure 4C, signature genes related to T cell differentiation and memory were enriched in TIL-mIL15-IL2 cells. As shown in Figure 4D, the expression of T cell differentiation and effector-related genes PRDM1 (encoding BLIMP-1), ID2, EOMES, IFNG, GZMB, and TNF in TIL-mIL15-IL2 cells was significantly reduced, while the expression of memory stem cell-like T cell marker TCF7 (encoding TCF1) was significantly increased; and the expression of survival genes BCL2 and BCL2L1 was significantly increased; and the expression of genes related to inhibitory regulatory factors LAG3, HAVCR2 (encoding TIM3), TIGIT, TOX, and CTLA4 in TIL-mIL15-IL2 cells was reduced. As shown in Figure 4E, the expression level of TCF1 in TIL-mIL15-IL2 cells was much higher than that in UN-TIL+IL2 and TIL-mIL15+IL2 cells.

[0180] Example 5: Anti-tumor activity of TIL cells overexpressing mIL-15

[0181] To verify whether overexpression of mIL-15 in TIL cells promotes their anti-tumor activity and the differences in the effects of CD80-TM and CD80-Cyto on mIL-15 compared with CD86-TM and CD86-Cyto, 001-TIL and 002-TIL cells were co-cultured with Calu6 tumor cells, and CCK8 and ELISA were used to detect tumor activity and IFN-γ secretion. The experimental methods are as follows:

[0182] 1. Detection of tumor cell activity by CCK8 assay

[0183] Take 1×10 5 Calu6 cells were seeded in 24-well plates. TIL cells were infected with lentivirus for 72 hours and then subjected to hypoxia. After 24 hours of hypoxia, TIL cells from the non-hypoxic and hypoxic groups were harvested 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-hypoxia and hypoxia TIL cell suspension (i.e. 1.5×10 6 After culturing in a 37°C, 5% CO2 incubator for 24 hours, the cell culture supernatant was collected and washed once with PBS. The activity of tumor cells was detected using the CCK8 assay.

[0184] 2. ELISA to detect IFN-γ secretion

[0185] Centrifuge the collected cell culture supernatant at 400g for 5 minutes and transfer the supernatant to a fresh centrifuge tube. Determine the number of strips required based on the number of experimental wells (blank and standard). Add 100μl of diluted standard and sample, and 100μl of 1× Dilution Buffer R as a control well. Add 100μl of biotinylated human IFN-γ antibody (Dayou, 1110002) working solution. Cover with film and incubate at room temperature (18-25°C) for 1 hour. Wash the plate: Remove all liquid from the well and add 300μl of 1× Wash Buffer working solution. Allow to stand for 1 minute, then discard the liquid. Repeat three times, patting dry on filter paper each time. Add 100μl of Streptavidin-HRP working solution. Cover with film and incubate at room temperature (18-25°C) for 20 minutes. Repeat the wash three times. Color development: Add 100 μl TMB and 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.

[0186] 3. Results

[0187] The results are shown in Figure 5, Table 1 and Table 2.

[0188] As shown in Figure 5A, 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.

[0189] As shown in Figure 5B, 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 was significantly increased; and compared with 001-TIL, the secretion of IFN-γ by 002-TIL cells was higher.

[0190] Table 1. Antitumor activity of different TIL cells

[0191] Table 2. IFN-γ secretion of different TIL cells

[0192] 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.

[0193] Example 6: Cytotoxic Effect

[0194] In clinical practice, in order to further improve the safety of TIL cell therapy that overexpresses mIL-15, the present invention provides a membrane surface domain (hEGFRt) as an immune brake element and / or recognition element. The hEGFRt-specific monoclonal antibody cetuximab can mediate complement-dependent cytotoxicity (CDC) and NK cell-mediated cytotoxicity (ADCC) to effectively lyse TIL cells that express mIL-15.

[0195] CDC involves the binding and activation of complement molecules by the Fc end of an antibody, followed by the binding of the Fab end of the antibody to target cells, forming a membrane attack complex, and ultimately leading to the lysis of the target cells. In the present invention, hEGFRt expressed by TIL cells binds to cetuximab and the added complement molecules, leading to the lysis of the TIL cells.

[0196] ADCC refers to the binding of the Fab segment of an antibody to the antigenic epitope 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 of action of anti-tumor therapeutic antibody drugs.

[0197] The experimental method is as follows:

[0198] (1) Complement-dependent cytotoxicity (CDC) assay

[0199] Lentivirus-infected TIL cells were co-cultured with K562-mIL21-4-1BBL cells for 18 days and then treated with hypoxia. Cells were collected and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were resuspended in the corresponding REP complete medium and counted. 5×10 5 The cells were used for CDC experiments; 48-well plates were used, the culture system was 200 μl, and the cells were divided into three 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 analysis.

[0200] (2) Antibody-dependent cellular cytotoxicity (ADCC) assay

[0201] Lentivirus-infected TIL cells were co-cultured with K562-mIL21-4-1BBL cells for 18 days and then treated with hypoxia. Cells were collected and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were resuspended in the corresponding REP complete medium and counted. 1×10 6 ADCC experiments were performed on cells; 24-well plates were used, the culture system was 1 ml, and the cells were divided into four experimental groups, namely: TIL cells, TIL cells + 100 μg / ml cetuximab, TIL cells + NK cells (effector-target ratio 1:10) + 100 μg / ml cetuximab, and TIL cells + NK cells (effector-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 analysis.

[0202] The experimental results are shown in Figure 6. 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 6A). 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 NK cells (1:10 and 1:20) and cetuximab for 24 hours (Figure 6B).

[0203] This shows that the molecular brake element based on the mechanism of action of the hEGFRt extracellular domain has higher safety and effectiveness.

[0204] Example 7: Different connection methods of the immune brake element hEGFRt

[0205] In addition to being linked to the constitutive promoter SFFV, the immune brake element hEGFRt in the present invention also features a peptide linker sequence, T2A, that can be cleaved in vivo. The DNA expression cassette for the hEGFRt fusion protein formed by T2A linkage is translated into a fusion polypeptide in vivo, which is then cleaved simultaneously with translation. hEGFRt is transported to the extracellular space via a signal peptide and anchored to the cell membrane surface via the transmembrane region of its C-terminal region. As shown in Figure 7A, construct 005 was constructed, in which the immune brake element is linked to the peptide linker sequence T2A. The SFFV promoter sequence in construct 002 was replaced with the T2A sequence through molecular cloning techniques.

[0206] As shown in Figure 7B, lentiviral packaging was performed, and TIL cells were infected for 5 days and then treated with hypoxia for 24 hours. Flow cytometry analysis of mIL-15 and hEGFRt was performed. The results showed that plasmid construction and lentiviral infection were successful. Compared with uninfected TIL, the proportion of mIL-15+ cells in TIL cells infected with 005 lentivirus was lower before hypoxia treatment; however, the proportion of mIL-15+ cells in TIL cells increased after hypoxia treatment.

[0207] As shown in Figure 7C, compared with uninfected TIL, the gMFI of mIL-15 in TIL cells infected with 005 lentivirus was lower without hypoxia treatment; after hypoxia treatment, the gMFI of mIL-15 in TIL was significantly increased; at the same time, the gMFI of hEGFRt in TIL cells infected with lentivirus was significantly increased after hypoxia compared with the non-hypoxia group. As shown in Figure 7D, under the condition of adding IL2, both uninfected TIL and TIL infected with 005 lentivirus could continue to proliferate to day 13, and the cell viability was ≥90%; under the condition of not adding IL2, TIL infected with 005 lentivirus could continue to proliferate to day 13, and the cell viability was ≥90% compared with uninfected TIL.

[0208] The results showed that the immune brake element hEGFRt can also be induced to express when linked to T2A. In the absence of IL2, TILs infected with the 005 lentivirus were able to proliferate continuously with a cell viability of ≥90%. These results also indicate that the immune brake element hEGFRt can also be induced to express when linked to T2A. In vitro, overexpression of mIL-15 can promote TIL cell proliferation and avoid the clinical side effects of IL2 use.

[0209] Example 8: Lentiviral infection of cervical cancer TIL cells

[0210] In this example, we further investigated whether expressing mIL-15 of the present invention could promote the stemness of cervical cancer TIL cells or reduce the expression of their exhaustion proteins. The method was as follows:

[0211] Preparation of cervical cancer TIL cells: Place the cervical cancer tumor in a 100mm 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, and then cut it into 1-3mm slices. 3Cervical cancer tumor pieces were cultured in RPMI1640 medium supplemented with 10% AB serum, 1% glutamine, 1% double-antibody, and 6000 IU / mL IL-2. Observe the cells under a microscope. If no adherent cells are evident, change the medium every other day. If there is no gradual increase in lymphocytes or decrease in cancer cells, continue changing the medium. If the lymphocyte density increases significantly, such as the appearance of cell clusters, expand the wells and continue culturing in a 37°C, 5% CO2 incubator. Continue culturing for no more than 10 days, then harvest the cells. Filter the harvested cell suspension through a 40μm filter to remove the tumor pieces. Centrifuge at 1000 rpm for 5 minutes, resuspend in an appropriate amount of REP complete medium, and set aside.

[0212] 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 depletion proteins were observed.

[0213] As shown in Figure 8A, cervical cancer TIL cells were infected with 005 lentivirus for 5 days and then treated with hypoxia for 24 hours. Flow cytometry analysis of mIL-15 and hEGFRt was performed. 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 before hypoxia treatment; after hypoxia treatment, the proportion of mIL-15+ and hEGFRt+ cells in TIL cells increased.

[0214] As shown in Figure 8B, compared with uninfected TIL, the gMFI of mIL-15 and hEGFRt in TIL cells infected with 005 lentivirus increased after hypoxia treatment.

[0215] As shown in Figure 8C, 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 cell ratio increased significantly.

[0216] As shown in Figure 8D, under non-hypoxic and hypoxic conditions, the proportion of IL7Ra+ cells in 005-TIL cells overexpressing mIL-15 in culture without IL2 was significantly increased compared with the control TIL cells cultured with IL2. SCM The proportion of IL7Ra+ cells increased, indicating that mIL-15 can enhance their stemness.

[0217] At the same time, as shown in Figure 8E, 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.

[0218] The results showed that the expression of mIL-15 in cervical cancer TIL cells can significantly promote their T SCM The generation of cells was significantly reduced and the expression of their depleted proteins was significantly decreased.

[0219] discuss

[0220] 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 greater therapeutic potential. Therefore, T cell subsets corresponding to immature differentiation states 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 necessary to prepare T cells suitable for cell therapy. SCM is crucial.

[0221] IL-15 is a pro-survival cytokine required for maintaining homeostasis of long-lived CD8+ memory T cells, inhibiting activation-induced cell death (AICD), enhancing anti-tumor activity in vivo, and reversing T cell anergy. In contrast to IL2, IL-15 does not lead to the expansion of Tregs. Furthermore, 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 either natural killer (NK) cells or T cells. Monomeric IL-15 is a small, unstable protein with a short serum half-life, requiring supraphysiological administration to achieve an in vivo response. Physiological trafficking of IL-15 requires the IL-15 receptor α chain (IL-15Rα), and recombinant soluble IL-15 binds to recombinant soluble IL-15Rα, enhancing the anti-tumor activity of T cells.

[0222] Based on the above research foundation, in order to improve the persistence and memory potential of infused T cells, the Tet-on system is used in patent application 202210316484.5, and the inducer tetracycline is added to induce the expression of IL-7 and / or IL-15. Although this method can increase the expansion of TIL cells, the Tet-on system contains the C-terminus of the herpes simplex virus (HSV) VP16 protein and 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 the addition of small molecule drugs, IL-15 can be expressed in multiple organs and tissues, lacks the selectivity of tumor microenvironment enrichment expression, and there are certain safety risks.

[0223] Hypoxia is a hallmark of solid tumors. Due to insufficient blood supply, 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, by utilizing the hypoxic microenvironment of solid tumors, the present invention uses HRE to induce the expression of membrane-bound IL-15 (mIL-15) in TIL cell transformation to maintain the long-term persistence of the memory stem cell phenotype. The use of CD86 transmembrane region and intracellular domain further increases the proportion of stem cells in TIL cells and enhances their anti-tumor activity; truncated hEGFRt is used in the molecular construction to further improve its safety. mIL-15 provides an ideal signaling pathway that prolongs the persistence of TIL cells infused into patients, thereby providing long-lasting immune surveillance and therapeutic potential.

[0224] All documents mentioned herein are incorporated herein by reference as if each document were individually incorporated by reference. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the appended claims.

Claims

1. A fusion polypeptide, characterized in that: The fusion polypeptide comprises the following elements: (i) interleukin 15 (IL-15) or a functionally active fragment thereof; (ii) a transmembrane domain; and (iii) intracellular domain; Wherein, the intracellular domain is the CD86 intracellular domain.

2. The fusion polypeptide according to claim 1, characterized in that The structural formula of the fusion polypeptide is shown in formula (I): X-IL15-LH-TM-Cyto (I) in, "-" each independently represents nothing or a peptide bond; X is none or signal peptide; IL15 is the interleukin 15 element; L is none or a linker; H is the hinge region; TM is the transmembrane domain; Cyto is an intracellular domain, wherein the intracellular domain is the CD86 intracellular domain.

3. The fusion polypeptide according to claim 1, wherein the amino acid sequence of the CD86 intracellular domain is shown in SEQ ID NO:

2.

4. The fusion polypeptide according to claim 2, wherein the transmembrane domain is a CD86 transmembrane domain, and / or the hinge region is a CD86 hinge region.

5. The fusion polypeptide according to claim 1, wherein 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. A carrier, characterized in that The vector comprises the polynucleotide according to claim 6.

9. 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 8.

10. The cell according to claim 9, characterized in that The cells are tumor infiltrating cells (TIL).

11. 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 8, and / or the cell of claim 9, and a pharmaceutically acceptable carrier.

12. 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 8, the cell of claim 9, and / or the composition of claim 11.

13. Use of the fusion polypeptide according to claim 1, the polynucleotide according to claim 6, the nucleic acid construct according to claim 7, the vector according to claim 8, the cell according to claim 9, the composition according to claim 11, and / or the kit according to claim 12 in the preparation of a medicament for preventing, alleviating and / or treating tumors.

14. The use according to claim 13, characterized in that The tumor is a solid tumor, a hematological tumor, or a combination thereof.

15. A method for treating a disease, characterized in that: The method comprises administering the immune cell of claim 9 or the composition of claim 11 to a subject.

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